MiR-200a调控卵巢上皮癌化疗敏感性、增殖及CSCs的作用及其机制研究
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摘要
研究背景
卵巢上皮癌(epithelial ovarian cancer, EOC)的病死率高居妇科恶性肿瘤之首,晚期患者的5年生存率仅约30%。目前,肿瘤细胞减灭术联合铂类药物为基础的化疗,对约70%的晚期患者初期治疗有效,但大部分患者最终会出现化疗耐药导致疾病复发。因此,深入认识EOC的进展机制,有效改善EOC的不良预后是目前临床亟待解决的难题。
新近的研究发现,EOC化疗耐药与上皮间质转化(epithelial mesenchymal transition, EMT)密切相关。EMT是指上皮细胞表面黏附分子表达降低、细胞间连接变得疏松、细胞骨架重塑,转变成间充质细胞表型过程。已有的研究证实EMT在肿瘤侵袭、转移和化疗耐药中具有重要的调控作用。更重要的是,有研究发现EOC中存在的肿瘤干细胞(cancer stem cells, CSCs)具有EMT的特征。CSCs学说认为：肿瘤是由异质性的细胞群体构成；其中存在极少量具有干细胞性质的癌细胞亚群,是肿瘤发生、进展、转移及耐药性产生的根源。因此,抑制EMT可能成为有效改善EOC预后的策略。
微小RNA(microRNA, miRNA, miR)是一类非编码的单链小分子RNA,长度约18-24个核苷酸(nucleotide, nt),其通过与mRNA3'端非翻译区(3'-untranslated region,3'-UTR)碱基识别配对,调控靶基因的表达。目前已发现miRNAs在调控EMT中具有重要作用,亦与多种肿瘤的发生及进展关系密切。Yang,等分析了459例EOC肿瘤组织的mRNA及miRNAs表达谱特征,证实间充质表型与EOC的不良预后相关,包括miR-20a、miR-141和miR-506在内的8个关键miRNA,调控了高达89%的间充质相关基因表达。
miR-200a和miR-141同属于miR-200家族(miR-200s,包括miR-200a/b/c/和miR-141/429共5个miRNA)。近年的研究发现miR-200s在多种肿瘤中具有阻碍EMT进程、抑制CSCs自我更新和去分化状态、调控细胞增殖及凋亡、逆转化疗耐药的作用。在EOC中已证实miR-200s的表达水平与化疗敏感性、无进展生存期(progression-free survival, PFS)以及总生存期(overall survival, OS)呈正相关。这些研究结果说明以miR-200s作为EOC潜在的治疗策略,具有理论上的可行性和积极的临床意义。
有研究证实,在EOC细胞株Hey中,过表达miR-200c通过靶向调控TUBB3增加了紫杉醇的敏感性。我们前期在卵巢浆液性腺癌细胞系--OVCAR-3中验证了CD133+细胞具有CSCs特征；进一步用miRNA芯片发现CD133+与CD133-细胞间存在miRNAs的差异表达谱；其中miR-200a在CD133+细胞中下调了约2.16倍,利用合成的miR-200a模拟物—miR-200a mimics,在CD133±细胞中瞬时上调miR-200a水平,发现miR-200a通过靶向下调ZEB2抑制了CD133+细胞的迁移和侵袭能力,初步证实miR-200a在EOC中具有抑制转移的积极作用。但目前关于miR-200a对EOC化疗敏感性和CSCs的调控作用及其相关机制尚未完全阐明。
我们发现瞬时转染,miR-200a mimics的方法仅适用于短期内观察,miR-200a对EOC细胞的影响；并且分选得到的CD133+细胞所占比例非常低(约0.2-1%)；另外,CD133+细胞在体外培养过程中逐渐分化为CD133-细胞的问题,都将为我们深入探索miR-200a在EOC中的生物学功能带来一定的局限性。近来,Wang,等用基因芯片鉴定了无血清培养OVCAR-3所形成的悬浮生长细胞球,具有CSCs的特征,适宜作为研究EOC中CSCs的体外模型。
综合考虑上述因素,本课题首先利用慢病毒表达载体成功构建了稳定上调miR-200a的EOC细胞模型,在普通贴壁培养和悬浮培养条件下,验证了miR-200a对EOC化疗敏感性的影响；进一步探索了miR-200a调控EOC增殖状态和CSCs的作用及其机制,以期为改善EOC的预后带来更新更有力的治疗策略。
第一章构建稳定高表达miR-200a的EOC细胞模型
目的：
利用慢病毒表达载体,构建稳定高表达miR-200a的贴壁及悬浮生长EOC细胞模型,为深入探索miR-200a对EOC的生物学作用及其机制提供平台。
方法：
1. miR-200a过表达慢病毒载体和对照空载体的鉴定。
miR-200a过表达慢病毒载体pLV-miR-200a和对照空载体pLV-control购自深圳市华安平康生物科技有限公司。通过质粒转化、质粒扩增、质粒提取、PCR扩增、DNA电泳以及DNA测序,鉴定miR-200a慢病毒表达载体和对照空载体。
2. miR-200a过表达慢病毒和对照慢病毒的包装。
慢病毒包装元件psPAX2和pMD2.G由南方医科大学肿瘤研究所提供,pLV-miR-200a或pLV-control、psPAX2和pMD2.G共同组成慢病毒包装系统。用Lipofectamine2000将慢病毒包装系统转染293T细胞,包装生产携带miR-200a过表达的慢病毒和对照慢病毒。
3. miR-200a过表达慢病毒和对照慢病毒感染EOC细胞。
用携带]miR-200a过表达的慢病毒和对照慢病毒,分别感染EOC细胞株OVCAR-3,在倒置荧光显微镜下观察报告基因一绿色荧光蛋白(Green Fluorescent Protein, GFP)的表达,以监测感染效率,应用流式细胞仪分选GFP+细胞。
4.验证miR-200a在感染后EOC细胞中的表达水平。
提取经流氏细胞仪分选后EOC细胞的总RNA, qRT-PCR检测miR-200a的表达水平,大量扩增并冻存高表达miR-200a的EOC细胞和对照细胞,用于后续实验。
5.建立miR-200a过表达的悬浮生长EOC细胞模型。
收集过表达miR-200a的贴壁生长EOC细胞和对照组细胞,分别用PBS洗涤2次,重悬于肿瘤球培养基(含EGF20ng/mL、bFGF10ng/mL、0.4%BSA、 insulin5μg/mL、100U/mL青霉素和100U/mL链霉素的DMEM/F12),接种于超低吸附培养皿,常规条件培养,间隔72h添加适量肿瘤球培养基,7d后收集球体细胞,用0.02%EDTA-2Na和0.25%胰蛋白酶1：1混合液消化球体细胞,继续用上述方法传代培养,得到悬浮生长的EOC细胞。qRT-PCR检测悬浮生长的EOC细胞中miR-200a的表达水平。
6.利用SPSS16.0统计软件对所有数据进行处理,采用单样本t检验分析贴壁及悬浮生长EOC细胞中miR-200a的表达差异,显著性水平定义α=0.05,P<0.05认为差异有统计学意义,数据用“均数士标准差”表示。
结果：
1.DNA测序证实miR-200a过表达慢病毒载体和对照空载体序列均正确。
2.成功生产了miR-200a过表达慢病毒和对照慢病毒,用以感染EOC细胞株OVCAR-3,感染后在倒置荧光显微镜下可见GFP十细胞的表达率约为20%-30%,进一步用流式细胞仪分选GFP+细胞,得到感染效率约为95%的EOC细胞。
3.成功构建了miR-200a过表达的贴壁生长EOC细胞模型。qRT-PCR验证携带miR-200a转基因的贴壁生长EOC细胞与对照细胞相比,miR-200a的表达水平升高(3.67±0.45)倍。统计分析结果提示,携带miR-200a转基因的贴壁生长EOC细胞和对照细胞,miR-200a的表达差异具有统计学意义(t值=10.187,P=0.009)。
4.成功构建miR-200a过表达的悬浮生长EOC细胞模型。qRT-PCR验证携带miR-200a转基因的悬浮生长EOC细胞与对照细胞相比,miR-200a的表达水平升高(2.05±0.39)倍。统计分析结果提示,携带miR-200a转基因的悬浮生长EOC细胞和对照细胞,niR-200a的表达差异具有统计学意义(t值=4.666,P=0.043)。
结论：
成功将目的基因miR-200a整合入EOC细胞中并实现高效表达,建立了稳定过表达miR-200a的贴壁及悬浮生长EOC细胞模型。
第二章MiR-200a对EOC化疗敏感性、增殖及CSCs的调控作用及其机制
第一节MiR-200a对EOC化疗敏感性的调控作用
目的：
在稳定过表达miR-200a的贴壁及悬浮生长EOC细胞模型中,验证miR-200a对EOC化疗敏感性的调控作用。
方法：
1.MTT检测miR-200a;对贴壁生长EOC细胞化疗敏感性的影响。
收集miR-200a过表达的贴壁生长EOC细胞和对照细胞,分别重悬于含10%FBS的RPMI1640培养基。按5x103细胞／孔接种于普通96孔板内,培养12-16h后,在细胞中分别加入不同浓度的紫杉醇(终浓度分别为：2nM,4nM,8nM,16nM,32nM)或顺铂(终浓度分别为：lug/mL,2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL),每个浓度设4个重复孔。药物作用48h后,每孔中加入201μL细胞活力检测试剂MTT(5mg/mL),继续常规培养4h后,小心弃去细胞上清液,每孔分别加入100uL DMSO,避光震荡混匀后,置酶联检测仪上测定各孔光密度(OD)值,检测波长490nm。细胞存活率(SR)按以下公式计算：加药孔吸光度平均值／空白对照组吸光度平均值x100%。实验重复三次。
2.MTT检测miR-200a;对悬浮生长EOC细胞化疗敏感性的影响。
收集miR-200a过表达的悬浮生长EOC细胞和对照细胞,分别重悬于肿瘤球培养基,按1x104细胞／孔接种于超低吸附96孔板内,培养24h后,在细胞中分别加入不同浓度紫杉醇(终浓度分别为：2nM,8nM,32nM,128nM,512nM)或顺铂(终浓度分别为：2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL),每个浓度设4个重复孔。药物作用72h后,每孔中加入20μL细胞活力检测试剂MTT(5mg/mL),继续培养4h后,箱式离心机3000rpm离心15min,此时可见肿瘤球细胞聚集于孔板的一侧,用1mL注射器小心吸弃上清液,每孔分别加入100uLDMSO,避光震荡混匀后,置酶联检测仪上测定各孔光密度(OD)值,检测波长490nm。细胞存活率(SR)按以下公式计算：加药孔吸光度平均值/空白对照组吸光度平均值x100%。实验重复三次。
3.利用SPSS16.0统计软件对所有数据进行处理,采用析因设计的方差分析和两个独立样本的t检验,分析细胞毒性试验中细胞存活率之间的差异。P<0.05认为差异有统计学意义,数据用“均数士标准差”表示。
结果：
1. miR-200a过表达的贴壁生长EOC细胞和对照组细胞,在紫杉醇的终浓度为：2nM,4nM,8nM,16nM,32nM时,细胞存活率(%)分别为：(85.86±4.16vs.87.80±2.22),(74.66±3.19vs.80.62±2.60),(65.85±4.24vs.76.15±4.46),(54.91±4.43vs.69.53±3.31),(54.62±1.76vs.66.91±3.41)。统计分析结果提示,miR-200a过表达的贴壁生长EOC细胞与对照组细胞,紫杉醇细胞毒性之间的差异总体有统计学意义(F值=63.464,P值<0.001)。
miR-200a过表达的贴壁生长EOC细胞和对照组细胞,在顺铂的终浓度为：1ug/mL,2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mLL时,细胞存活率(%)分别为：(90.24±5.00vs.85.60±3.60),(66.96±3.57vs.71.95±6.68),(62.02±2.57vs.66.46±2.89),(54.45±2.63vs.56.89±2.80),(49.39±4.21vs.46.61±2.75),(43.54±4.34vs.42.38±2.18)。统计分析结果提示,miR-200a过表达的贴壁生长EOC细胞与对照组细胞,顺铂细胞毒性之间的差异总体没有统计学意义(F值=0.247,P值=0.622)。
2. miR-200a过表达的悬浮生长EOC细胞和对照组细胞,在紫杉醇的终浓度为：2nM,8nM,32nM,128nM,512nM时,细胞存活率(%)分别为：(69.27±3.63vs.83.43±4.01),(57.03±2.88vs.78.91±0.86),(57.47±1.31vs.80.89±1.29),(57.69±1.35vs.80.11±1.80),(55.17±0.60vs.77.96±3.75)。统计分析结果提示,miR-200a过表达的悬浮生长EOC细胞与对照组细胞,紫杉醇细胞毒性之间的差异总体有统计学意义(F值=721.813,P值<0.001)。
miR-200a过表达的悬浮生长EOC细胞和对照组细胞,在顺铂的终浓度为：2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL时,细胞存活率(%)分别为：(50.80±2.35vs.54.37±1.86),(42.64±2.80vs.41.71±1.62),(49.66±2.03vs.46.92±6.37),(46.84±7.72vs.51.57±5.25),(42.66±2.98vs.45.54±5.36)。统计分析结果提示,miR-200a过表达的悬浮生长EOC细胞与对照组细胞,顺铂细胞毒性之间的差异总体没有统计学意义(F值=1.191,P值=0.284)。
结论：
miR-200a过表达增加了贴壁和悬浮生长的EOC细胞对紫杉醇的敏感性,没有影响贴壁和悬浮生长的EOC细胞对顺铂的敏感性。
第二节MiR-200a对EOC细胞增殖状态的调控作用
目的：
miR-200a具有提高贴壁和悬浮生长的EOC细胞对紫杉醇敏感性的作用,而对顺铂的敏感性没有影响；考虑到紫杉醇是靶向细胞增殖周期的药物,而顺铂为非细胞周期依赖药物,所以有必要进一步探索miR-200a对EOC细胞增殖状态的影响。本部分通过一系列体内、外实验验证了miR-200a对EOC细胞增殖状态的调控作用。
方法：
1.平板克隆形成实验检测]miR-200a;对贴壁生长EOC细胞增殖状态的影响。
收集lniR-200a过表达的贴壁生长EOC细胞和对照细胞,重悬于含10%FBS的RPMI1640培养基,按1.5x102细胞/孔接种于普通6孔板内,每组设3个复孔,常规培养10d,观察细胞克隆的形态,计数大于50个细胞的克隆数,弃掉培基,PBS小心漂洗3遍,甲醇固定10min,苏木素染色5-10min,自来水小心漂洗后,拍照记录克隆外观。计算克隆形成率=(克隆数／接种细胞数)x100%。
2.生长曲线(CCK8法)实验检测]miR-200a对贴壁及悬浮生长EOC细胞增殖状态的影响。
收集miR-200a过表达的贴壁生长EOC细胞和对照细胞,重悬于含10%FBS的RPMI1640培养基,按2.0×103细胞／孔接种于普通96孔培养板中,每组设6个复孔,培养6h、1、2、3、4、5、6d时,加入CCK8与培基混合液110μL(比例为1：10),继续常规培养1.5h,以空白对照孔调零,置酶联检测仪上测定各孔光密度(OD)值,检测波长450nm,以相对应OD值代表细胞增殖状态,各组取6孔OD值的平均值,绘制细胞增殖曲线。
收集miR-200a过表达的悬浮生长EOC细胞和对照细胞,重悬于肿瘤球培养基,按4.0×103细胞／孔接种于超低吸附96孔培养板中,每组设6个复孔,培养1、2、3、4、5、6、7d时,加入CCK8液10μL,继续常规培养4h,以空白对照孔调零,置酶联检测仪上测定各孔光密度(OD)值,检测波长450nm,以相对应OD值代表细胞增殖状态,各组取6孔OD值的平均值,绘制细胞增殖曲线。
3.流式细胞仪检测miR-200对贴壁及悬浮生长EOC细胞细胞周期分布的影响。
分别收集]miR-200a过表达及对照的贴壁和悬浮生长EOC细胞,用预冷PBS漂洗细胞两次,加入预冷70%乙醇,4℃固定过夜或-20℃长期固定,检测前PBS漂洗细胞一次,加入含50μg/mL溴化乙锭(PI)、100μg/mL RNase A、0.2%TritonX-100的PBS500μL,室温避光孵育30分钟,以标准程序用流式细胞仪检测,计数1～2万个细胞,结果用细胞周期拟和软件ModFit分析结果。
4.体内验证miR-200a对EOC细胞增殖状态的影响。
选取4-5周龄雌性BALB/c-nu/nu裸鼠作为实验对象,将miR-200a过表达的贴壁生长EOC细胞和对照组细胞,分别皮下注射移植入裸鼠背部的右侧及左侧,每侧移植1.5x106个细胞,细胞悬液总体积为100μL。实验共用8只裸鼠,每3日观察裸鼠一般情况、测量接种部位包块生长情况并记录裸鼠体重,依据移植瘤生长情况适时取材并作相关分析。
5.取移植瘤组织免疫组化检测Ki67的表达情况,光学显微镜下观察拍照,随机选取3个高倍视野,计算每个视野阳性细胞比例。阳性细胞比例=每个视野下阳性细胞数/每个视野下细胞总数×100%。
6.利用SPSS16.0统计软件对所有数据进行处理,采用两个独立样本t检验分析平板克隆形成率差异、移植瘤组织中Ki67阳性率差异、细胞周期分布差异。采用析因设计的方差分析,分析生长曲线(CCK8法)及裸鼠皮下移植瘤生长差异,P<0.05认为差异有统计学意义,数据用“均数士标准差”表示。
结果：
1.平板克隆实验中,miR-200过表达的贴壁生长EOC细胞较对照组细胞,体积缩小、细胞排列更紧密,更倾向于上皮细胞表型；miR-200a过表达的贴壁生长EOC细胞与对照组细胞的克隆形成率(%)分别为(92.52±5.60vs.79.12±4.41)。统计分析结果提示,miR-200a过表达的贴壁生长EOC细胞与对照组细胞,克隆形成率的差异具有统计学意义(t值=-4.461,P值=0.001)。
2.生长曲线(CCK8法)实验中,miR-200a过表达的贴壁生长EOC细胞和对照组细胞,在培养8h、1、2、3、4、5、6d时,吸光度值(OD)分别为(0.278±0.008vs.0.275±0.002),(0.500±0.096vs.0.433±0.035),(0.624±0.058vs.0.528±0.046),(1.028±0.115vs.0.710±0.048),(1.530±0.160vs.1.000±0.171),(1.993±0.206vs.1.239±0.159),(2.450±0.233vs.1.428±0.161)。统计分析结果提示,miR-200a过表达的贴壁生长EOC细胞与对照组细胞,细胞增殖的差异总体具有统计学意义(F值=199.507,P值<0.001)。
miR-200过表达的悬浮生长EOC细胞和对照组细胞,在培养1、2、3、4、5、6、7d时,吸光度值(OD)分别为(0.218±0.015vs.0.216±0.013),(0.333±0.013vs.0.323±0.014),(0.462±0.028vs.0.437±0.021),(0.720±0.078vs.0.565±0.050),(0.952±0.081vs.0.688±0.062),(1.149±0.116vs.0.803±0.054),(1.434±0.135vs.0.957±0.080)。统计分析结果提示,miR-200a过表达的悬浮生长EOC细胞与对照组细胞,细胞增殖的差异总体具有统计学意义(F值=159.036,P值<0.001)。
3.流式检测细胞周期实验中,miR-200过表达的贴壁生长EOC细胞和对照组细胞,G0/G1、S、G2/M期所占比例(%)分别为(55.86±2.55vs.72.41±3.22),(26.15±2.30vs.21.76±2.11),(13.52±1.99vs.6.82±0.76)。统计分析结果提示,miR-200a过表达的贴壁生长EOC细胞与对照组细胞,G0/G1、S、G2/M期比例的差异分别为(t值=9.861,P值<0.001)、(t值=-3.445,P值=0.006)、(t值=-7.686,P值<0.001),均具有统计学意义。
miR-200a过表达的悬浮生长EOC细胞和对照组细胞,Go/G1、S、G2/M期所占比例(%)分别为(69.52±3.24vs.90.48±2.36),(21.35±2.10vs.5.49±0.52),(11.31±1.43vs.3.32±0.28)。统计分析结果提示,miR-200a过表达的悬浮生长EOC细胞与对照组细胞,G0/G1、S、G2/M期比例的差异分别为(t值=11.470,P值<0.001)、(t值=-17.926,P值=0.006)、(t值=-13.386,P值<0.001),均具有统计学意义。
4.裸鼠皮下移植瘤实验中,miR-200过表达的EOC细胞和对照细胞均能在裸鼠皮下生长形成肿瘤。miR-200a过表达的皮下移植瘤和对照移植瘤体积(mm3),在皮下接种移植后50、53、56、59、62d分别为(110.27±57.84vs.58.19±19.55),(146.88±73.64vs.67.44±32.58),(167.03±75.68vs.82.25±48.15),(234.01±80.85vs.108.74±42.98),(366.36±99.87vs.137.44±43.13)。统计分析结果提示,miR-200a过表达的EOC细胞与对照组细胞,裸鼠皮下移植瘤增长的差异总体具有统计学意义(F值=63.441,P值<0.001)。
5.免疫组化检测移植瘤组织中Ki67表达的实验中,miR-200a过表达和对照移植瘤组织中Ki67阳性表达率(%)为(70.91±5.18vs.15.21±3.52)。统计分析结果提示,miR-200a过表达和对照移植瘤组织中,Ki67阳性表达率差异具有统计学意义(t值=-25.156,P值<0.001)。
结论：
1.过表达miR-200a增加了EOC细胞平板克隆形成率；促进了贴壁及悬浮生长EOC细胞的生长、诱导其进入增殖周期；有益于EOC细胞裸鼠皮下移植瘤生长；说明miR-200a具有促进EOC细胞的增殖的作用。这种促增殖效应可能是miR-200a增加EOC细胞对紫杉醇敏感性的作用机制之一。
第三节MiR-200a调控EOC中CSCs的作用及其机制
目的：
以上研究发现miR-200a对EOC细胞株紫杉醇敏感性及增殖状态具有一定的调控作用,接下来我们拟探索miR-200a调控EOC中CSCs的作用及其机制。通过肿瘤球形成实验、流氏细胞仪检测侧群(side population,SP)细胞比例、检测CSCs特性相关基因表达水平,验证miR-200a对EOC中CSCs干性特征的调控作用及其机制。
方法：
1.肿瘤球形成实验检测miR-200a对EOC细胞自我更新能力的影响。
收集miR-200a过表达的EOC细胞和对照细胞,重悬于肿瘤球培养基,按1×103细胞/孔分别接种于超低吸附24孔板内,每组三个复孔,常规培养7d,倒置荧光显微镜下观察肿瘤球形态,计数直径≥-70μm的肿瘤球个数。实验重复三次。
2.流氏细胞仪检测SP细胞比例,验证miR-200a对EOC中CSCs比例的影响。
收集miR-200a过表达的贴壁生长EOC细胞和对照组细胞,重悬于已预热为37℃含2%FBS的RPMI1640培养基,调整细胞密度至1×106/mL,每组各准备两管细胞,四管细胞中均加入Hoeehst33342终浓度为5ug/mL,在每组细胞中随机选择一管再加入维拉帕米终浓度为50μmol/L,37℃摇床避光孵育90min后放于冰上终止反应。4℃1000rpm离心3min,弃上清,预冷PBS洗涤一次。40μm滤器过滤待检测细胞,检测前加入碘化丙淀(PI)终浓度1μg/mL标记死细胞。流式细胞仪检测SP比例,Hoeehst33342激发光波长为350nm,405/30带通收集蓝光,570/20带通收集红光,PI用488nm蓝光激发,630/30带通收集红光。去除PI直接染色阳性的死细胞,以Hoechst Red为X轴,Hoechst Blue为Y轴作二维散点图。设门选低Hoechst Red及低Hoechst Blue且维拉帕米组缺失的区域为SP细胞。比较SP细胞比例在miR-200a过表达的EOC细胞与对照组细胞间的差别。
3. qRT-PCR和Western Blot检测miR-200a对EOC细胞中CSCs相关基因表达的调控作用。
qRT-PCR检测方法如下,提取miR-200a过表达的EOC细胞和对照组细胞总RNA,将mRNA逆转为cDNA, qRT-PCR检测(SYBR法)SOX2和OCT4mRNA水平,以GAPDH作为内源性对照。
Western Blot检测方法如下,提取miR-200a过表达的EOC细胞和对照组细胞总蛋白,BCA蛋白试剂盒测定蛋白浓度,每个泳道加30μg蛋白样品,SDS-PAGE将蛋白分离后转移到PVDF膜上,3%BSA拮抗非特异性位点,分别用SOX2和OCT4的一抗(稀释比例1：1000),4℃孵育过夜,相应二抗室温孵育1h,增强型化学发光系统(ECL)观察蛋白印迹,以P-actin作为内源性对照。
4.利用SPSS16.0统计软件对所有数据进行处理,采用两个独立样本的t检验分析肿瘤球形成差异、SP细胞比例差异,单样本的t检验分析qRT-PCR和Western Blot检测SOX2和OCT4表达差异。P<0.05认为差异有统计学意义,数据用“均数士标准差”表示。
结果：
1.肿瘤球形成实验中,我们观察到有部分miR-200a过表达的EOC细胞,在超低吸附培养皿中亦可贴壁生长,并以贴壁方式逐渐增殖,而对照组中几乎没有肿瘤球细胞贴壁生长的现象。miR-200a过表达的EOC细胞和对照细胞,肿瘤球形成个数(/1000个细胞)分别为(7.17±1.17vs.17.50±1.87)。统计分析结果提示,miR-200a过表达的EOC细胞与对照组细胞,肿瘤球形成个数差异具有统计学意义(t值=11.474,P值<0.001)。
2.流式细胞仪检测SP细胞比例的实验中,miR-200a过表达的EOC细胞和对照细胞SP细胞比例(%)为(0.233±0.076vs.0.850±0.100)。统计分析结果提示,miR-200a过表达的EOC细胞和对照细胞,SP细胞比例差异具有统计学意义(t值=8.488,P值=0.001)。
3. qRT-PCR检测miR-200a过表达的EOC细胞中SOX2和OCT4mRNA水平分别下降至对照细胞的0.467和0.361倍。统计分析结果提示,miR-200a过表达的EOC细胞与对照组细胞,SOX2和OCT4mRNA水平差异均具有统计学意义(t值=-10.932,P=0.008)和(t值=-17.416,P值=0.003)。
Western Blot结果提示miR-200过表达的EOC中细胞中,SOX2和OCT4蛋白表达水平分别下降至对照细胞的0.546和0.518倍。统计分析结果提示,miR-200a过表达的EOC细胞与对照组细胞,SOX2和OCT4蛋白水平差异均具有统计学意义(t值=-10.191,P值=0.009)和(t值=-9.659,P值=0.011)。
结论：
过表达miR-200a通过抑制EOC细胞自我更新能力、诱导分化、降低SP细胞比例、下调干性相关基因SOX2和OCT4的表达,弱化了EOC中CSCs的干性特征。
Background:
Epithelial ovarian cancer (EOC) is the most lethal gynaecologic malignancy, the5-year survival rate is only about30%in patients with advanced stage. Currently, cytoreductive surgery combined with platinum-based chemotherapy were efficient in almost70%of advanced cases at initial treatment, but most of these patients will eventually develop chemo-resistance and recurrence. Therefore, an in-depth understanding of EOC and effectively improve its poor prognosis is needed.
Recent studies have identified the close relationship between chemo-resistance and epithelial mesenchymal transition (EMT) in EOC. EMT consists of a reversible change of cell phenotype during which epithelial cells loosen cell-cell adhesion structures including adherens junctions and desmosomes, modulate their polarity and rearrange their cytoskeleton, transformed into mesenchymal cell phenotype. Previous studies have demonstrated EMT played a central role in regulating tumor invasion, metastasis and chemoresistance. Most importantly, recent studies have found that EOC cancer stem cells (CSCs) harboured post-EMT characteristics. CSCs theory believes:tumor is composed of a heterogeneous population of cells; in which very small amount of subpopulation with stem cell properties were the root of tumor progression, metastasis and chemoresistance. Therefore, inhibition of EMT may be a promising strategy to improve the prognosis of EOC.
MicroRNA (miRNA, miR) is a large class of small noncoding RNAs; approximately18-24nucleotides (nt) in length, as critical post-transcriptional regulators of gene expression through targeting mRNA3'untranslated region (3'-untranslated region,3'-UTR) base pair recognition. A growing body of evidence indicated that miRNAs played an important role in the regulation of EMT, and aberrant miRNAs profile were the defining feature in many types of tumours. Integrated genomic analyses confirmed that mesenchymal phenotype was associated with poor prognosis of EOC, and miR-200a, miR-141and miR-506were included in the eight key miRNAs predicted to target the mesenchymal subtype in459cases of serous ovarian cancer.
MiR-200a and miR-141are belong to the miR-200family (miR-200s, including miR-200a/b/c, and miR-141/429of5miRNA). MiR-200s, which are significantly involved in inhibition of EMT, repression of CSCs self-renewal, differentiation, modulation of cell division, apoptosis and reversal of chemoresistance in many types of human cancer. Accumulating evidences showed that miR-200s were associated with progression-free survival (PFS), overall survival(OS) and chemo-sensitivity in EOC. These foundings showed that investigation miR-200s as potential therapeutic strategy for EOC, has theoretical feasibility and positive clinical significance.
Studies have reported that overexpression of miR-200c may increase EOC cell line Hey's. sensitivity to paclitaxel by targeting TUBB3. Previously, we verified CD133+cells have characteristics of CSCs in EOC cell lines-OVCAR-3; further demonstrated that the profile of miRNAs was different between CD133+and CD133-cells; in which miR-200a was down-regulated about2.16times in CD133+cells compared with CD133-cells, additionally, restoration of miR-200a expression by synthetic miR-200a mimics could reduce the ZEB2-mediated migration and invasion of CD133+ovarian CSCs, which initially confirmed miR-200a has a positive effect in inhibiting metastasis in EOC. However, miR-200a regulation of chemotoxicity, CSCs phenotype and its clinical significance in EOC remains unclear.
We found transient transfection of miR-200a mimics was only suitable to observe the effects of miR-200a on ovarian cancer cells in a short term; additionally, the percentage of CD133+cells by sorting is very low (about0.2%～1%); furthermore, CD133+cells will gradually differentiate into CD133-cells in vitro, these limitations will impact our in-depth explore the biological function of miR-200a in the chemosensitivity of EOC. Recently, Wang et al. found that SDCs (spheriod derived cells) formed from OVCAR-3cells growth in serum-free culture medium, maintained CSCs characteristics and were ideal models in vitro for CSCs-targeted investigations in EOC.
Considering the above factors, herein we first constructed EOC cells model, in which miR-200a was stably over-expressed via a lentiviral expression vector, then verified the role of miR-200a on chemosensitivity in both normal2D and3D culture; then further explored the role of miR-200a in regulation proliferation, CSCs and its mechanisms, in order to bring more powerful treatment strategies resulting in improving the prognosis of EOC.
Chapter1Establishment miR-200a over-expression EOC cells model Objective:
Establishment of miR-200a over-expression EOC cells via a lentiviral expression vector in both2D and3D culture, for further exploring the biological role and mechanism of miR-200a in EOC.
Methods:
1. Identification of miR-200a over-expression lentiviral vector and control empty vector.
Over-expression of miR-200a lentivirus vector pLV-miR-200a and control empty vector pLV-control were purchased from Shenzhen, China Anping Kang Biotechnology Co., Ltd., through plasmid transformation, plasmid amplification, plasmid extraction, PCR amplification, DNA electrophoresis and DNA sequencing, identification miR-200a expression lentiviral vector and control empty vector.
2. Packaging miR-200a over-expression and control lentivirus.
Lentiviral packaging components psPAX2and pMD2.G were kept by the Southern Medical University Cancer Institute. Lentiviral packaging systems was composed by three plasmids pLV-miR-200a or pLV-control, psPAX2and pMD2.G.293T cells transfected with lentiviral packaging system by Lipofectamine2000, were utilized to produce lentivirus carrying the vector over-expression of miR-200a and control empty vector.
3. Infection EOC cells by miR-200a over-expression and control lentivirus.
Infection EOC cell line OVCAR-3by miR-200a over-expression and control lentivirus, then monitor the infection efficiency by green fluorescent protein (GFP) expression under inverted fluorescence microscope, and sorting GFP+cells by flow cytometry.
4. Verify miR-200a expression level in EOC cells after infection.
Extraction total RNA from infected EOC cells, then detection the level of miR-200a by qRT-PCR. Frozen and expanded miR-200a over-expression ovarian cancer cells and control cells for subsequent experiments.
5. Establish suspended growth model of EOC cells over-expression of miR-200a.
Collecting miR-200a over-expression EOC cells and control cells, washing twice with PBS, resuspending in tumor sphere medium (containing EGF20ng/ml, bFGF lOng/ml,0.4%BSA, insulin5μg/ml,100U/ml penicillin and100U/ml streptomycin DMEM/F12), inoculated at ultra-low petri dishes with conventional conditions, adding appropriate amount of tumor sphere medium72h later, collecting spheroid cells7days later. The collected cells were digested with0.02%EDTA-2Na and a1:1mixture of0.25%trypsin at37℃at about lmin to form single cells, re-suspend in tumor sphere medium and continue culture with the above method, then obtaining suspended growth of EOC cells. Detected the level of miR-200a in EOC cells grown in suspension by qRT-PCR.
6. Statistical Analysis. Statistical analyses were performed using SPSS version16.0, one-sample t-test was utilized to analyze the differential expression of miR-200a in adherent and suspension growth of ovarian cancer cells, the definition of significant level α=0.05, P<0.05was considered statistically significant, all data are presented as "mean values±SD".
Results:
1. DNA sequencing confirmed that the sequence of miR-200a over-expression lentiviral vector and control empty vector were correct.
2. Successfully produced miR-200a over-expression and control lentivirus for infection EOC cell line OVCAR-3. The expression of GFP was approximately20%-30%under inverted fluorescence microscope, after sorting GFP+cells by FACS, the infection efficiency was approximately95%.
3. Obtained miR-200a over-expression adhesive cells model of EOC. qRT-PCR validation the expression level of miR-200a increased about (3.67±0.45) times compared with the control, and the difference between the two group was statistically significant(t=10.187, P=0.009).
4. Obtained miR-200a over-expression suspended spheroid cells model of EOC. qRT-PCR validation the expression level of miR-200a increased about (2.05±0.39) times compared with the control, and the difference between the two group was statistically significant (t=4.666, P=0.043).
Conclusion:
1. Successfully integrated miR-200a into EOC cells, and achieved its high expression. Establishing a stable miR-200a over-expression EOC cells in the presence of adherent and suspended growth.
Chapter2The roles and mechanisms of miR-200a regulation on chemosensitivity, proliferation and CSCs in EOC
Section1The role of miR-200a regulation on chemosensitivity in EOC Objective:
Verifying the relationship between miR-200a and the chemo-sensitivity of paclitaxel and cisplatin in adherent and suspension growth of EOC cells model.
Method:
1. MTT detection of miR-200a regulation on chemosensitivity in adherent growth of EOC cells.
MiR-200a over-expressing or control adherent growth of EOC cells were collected and resuspended in RPMI1640containing10%FBS medium.5×103miR-200a over-expressing or control adherent growth of EOC cells were seeded in normal96-well plates, respectively, routine cultured12-16h later, paclitaxel (final concentration:2nM,4nM,8nM,16nM,32nM) or cisplatin (final concentration:1ug/mL,2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL), were added in the cells, each concentration was repeated four holes,48h after drug treatment, added20νL of cell viability assay MTT (5mg/mL) to each well, continue regular culture4h, the supernatant was carefully discarded and added100uL DMSO in each well, after mixing in dark, absorbance values (OD) of each well was measured by enzyme-linked detector, detection wavelength was490nm. Cell viability (SR) according to the following formula:(the average OD of dosing wells/the average OD of control wells) x100%. Draw viability curves with SR value in each group. This experiment was repeated three times.
2. MTT detection of miR-200a regulation on chemosensitivity in suspension growth of EOC cells.
MiR-200a over-expressing or control suspension growth of EOC cells were collected and resuspended in tumour spheroid medium,1×104miR-200a over-expressing or control suspension growth of EOC cells were seeded in ultra-low attach96-well plates, respectively, routine cultured24h later, paclitaxel (final concentration:2nM,8nM,32nM,128nM,512nM) or cisplatin (final concentration:2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL), were added in the cells, each concentration was repeated four holes.72h after drug treatment, added20μL of cell viability assay MTT (5mg/mL) to each well, continue regular culture4h, box-type centrifuge3000rpm,15min, then visible tumor cells aggregates in the side plates, using1mL syringe suction supernatant carefully, added100uL DMSO in each well, after mixing in dark, absorbance values (OD) of each well was measured by enzyme-linked detector, detection wavelength was490nm. Cell viability (SR) according to the following formula:(the average OD of dosing wells/the average OD of control wells) x100%. Draw viability curves with SR value in each group. This experiment was repeated three times.
3. Statistical Analysis. Statistical analyses were performed using SPSS version16.0, factorial design analysis of variance and two independent samples t-test were utilized to analyze the differences of cell viability in cytotoxicity tests. P<0.05was considered statistically significant, data are presented as "mean values±SD".
Results:
1. The cell survival rate (%) of miR-200a over-expressing adherent growth of EOC cells and control cells, after treated with a final concentration of paclitaxel:2nM,4nM,8nM,16nM,32nM, were (85.86±4.16vs.87.80±2.22),(74.66±3.19vs.80.62±2.60),(65.85±4.24vs.76.15±4.46),(54.91±4.43vs.69.53±3.31),(54.62±1.76vs.66.91±3.41), respectively. These results showed that, the difference of paclitaxel cytotoxicity between miR-200a over-expressing adherent growth of EOC cells and control cells, was statistically significant (F=63.464, P<0.001).
The cell survival rate (%) of miR-200a over-expressing adherent growth of EOC cells and control cells, after treated with a final concentration of cisplatin (final concentration:1ug/mL,2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL), were (84.26±5.50vs.90.73±5.19),(71.95±6.68vs.66.96±3.57),(66.46±2.89vs.60.52±4.93),(57.48±3.70vs.54.45±2.63),(48.91±2.40vs.51.48±1.52),(43.54±4.34vs.45.87±4.19), respectively. These results showed that, the difference of cisplatin cytotoxicity between miR-200a over-expressing adherent growth of EOC cells and control cells, was not statistically significant (F=0.247, P=0.622).
2. The cell survival rate (%) of miR-200a over-expressing suspension growth of EOC cells and control cells, after treated with a final concentration of paclitaxel:2nM,8nM,32nM,128nM,512nM, were(69.27±3.63vs.83.43±4.01),(57.03±2.88vs.78.91±0.86),(57.47±1.31vs.80.89±1.29),(57.69±1.35vs.80.1±1.80),(55.17±0.60vs.77.96±3.75), respectively. These results showed that, the difference of paclitaxel cytotoxicity between miR-200a over-expressing suspension growth of EOC cells and control cells, was statistically significant (F=721.813, P<0.001).
The cell survival rate (%) of miR-200a over-expressing suspension growth of EOC cells and control cells, after treated with a final concentration of cisplatin (final concentration:2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL), were (50.80±2.35vs.54.37±1.86),(42.64±2.80vs.41.71±11.62),(49.66±2.03vs.46.92±6.37),(46.84±7.72vs.51.57±5.25),(42.66±2.98vs.45.54±5.36), respectively. These results showed that, the difference of cisplatin cytotoxicity between miR-200a over-expressing suspension growth of EOC cells and control cells, was not statistically significant (F=1.191, P=0.284).
Conclusion:
Over-expression of miR-200a increased the sensitivity of adherent and suspension growth of EOC cells to paclitaxel, without affecting the sensitivity of adherent and suspension growth of EOC cells to cisplatin.
Section2The role of miR-200a regulation on proliferation in EOC
Objective:
Given miR-200a enhanced paclitaxel sensitivity in adherent and suspension growth of EOC cells, but no such effect to cisplatin. Considering paclitaxel is a drug targeting cell cycle, while cisplatin is not a cell cycle dependent drug, we intended to further explore the effects of miR-200a on proliferation in EOC through a series in vitro and in vivo assays.
Methods:
1. Colony forming assay was utilized to detect the impact of miR-200a on the growth state of adherent EOC cells.
MiR-200a over-expressing or control adherent growth of EOC cells were collected and resuspended in RPMI1640containing10%FBS medium.1.5×102over-expression miR-200a or control ovarian cancer cells, were seeded in normal6-well plates, respectively, three wells in each group, routine cultured10d, observed the morphology of cell clones, calculating the number of clone more than50cells, cloning efficiency=(clone number/seeded cells)×100%. Discard cells medium, carefully rinsed cells with PBS three times, fixed with methanol lOmin, hematoxylin5-10min, carefully rinsed cells with water, photographed cloning appearance.
2. Growth curve (CCK8) assay was utilized to detect the impact of miR-200a on the growth state of adherent and suspended EOC cells.
MiR-200a over-expressing or control adherent growth of EOC cells were collected and resuspended in RPMI1640containing10%FBS medium.2.0×103over-expression miR-200a or control adherent growth of EOC cells were seeded in normal96-well culture plates, respectively, six wells in each group, when cultured6h,1,2,3,4,5,6d, adding CCK8with culture medium mixture110μL (ratio of1:10),37℃cultured1.5h, absorbance values (OD) of each well was measured by enzyme-linked detector, detection wavelength was450nm, blank control wells as zero, draw growth curves with the average OD value from6wells in each group.
MiR-200a over-expressing or control suspension growth of EOC cells were collected and resuspended in tumour spheroid medium.4.0×103over-expression miR-200a or control suspended growth of ovarian cancer cells were seeded in ultra-low attach96-well culture plates, respectively, six wells in each group, when cultured1,2,3,4,5,6,7d, adding10μL CCK8,37℃cultured4h, absorbance values (OD) of each well was measured by enzyme-linked detector, detection wavelength was450nm, blank control wells as zero, draw growth curves with the average OD value from6wells in each group.
3. Flow cytometry was utilized to detect the impact of miR-200a on the cell cycle progression of adherent and suspended EOC cells.
Collecting over-expression miR-200a and control EOC cells growth in adherent and suspension, respectively, rinsed twice with cold PBS, adding cold70%ethanol,4℃fixed overnight or-20℃long-term fixed, cells were rinsed once with PBS before detection, incubated with a solution containing50μg/mL ethidium bromide (PI),100μg/mL RNase A,0.2%Triton X-100in500μL PBS for30min at room temperature in the dark, using a standard procedure by flow cytometry, counting from10,000to20,000cells, the results were intended by the cell cycle software ModFit analysis.
4. In vivo xenograft experiment was utilized to detect the impact of miR-200a on the proliferation of EOC cells.
Selected4-5week-old female BALB/c-nu/nu mice as transplant objects, subcutaneously transplanted nude mice back at right and left side with over-expression miR-200a and control EOC cells growth in adherent, respectively. Each point transplantation1.5x106cells, the total volume of the cell suspension was1100μL. The number of nude mice used in this experiment was eight. Observed the general status, measured the growth of mass in vaccination site, and record the weight of nude mice every three days. Timely drawn and correlation analysis based on tumor growth.
5. Immunohistochemistry assay was utilized to detect the expression of Ki67in transplanted tumor tissue, photographed under an optical microscope, randomly selected three high-power fields, calculate the proportion of positive cells per field. The percentage of positive cells per field=the number of positive cells/total cells per field×100%.
6. Statistical Analysis. Statistical analyses were performed using SPSS version16.0, two independent samples t-test was utilized to analyze the differences of colony formation rate, Ki67-positive rate and cell cycle distribution, and factorial design analysis of variance was utilized to analyze the differences of growth curves (CCK8) and xenograft in nude mice. P<0.05was considered statistically significant, all data are presented as "mean values±SD".
Results:
1. Colony forming assay shown, over-expression miR-200a adherent growth of EOC cells became smaller in size, arranged closer, more inclined epithelial phenotype compared with control group. The colony formation rate(%) of over-expression miR-200a adherent growth of ovarian cancer cells and control cells was (92.52±5.60vs.79.12±4.41). Statistical analysis showed that the difference of colony formation rate between miR-200a over-expressing adherent growth of EOC cells and control cells, was statistically significant (t=-4.461, P=0.001).
2. Growth curves (CCK8) experiment shown, the OD value of miR-200a over-expressing adherent growth of EOC cells and control cells, cultured in8h,1,2,3,4,5,6d, were (0.278±0.008vs.0.275±0.002),(0.500±0.096vs.0.433±0.035), (0.624±0.058vs.0.528±0.046),(1.028±0.115vs.0.710±0.048),(1.530±0.160vs.1.000±0.171),(1.993±0.206vs.1.239±0.159),(2.450±0.233vs.1.428±0.161), respectively. Statistical analysis showed that, the difference of cell growth between miR-200a over-expressing adherent growth of EOC cells and control cells, was statistically significant (F=199.507, P<0.001).
The OD value of miR-200a over-expressing suspended growth of EOC cells and control cells, cultured in1,2,3,4,5,6,7d, were (0.218±0.015vs.0.216±0.013),(0.333±0.013vs.0.323±0.014),(0.462±0.028vs.0.437±0.021),(0.720±0.078vs.0.565±0.050),(0.952±0.081vs.0.688±0.062),(1.149±0.116vs.0.803±0.054),(1.434±0.135vs.0.957±0.080), respectively. Statistical analysis showed that, the difference of cell growth between miR-200a over-expressing suspended growth of EOC cells and control cells, was statistically significant (F=159.036, P<0.001).
3. Flow cytometry cell cycle analysis shown, G0/G1, S, G2/M proportion (%) in over-expression miR-200a adherent growth of EOC cells and control cells, were (55.86±2.55vs.72.41±3.22),(26.15±2.30vs.21.76±2.11),(13.52±1.99vs.6.82±0.76), respectively. Statistical analysis showed that, the difference of G0/G1,S, G2/M ratio between miR-200a over-expressing adherent growth of EOC cells and control cells (t=9.861, P<0.001),(t=-3.445, P=0.006),(t=-7.686, P<0.001) were statistically significant.
The G0/G1, S, G2/M proportion (%) in over-expression miR-200a suspended growth of EOC cells and control cells, were (69.52±3.24vs.90.48±2.36),(21.35±2.10vs.5.49±0.52),(11.31±1.43vs.3.32±0.28), respectively. Statistical analysis showed that, the difference of G0/G1, S, G2/M ratio between over-expression miR-200a suspended growth of EOC cells and control cells (t=11.470, P<0.001),(t=-17.926, P=0.006),(t=-13.386, P<0.001) were statistically significant.
4. In vivo xenograft experiment shown, both miR-200a overexpression EOC cells and control cells could form xenograft in nude mice. The tumor volume (mm3) of over-expression miR-200a and control group after subcutaneous inoculation transplantation50,53,56,59,62d, were (110.27±57.84vs.58.19±19.55),(146.88±73.64vs.67.44±32.58),(167.03±75.68vs.82.25±48.15),(234.01±80.85vs.108.74±42.98),(366.36±99.87vs.137.44±43.13), respectively. Statistical analysis showed that the difference of tumor volume between over-expression miR-200a and control group, was statistically significant (F=63.44, P<0.001).
5. Immunohistochemistry assay shown, the positive expression rate of Ki67(%) between over-expression miR-200a xenograft and control was (70.91±5.18vs.15.21±3.52). Statistical analysis showed that the difference positive expression rate of Ki67between over-expression miR-200a and control group, was statistically significant (t=-21.156, P<0.001).
Conclusion:
Over-expression miR-200a increased EOC cells cloning efficiency, promoted cells growth, induced cells enter into cell cycle, and promoted the subcutaneous xenograft growth in nud mice, suggesting miR-200a faciliated the proliferation of EOC cells, and this effect of promoting proliferation may be one of the mechanisms involved in enhancing the chemosensivitity of paclitaxel regulated by miR-200a.
Section3The roles and mechanisms of miR-200a regulation on CSCs in EOC
Objective:
The above findings showed miR-200a has a certain influence on paclitaxel sensitivity and proliferation in EOC cells. Then we intended to further explore the effects of miR-200a regulation on CSCs in EOC through tumor sphere formation assay, detection the side population (SP) by FACS, and validation stem-related genes expression regulated by miR-200a.
Methods:
1. Tumor sphere formation assay was utilized to detect the impact of miR-200a on the self-renewal capacity in EOC cells.
MiR-200a over-expressing or control suspension growth of EOC cells were collected and resuspended in tumour spheroid medium,1×103over-expression miR-200a or control EOC cells were seeded in ultra-low attach24-well culture plates, respectively, three wells in each group, routine cultured7d, observed the morphology of tumor spheroids, calculating the number of tumor spheroids diameter≥70μm. The experiment was repeated three times.
2. Investigation the proportion of side population (SP) by FACS was utilized to detect the impact of miR-200a on CSCs ratio.
Collecting miR-200a over-expression EOC cells and control cells, resuspended in RPMI1640medium containing2%FBS preheated to37℃, cells'density was adjusted to1×106/mL. Prepare two bottles of cells in each group, Hoeehst33342was added with final concentration of5ug/mL in all four bottles of cells, randomly selected one bottle of cells in each group and verapamil was added with final concentration of50μmol/L, incubated at37℃shaker for90min in the dark, and terminated the reaction on ice.4℃1000rpm centrifuge3min, supernatant was removed, washed with pre-cooling PBS once.40μm filter filtering cells before detection, propionate iodide (PI) was added with final concentration1μg/mL to label dead cells. SP ratio was detected by FACS, Hoeehst33342excitation wavelength350nm,405/30bandpass collected blue,570/20bandpass collect red, PI was excitated by488nm blue,630/30bandpass collected red. Dead cells were directly removed by PI staining, Hoechst Red X-axis, Hoechst Blue Y-axis for the two-dimensional scatter plot. Low Hoechst Red and low Hoechst Blue and verapamil missing region was SP. Comparison the proportion of SP between miR-200a over-expression EOC cells and control cells.
3. qRT-PCR and Western Blot were utilized to detect the impact of miR-200a on regulation CSCs related genes expression.
qRT-PCR detection method is as follows, total RNA in miR-200a over-expression EOC cells and control cells was extracted, then reversed to cDNA, and qRT-PCR detected (SYBR method) SOX2and OCT4mRNA levels, GAPDH used as an endogenous control.
Western blot methods is as follows:the proteins in over-expression miR-200a or control EOC cells were extracted with RIPA lysis buffer, BCA protein assay kit was employed to assess protein concentrations, protein samples (30μg per lane) were resolved using SDS-PAGE, and then transferred to PVDF membranes, after blocking the nonspecific binding sites with3%BSA, PVDF membranes were incubated with primary antibodies at4℃overnight, specifically, rabbit monoclonal antibody to SOX2and OCT4, followed by incubation with the corresponding secondary antibodies at room temperature for1h, immunoblots were visualized using an enhanced chemiluminescence (ECL) Western Blot Detection System, P-actin used as an endogenous control.
4. Statistical Analysis. Statistical analyses were performed using SPSS version16.0, two independent samples t-test was utilized to analyze the differences of tumor sphere formation and the ratio of SP, one-sample t-test was utilized to analyze the different expression of SOX2and OCT4by qRT-PCR and Western Blot. P<0.05was considered statistically significant, all data are presented as "mean values±SD".
Results:
1. Tumor sphere formation assay showed, over-expression of miR-200a induced the attachment of suspended tumor spheroid cells, even in ultralow attachment dishes, and showed a gradual proliferation state, which could not be seen in control groups. The number of tumor spheroids(/1,000cells) in over-expression miR-200a and control EOC cells was (7.17±1.17vs.17.50±1.87). Statistical analysis showed that, the difference number of tumor spheroids between over-expression miR-200a and control EOC cells was statistically significant (t=11.474, P<0.001).
2. SP ratio by FACS experiments show that two groups of cells, the lower left corner show low fluorescence or negative cell populations, after adding verapamil these cells disappear. SP ratio (%) of over-expression miR-200a and control EOC cells was (0.233±0.076vs.0.850±0.100). Statistical analysis showed that, the differences of SP ratio between over-expression miR-200a and control EOC cells was statistically significant (t=8.488, P=0.001).
3. qRT-PCR showed that SOX2and OCT4mRNA levels in over-expression miR-200a EOC cells, decreased to (0.467±0.085) and (0.361±0.064) times, respectively, compared with control. Statistical analysis showed that, the difference mRNA levels of SOX2and OCT4between over-expression miR-200a EOC cells and control cells (t=10.932, P<0.001),(t=17.416, P=0.003) were statistically significant.
Western Blot results Show that SOX2and OCT4levels in over-expression miR-200a EOC cells, decreased to (0.546±0.077) and (0.518±0.086) times, respectively, compared with control. Statistical analysis showed that, the difference levels of SOX2and OCT4between over-expression miR-200a EOC cells and control cells (t=10.191, P=0.001),(t=9.659, P=0.011) were statistically significant.
Conclusion:
miR-200a weakened the stem characteristics of CSCs in EOC by blocking the formation of tumour spheroids, inducing the differentiation process in3D culture, reducing the proportion of CSCs and down-regulating the CSCs related genes SOX2and OCT4.
卵巢上皮癌(epithelial ovarian cancer, EOC)的病死率高居妇科恶性肿瘤之首,晚期患者的5年生存率仅约30%。目前,肿瘤细胞减灭术联合铂类药物为基础的化疗,对约70%的晚期患者初期治疗有效,但大部分患者最终会出现化疗耐药导致疾病复发。因此,深入认识EOC的进展机制,有效改善EOC的不良预后是目前临床亟待解决的难题。
新近的研究发现,EOC化疗耐药与上皮间质转化(epithelial mesenchymal transition, EMT)密切相关。EMT是指上皮细胞表面黏附分子表达降低、细胞间连接变得疏松、细胞骨架重塑,转变成间充质细胞表型过程。已有的研究证实EMT在肿瘤侵袭、转移和化疗耐药中具有重要的调控作用。更重要的是,有研究发现EOC中存在的肿瘤干细胞(cancer stem cells, CSCs)具有EMT的特征。CSCs学说认为：肿瘤是由异质性的细胞群体构成；其中存在极少量具有干细胞性质的癌细胞亚群,是肿瘤发生、进展、转移及耐药性产生的根源。因此,抑制EMT可能成为有效改善EOC预后的策略。
微小RNA(microRNA, miRNA, miR)是一类非编码的单链小分子RNA,长度约18-24个核苷酸(nucleotide, nt),其通过与mRNA3'端非翻译区(3'-untranslated region,3'-UTR)碱基识别配对,调控靶基因的表达。目前已发现miRNAs在调控EMT中具有重要作用,亦与多种肿瘤的发生及进展关系密切。Yang,等分析了459例EOC肿瘤组织的mRNA及miRNAs表达谱特征,证实间充质表型与EOC的不良预后相关,包括miR-20a、miR-141和miR-506在内的8个关键miRNA,调控了高达89%的间充质相关基因表达。
miR-200a和miR-141同属于miR-200家族(miR-200s,包括miR-200a/b/c/和miR-141/429共5个miRNA)。近年的研究发现miR-200s在多种肿瘤中具有阻碍EMT进程、抑制CSCs自我更新和去分化状态、调控细胞增殖及凋亡、逆转化疗耐药的作用。在EOC中已证实miR-200s的表达水平与化疗敏感性、无进展生存期(progression-free survival, PFS)以及总生存期(overall survival, OS)呈正相关。这些研究结果说明以miR-200s作为EOC潜在的治疗策略,具有理论上的可行性和积极的临床意义。
有研究证实,在EOC细胞株Hey中,过表达miR-200c通过靶向调控TUBB3增加了紫杉醇的敏感性。我们前期在卵巢浆液性腺癌细胞系--OVCAR-3中验证了CD133+细胞具有CSCs特征；进一步用miRNA芯片发现CD133+与CD133-细胞间存在miRNAs的差异表达谱；其中miR-200a在CD133+细胞中下调了约2.16倍,利用合成的miR-200a模拟物—miR-200a mimics,在CD133±细胞中瞬时上调miR-200a水平,发现miR-200a通过靶向下调ZEB2抑制了CD133+细胞的迁移和侵袭能力,初步证实miR-200a在EOC中具有抑制转移的积极作用。但目前关于miR-200a对EOC化疗敏感性和CSCs的调控作用及其相关机制尚未完全阐明。
我们发现瞬时转染,miR-200a mimics的方法仅适用于短期内观察,miR-200a对EOC细胞的影响；并且分选得到的CD133+细胞所占比例非常低(约0.2-1%)；另外,CD133+细胞在体外培养过程中逐渐分化为CD133-细胞的问题,都将为我们深入探索miR-200a在EOC中的生物学功能带来一定的局限性。近来,Wang,等用基因芯片鉴定了无血清培养OVCAR-3所形成的悬浮生长细胞球,具有CSCs的特征,适宜作为研究EOC中CSCs的体外模型。
综合考虑上述因素,本课题首先利用慢病毒表达载体成功构建了稳定上调miR-200a的EOC细胞模型,在普通贴壁培养和悬浮培养条件下,验证了miR-200a对EOC化疗敏感性的影响；进一步探索了miR-200a调控EOC增殖状态和CSCs的作用及其机制,以期为改善EOC的预后带来更新更有力的治疗策略。
第一章构建稳定高表达miR-200a的EOC细胞模型
目的：
利用慢病毒表达载体,构建稳定高表达miR-200a的贴壁及悬浮生长EOC细胞模型,为深入探索miR-200a对EOC的生物学作用及其机制提供平台。
方法：
1. miR-200a过表达慢病毒载体和对照空载体的鉴定。
miR-200a过表达慢病毒载体pLV-miR-200a和对照空载体pLV-control购自深圳市华安平康生物科技有限公司。通过质粒转化、质粒扩增、质粒提取、PCR扩增、DNA电泳以及DNA测序,鉴定miR-200a慢病毒表达载体和对照空载体。
2. miR-200a过表达慢病毒和对照慢病毒的包装。
慢病毒包装元件psPAX2和pMD2.G由南方医科大学肿瘤研究所提供,pLV-miR-200a或pLV-control、psPAX2和pMD2.G共同组成慢病毒包装系统。用Lipofectamine2000将慢病毒包装系统转染293T细胞,包装生产携带miR-200a过表达的慢病毒和对照慢病毒。
3. miR-200a过表达慢病毒和对照慢病毒感染EOC细胞。
用携带]miR-200a过表达的慢病毒和对照慢病毒,分别感染EOC细胞株OVCAR-3,在倒置荧光显微镜下观察报告基因一绿色荧光蛋白(Green Fluorescent Protein, GFP)的表达,以监测感染效率,应用流式细胞仪分选GFP+细胞。
4.验证miR-200a在感染后EOC细胞中的表达水平。
提取经流氏细胞仪分选后EOC细胞的总RNA, qRT-PCR检测miR-200a的表达水平,大量扩增并冻存高表达miR-200a的EOC细胞和对照细胞,用于后续实验。
5.建立miR-200a过表达的悬浮生长EOC细胞模型。
收集过表达miR-200a的贴壁生长EOC细胞和对照组细胞,分别用PBS洗涤2次,重悬于肿瘤球培养基(含EGF20ng/mL、bFGF10ng/mL、0.4%BSA、 insulin5μg/mL、100U/mL青霉素和100U/mL链霉素的DMEM/F12),接种于超低吸附培养皿,常规条件培养,间隔72h添加适量肿瘤球培养基,7d后收集球体细胞,用0.02%EDTA-2Na和0.25%胰蛋白酶1：1混合液消化球体细胞,继续用上述方法传代培养,得到悬浮生长的EOC细胞。qRT-PCR检测悬浮生长的EOC细胞中miR-200a的表达水平。
6.利用SPSS16.0统计软件对所有数据进行处理,采用单样本t检验分析贴壁及悬浮生长EOC细胞中miR-200a的表达差异,显著性水平定义α=0.05,P<0.05认为差异有统计学意义,数据用“均数士标准差”表示。
结果：
1.DNA测序证实miR-200a过表达慢病毒载体和对照空载体序列均正确。
2.成功生产了miR-200a过表达慢病毒和对照慢病毒,用以感染EOC细胞株OVCAR-3,感染后在倒置荧光显微镜下可见GFP十细胞的表达率约为20%-30%,进一步用流式细胞仪分选GFP+细胞,得到感染效率约为95%的EOC细胞。
3.成功构建了miR-200a过表达的贴壁生长EOC细胞模型。qRT-PCR验证携带miR-200a转基因的贴壁生长EOC细胞与对照细胞相比,miR-200a的表达水平升高(3.67±0.45)倍。统计分析结果提示,携带miR-200a转基因的贴壁生长EOC细胞和对照细胞,miR-200a的表达差异具有统计学意义(t值=10.187,P=0.009)。
4.成功构建miR-200a过表达的悬浮生长EOC细胞模型。qRT-PCR验证携带miR-200a转基因的悬浮生长EOC细胞与对照细胞相比,miR-200a的表达水平升高(2.05±0.39)倍。统计分析结果提示,携带miR-200a转基因的悬浮生长EOC细胞和对照细胞,niR-200a的表达差异具有统计学意义(t值=4.666,P=0.043)。
结论：
成功将目的基因miR-200a整合入EOC细胞中并实现高效表达,建立了稳定过表达miR-200a的贴壁及悬浮生长EOC细胞模型。
第二章MiR-200a对EOC化疗敏感性、增殖及CSCs的调控作用及其机制
第一节MiR-200a对EOC化疗敏感性的调控作用
目的：
在稳定过表达miR-200a的贴壁及悬浮生长EOC细胞模型中,验证miR-200a对EOC化疗敏感性的调控作用。
方法：
1.MTT检测miR-200a;对贴壁生长EOC细胞化疗敏感性的影响。
收集miR-200a过表达的贴壁生长EOC细胞和对照细胞,分别重悬于含10%FBS的RPMI1640培养基。按5x103细胞／孔接种于普通96孔板内,培养12-16h后,在细胞中分别加入不同浓度的紫杉醇(终浓度分别为：2nM,4nM,8nM,16nM,32nM)或顺铂(终浓度分别为：lug/mL,2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL),每个浓度设4个重复孔。药物作用48h后,每孔中加入201μL细胞活力检测试剂MTT(5mg/mL),继续常规培养4h后,小心弃去细胞上清液,每孔分别加入100uL DMSO,避光震荡混匀后,置酶联检测仪上测定各孔光密度(OD)值,检测波长490nm。细胞存活率(SR)按以下公式计算：加药孔吸光度平均值／空白对照组吸光度平均值x100%。实验重复三次。
2.MTT检测miR-200a;对悬浮生长EOC细胞化疗敏感性的影响。
收集miR-200a过表达的悬浮生长EOC细胞和对照细胞,分别重悬于肿瘤球培养基,按1x104细胞／孔接种于超低吸附96孔板内,培养24h后,在细胞中分别加入不同浓度紫杉醇(终浓度分别为：2nM,8nM,32nM,128nM,512nM)或顺铂(终浓度分别为：2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL),每个浓度设4个重复孔。药物作用72h后,每孔中加入20μL细胞活力检测试剂MTT(5mg/mL),继续培养4h后,箱式离心机3000rpm离心15min,此时可见肿瘤球细胞聚集于孔板的一侧,用1mL注射器小心吸弃上清液,每孔分别加入100uLDMSO,避光震荡混匀后,置酶联检测仪上测定各孔光密度(OD)值,检测波长490nm。细胞存活率(SR)按以下公式计算：加药孔吸光度平均值/空白对照组吸光度平均值x100%。实验重复三次。
3.利用SPSS16.0统计软件对所有数据进行处理,采用析因设计的方差分析和两个独立样本的t检验,分析细胞毒性试验中细胞存活率之间的差异。P<0.05认为差异有统计学意义,数据用“均数士标准差”表示。
结果：
1. miR-200a过表达的贴壁生长EOC细胞和对照组细胞,在紫杉醇的终浓度为：2nM,4nM,8nM,16nM,32nM时,细胞存活率(%)分别为：(85.86±4.16vs.87.80±2.22),(74.66±3.19vs.80.62±2.60),(65.85±4.24vs.76.15±4.46),(54.91±4.43vs.69.53±3.31),(54.62±1.76vs.66.91±3.41)。统计分析结果提示,miR-200a过表达的贴壁生长EOC细胞与对照组细胞,紫杉醇细胞毒性之间的差异总体有统计学意义(F值=63.464,P值<0.001)。
miR-200a过表达的贴壁生长EOC细胞和对照组细胞,在顺铂的终浓度为：1ug/mL,2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mLL时,细胞存活率(%)分别为：(90.24±5.00vs.85.60±3.60),(66.96±3.57vs.71.95±6.68),(62.02±2.57vs.66.46±2.89),(54.45±2.63vs.56.89±2.80),(49.39±4.21vs.46.61±2.75),(43.54±4.34vs.42.38±2.18)。统计分析结果提示,miR-200a过表达的贴壁生长EOC细胞与对照组细胞,顺铂细胞毒性之间的差异总体没有统计学意义(F值=0.247,P值=0.622)。
2. miR-200a过表达的悬浮生长EOC细胞和对照组细胞,在紫杉醇的终浓度为：2nM,8nM,32nM,128nM,512nM时,细胞存活率(%)分别为：(69.27±3.63vs.83.43±4.01),(57.03±2.88vs.78.91±0.86),(57.47±1.31vs.80.89±1.29),(57.69±1.35vs.80.11±1.80),(55.17±0.60vs.77.96±3.75)。统计分析结果提示,miR-200a过表达的悬浮生长EOC细胞与对照组细胞,紫杉醇细胞毒性之间的差异总体有统计学意义(F值=721.813,P值<0.001)。
miR-200a过表达的悬浮生长EOC细胞和对照组细胞,在顺铂的终浓度为：2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL时,细胞存活率(%)分别为：(50.80±2.35vs.54.37±1.86),(42.64±2.80vs.41.71±1.62),(49.66±2.03vs.46.92±6.37),(46.84±7.72vs.51.57±5.25),(42.66±2.98vs.45.54±5.36)。统计分析结果提示,miR-200a过表达的悬浮生长EOC细胞与对照组细胞,顺铂细胞毒性之间的差异总体没有统计学意义(F值=1.191,P值=0.284)。
结论：
miR-200a过表达增加了贴壁和悬浮生长的EOC细胞对紫杉醇的敏感性,没有影响贴壁和悬浮生长的EOC细胞对顺铂的敏感性。
第二节MiR-200a对EOC细胞增殖状态的调控作用
目的：
miR-200a具有提高贴壁和悬浮生长的EOC细胞对紫杉醇敏感性的作用,而对顺铂的敏感性没有影响；考虑到紫杉醇是靶向细胞增殖周期的药物,而顺铂为非细胞周期依赖药物,所以有必要进一步探索miR-200a对EOC细胞增殖状态的影响。本部分通过一系列体内、外实验验证了miR-200a对EOC细胞增殖状态的调控作用。
方法：
1.平板克隆形成实验检测]miR-200a;对贴壁生长EOC细胞增殖状态的影响。
收集lniR-200a过表达的贴壁生长EOC细胞和对照细胞,重悬于含10%FBS的RPMI1640培养基,按1.5x102细胞/孔接种于普通6孔板内,每组设3个复孔,常规培养10d,观察细胞克隆的形态,计数大于50个细胞的克隆数,弃掉培基,PBS小心漂洗3遍,甲醇固定10min,苏木素染色5-10min,自来水小心漂洗后,拍照记录克隆外观。计算克隆形成率=(克隆数／接种细胞数)x100%。
2.生长曲线(CCK8法)实验检测]miR-200a对贴壁及悬浮生长EOC细胞增殖状态的影响。
收集miR-200a过表达的贴壁生长EOC细胞和对照细胞,重悬于含10%FBS的RPMI1640培养基,按2.0×103细胞／孔接种于普通96孔培养板中,每组设6个复孔,培养6h、1、2、3、4、5、6d时,加入CCK8与培基混合液110μL(比例为1：10),继续常规培养1.5h,以空白对照孔调零,置酶联检测仪上测定各孔光密度(OD)值,检测波长450nm,以相对应OD值代表细胞增殖状态,各组取6孔OD值的平均值,绘制细胞增殖曲线。
收集miR-200a过表达的悬浮生长EOC细胞和对照细胞,重悬于肿瘤球培养基,按4.0×103细胞／孔接种于超低吸附96孔培养板中,每组设6个复孔,培养1、2、3、4、5、6、7d时,加入CCK8液10μL,继续常规培养4h,以空白对照孔调零,置酶联检测仪上测定各孔光密度(OD)值,检测波长450nm,以相对应OD值代表细胞增殖状态,各组取6孔OD值的平均值,绘制细胞增殖曲线。
3.流式细胞仪检测miR-200对贴壁及悬浮生长EOC细胞细胞周期分布的影响。
分别收集]miR-200a过表达及对照的贴壁和悬浮生长EOC细胞,用预冷PBS漂洗细胞两次,加入预冷70%乙醇,4℃固定过夜或-20℃长期固定,检测前PBS漂洗细胞一次,加入含50μg/mL溴化乙锭(PI)、100μg/mL RNase A、0.2%TritonX-100的PBS500μL,室温避光孵育30分钟,以标准程序用流式细胞仪检测,计数1～2万个细胞,结果用细胞周期拟和软件ModFit分析结果。
4.体内验证miR-200a对EOC细胞增殖状态的影响。
选取4-5周龄雌性BALB/c-nu/nu裸鼠作为实验对象,将miR-200a过表达的贴壁生长EOC细胞和对照组细胞,分别皮下注射移植入裸鼠背部的右侧及左侧,每侧移植1.5x106个细胞,细胞悬液总体积为100μL。实验共用8只裸鼠,每3日观察裸鼠一般情况、测量接种部位包块生长情况并记录裸鼠体重,依据移植瘤生长情况适时取材并作相关分析。
5.取移植瘤组织免疫组化检测Ki67的表达情况,光学显微镜下观察拍照,随机选取3个高倍视野,计算每个视野阳性细胞比例。阳性细胞比例=每个视野下阳性细胞数/每个视野下细胞总数×100%。
6.利用SPSS16.0统计软件对所有数据进行处理,采用两个独立样本t检验分析平板克隆形成率差异、移植瘤组织中Ki67阳性率差异、细胞周期分布差异。采用析因设计的方差分析,分析生长曲线(CCK8法)及裸鼠皮下移植瘤生长差异,P<0.05认为差异有统计学意义,数据用“均数士标准差”表示。
结果：
1.平板克隆实验中,miR-200过表达的贴壁生长EOC细胞较对照组细胞,体积缩小、细胞排列更紧密,更倾向于上皮细胞表型；miR-200a过表达的贴壁生长EOC细胞与对照组细胞的克隆形成率(%)分别为(92.52±5.60vs.79.12±4.41)。统计分析结果提示,miR-200a过表达的贴壁生长EOC细胞与对照组细胞,克隆形成率的差异具有统计学意义(t值=-4.461,P值=0.001)。
2.生长曲线(CCK8法)实验中,miR-200a过表达的贴壁生长EOC细胞和对照组细胞,在培养8h、1、2、3、4、5、6d时,吸光度值(OD)分别为(0.278±0.008vs.0.275±0.002),(0.500±0.096vs.0.433±0.035),(0.624±0.058vs.0.528±0.046),(1.028±0.115vs.0.710±0.048),(1.530±0.160vs.1.000±0.171),(1.993±0.206vs.1.239±0.159),(2.450±0.233vs.1.428±0.161)。统计分析结果提示,miR-200a过表达的贴壁生长EOC细胞与对照组细胞,细胞增殖的差异总体具有统计学意义(F值=199.507,P值<0.001)。
miR-200过表达的悬浮生长EOC细胞和对照组细胞,在培养1、2、3、4、5、6、7d时,吸光度值(OD)分别为(0.218±0.015vs.0.216±0.013),(0.333±0.013vs.0.323±0.014),(0.462±0.028vs.0.437±0.021),(0.720±0.078vs.0.565±0.050),(0.952±0.081vs.0.688±0.062),(1.149±0.116vs.0.803±0.054),(1.434±0.135vs.0.957±0.080)。统计分析结果提示,miR-200a过表达的悬浮生长EOC细胞与对照组细胞,细胞增殖的差异总体具有统计学意义(F值=159.036,P值<0.001)。
3.流式检测细胞周期实验中,miR-200过表达的贴壁生长EOC细胞和对照组细胞,G0/G1、S、G2/M期所占比例(%)分别为(55.86±2.55vs.72.41±3.22),(26.15±2.30vs.21.76±2.11),(13.52±1.99vs.6.82±0.76)。统计分析结果提示,miR-200a过表达的贴壁生长EOC细胞与对照组细胞,G0/G1、S、G2/M期比例的差异分别为(t值=9.861,P值<0.001)、(t值=-3.445,P值=0.006)、(t值=-7.686,P值<0.001),均具有统计学意义。
miR-200a过表达的悬浮生长EOC细胞和对照组细胞,Go/G1、S、G2/M期所占比例(%)分别为(69.52±3.24vs.90.48±2.36),(21.35±2.10vs.5.49±0.52),(11.31±1.43vs.3.32±0.28)。统计分析结果提示,miR-200a过表达的悬浮生长EOC细胞与对照组细胞,G0/G1、S、G2/M期比例的差异分别为(t值=11.470,P值<0.001)、(t值=-17.926,P值=0.006)、(t值=-13.386,P值<0.001),均具有统计学意义。
4.裸鼠皮下移植瘤实验中,miR-200过表达的EOC细胞和对照细胞均能在裸鼠皮下生长形成肿瘤。miR-200a过表达的皮下移植瘤和对照移植瘤体积(mm3),在皮下接种移植后50、53、56、59、62d分别为(110.27±57.84vs.58.19±19.55),(146.88±73.64vs.67.44±32.58),(167.03±75.68vs.82.25±48.15),(234.01±80.85vs.108.74±42.98),(366.36±99.87vs.137.44±43.13)。统计分析结果提示,miR-200a过表达的EOC细胞与对照组细胞,裸鼠皮下移植瘤增长的差异总体具有统计学意义(F值=63.441,P值<0.001)。
5.免疫组化检测移植瘤组织中Ki67表达的实验中,miR-200a过表达和对照移植瘤组织中Ki67阳性表达率(%)为(70.91±5.18vs.15.21±3.52)。统计分析结果提示,miR-200a过表达和对照移植瘤组织中,Ki67阳性表达率差异具有统计学意义(t值=-25.156,P值<0.001)。
结论：
1.过表达miR-200a增加了EOC细胞平板克隆形成率；促进了贴壁及悬浮生长EOC细胞的生长、诱导其进入增殖周期；有益于EOC细胞裸鼠皮下移植瘤生长；说明miR-200a具有促进EOC细胞的增殖的作用。这种促增殖效应可能是miR-200a增加EOC细胞对紫杉醇敏感性的作用机制之一。
第三节MiR-200a调控EOC中CSCs的作用及其机制
目的：
以上研究发现miR-200a对EOC细胞株紫杉醇敏感性及增殖状态具有一定的调控作用,接下来我们拟探索miR-200a调控EOC中CSCs的作用及其机制。通过肿瘤球形成实验、流氏细胞仪检测侧群(side population,SP)细胞比例、检测CSCs特性相关基因表达水平,验证miR-200a对EOC中CSCs干性特征的调控作用及其机制。
方法：
1.肿瘤球形成实验检测miR-200a对EOC细胞自我更新能力的影响。
收集miR-200a过表达的EOC细胞和对照细胞,重悬于肿瘤球培养基,按1×103细胞/孔分别接种于超低吸附24孔板内,每组三个复孔,常规培养7d,倒置荧光显微镜下观察肿瘤球形态,计数直径≥-70μm的肿瘤球个数。实验重复三次。
2.流氏细胞仪检测SP细胞比例,验证miR-200a对EOC中CSCs比例的影响。
收集miR-200a过表达的贴壁生长EOC细胞和对照组细胞,重悬于已预热为37℃含2%FBS的RPMI1640培养基,调整细胞密度至1×106/mL,每组各准备两管细胞,四管细胞中均加入Hoeehst33342终浓度为5ug/mL,在每组细胞中随机选择一管再加入维拉帕米终浓度为50μmol/L,37℃摇床避光孵育90min后放于冰上终止反应。4℃1000rpm离心3min,弃上清,预冷PBS洗涤一次。40μm滤器过滤待检测细胞,检测前加入碘化丙淀(PI)终浓度1μg/mL标记死细胞。流式细胞仪检测SP比例,Hoeehst33342激发光波长为350nm,405/30带通收集蓝光,570/20带通收集红光,PI用488nm蓝光激发,630/30带通收集红光。去除PI直接染色阳性的死细胞,以Hoechst Red为X轴,Hoechst Blue为Y轴作二维散点图。设门选低Hoechst Red及低Hoechst Blue且维拉帕米组缺失的区域为SP细胞。比较SP细胞比例在miR-200a过表达的EOC细胞与对照组细胞间的差别。
3. qRT-PCR和Western Blot检测miR-200a对EOC细胞中CSCs相关基因表达的调控作用。
qRT-PCR检测方法如下,提取miR-200a过表达的EOC细胞和对照组细胞总RNA,将mRNA逆转为cDNA, qRT-PCR检测(SYBR法)SOX2和OCT4mRNA水平,以GAPDH作为内源性对照。
Western Blot检测方法如下,提取miR-200a过表达的EOC细胞和对照组细胞总蛋白,BCA蛋白试剂盒测定蛋白浓度,每个泳道加30μg蛋白样品,SDS-PAGE将蛋白分离后转移到PVDF膜上,3%BSA拮抗非特异性位点,分别用SOX2和OCT4的一抗(稀释比例1：1000),4℃孵育过夜,相应二抗室温孵育1h,增强型化学发光系统(ECL)观察蛋白印迹,以P-actin作为内源性对照。
4.利用SPSS16.0统计软件对所有数据进行处理,采用两个独立样本的t检验分析肿瘤球形成差异、SP细胞比例差异,单样本的t检验分析qRT-PCR和Western Blot检测SOX2和OCT4表达差异。P<0.05认为差异有统计学意义,数据用“均数士标准差”表示。
结果：
1.肿瘤球形成实验中,我们观察到有部分miR-200a过表达的EOC细胞,在超低吸附培养皿中亦可贴壁生长,并以贴壁方式逐渐增殖,而对照组中几乎没有肿瘤球细胞贴壁生长的现象。miR-200a过表达的EOC细胞和对照细胞,肿瘤球形成个数(/1000个细胞)分别为(7.17±1.17vs.17.50±1.87)。统计分析结果提示,miR-200a过表达的EOC细胞与对照组细胞,肿瘤球形成个数差异具有统计学意义(t值=11.474,P值<0.001)。
2.流式细胞仪检测SP细胞比例的实验中,miR-200a过表达的EOC细胞和对照细胞SP细胞比例(%)为(0.233±0.076vs.0.850±0.100)。统计分析结果提示,miR-200a过表达的EOC细胞和对照细胞,SP细胞比例差异具有统计学意义(t值=8.488,P值=0.001)。
3. qRT-PCR检测miR-200a过表达的EOC细胞中SOX2和OCT4mRNA水平分别下降至对照细胞的0.467和0.361倍。统计分析结果提示,miR-200a过表达的EOC细胞与对照组细胞,SOX2和OCT4mRNA水平差异均具有统计学意义(t值=-10.932,P=0.008)和(t值=-17.416,P值=0.003)。
Western Blot结果提示miR-200过表达的EOC中细胞中,SOX2和OCT4蛋白表达水平分别下降至对照细胞的0.546和0.518倍。统计分析结果提示,miR-200a过表达的EOC细胞与对照组细胞,SOX2和OCT4蛋白水平差异均具有统计学意义(t值=-10.191,P值=0.009)和(t值=-9.659,P值=0.011)。
结论：
过表达miR-200a通过抑制EOC细胞自我更新能力、诱导分化、降低SP细胞比例、下调干性相关基因SOX2和OCT4的表达,弱化了EOC中CSCs的干性特征。
Background:
Epithelial ovarian cancer (EOC) is the most lethal gynaecologic malignancy, the5-year survival rate is only about30%in patients with advanced stage. Currently, cytoreductive surgery combined with platinum-based chemotherapy were efficient in almost70%of advanced cases at initial treatment, but most of these patients will eventually develop chemo-resistance and recurrence. Therefore, an in-depth understanding of EOC and effectively improve its poor prognosis is needed.
Recent studies have identified the close relationship between chemo-resistance and epithelial mesenchymal transition (EMT) in EOC. EMT consists of a reversible change of cell phenotype during which epithelial cells loosen cell-cell adhesion structures including adherens junctions and desmosomes, modulate their polarity and rearrange their cytoskeleton, transformed into mesenchymal cell phenotype. Previous studies have demonstrated EMT played a central role in regulating tumor invasion, metastasis and chemoresistance. Most importantly, recent studies have found that EOC cancer stem cells (CSCs) harboured post-EMT characteristics. CSCs theory believes:tumor is composed of a heterogeneous population of cells; in which very small amount of subpopulation with stem cell properties were the root of tumor progression, metastasis and chemoresistance. Therefore, inhibition of EMT may be a promising strategy to improve the prognosis of EOC.
MicroRNA (miRNA, miR) is a large class of small noncoding RNAs; approximately18-24nucleotides (nt) in length, as critical post-transcriptional regulators of gene expression through targeting mRNA3'untranslated region (3'-untranslated region,3'-UTR) base pair recognition. A growing body of evidence indicated that miRNAs played an important role in the regulation of EMT, and aberrant miRNAs profile were the defining feature in many types of tumours. Integrated genomic analyses confirmed that mesenchymal phenotype was associated with poor prognosis of EOC, and miR-200a, miR-141and miR-506were included in the eight key miRNAs predicted to target the mesenchymal subtype in459cases of serous ovarian cancer.
MiR-200a and miR-141are belong to the miR-200family (miR-200s, including miR-200a/b/c, and miR-141/429of5miRNA). MiR-200s, which are significantly involved in inhibition of EMT, repression of CSCs self-renewal, differentiation, modulation of cell division, apoptosis and reversal of chemoresistance in many types of human cancer. Accumulating evidences showed that miR-200s were associated with progression-free survival (PFS), overall survival(OS) and chemo-sensitivity in EOC. These foundings showed that investigation miR-200s as potential therapeutic strategy for EOC, has theoretical feasibility and positive clinical significance.
Studies have reported that overexpression of miR-200c may increase EOC cell line Hey's. sensitivity to paclitaxel by targeting TUBB3. Previously, we verified CD133+cells have characteristics of CSCs in EOC cell lines-OVCAR-3; further demonstrated that the profile of miRNAs was different between CD133+and CD133-cells; in which miR-200a was down-regulated about2.16times in CD133+cells compared with CD133-cells, additionally, restoration of miR-200a expression by synthetic miR-200a mimics could reduce the ZEB2-mediated migration and invasion of CD133+ovarian CSCs, which initially confirmed miR-200a has a positive effect in inhibiting metastasis in EOC. However, miR-200a regulation of chemotoxicity, CSCs phenotype and its clinical significance in EOC remains unclear.
We found transient transfection of miR-200a mimics was only suitable to observe the effects of miR-200a on ovarian cancer cells in a short term; additionally, the percentage of CD133+cells by sorting is very low (about0.2%～1%); furthermore, CD133+cells will gradually differentiate into CD133-cells in vitro, these limitations will impact our in-depth explore the biological function of miR-200a in the chemosensitivity of EOC. Recently, Wang et al. found that SDCs (spheriod derived cells) formed from OVCAR-3cells growth in serum-free culture medium, maintained CSCs characteristics and were ideal models in vitro for CSCs-targeted investigations in EOC.
Considering the above factors, herein we first constructed EOC cells model, in which miR-200a was stably over-expressed via a lentiviral expression vector, then verified the role of miR-200a on chemosensitivity in both normal2D and3D culture; then further explored the role of miR-200a in regulation proliferation, CSCs and its mechanisms, in order to bring more powerful treatment strategies resulting in improving the prognosis of EOC.
Chapter1Establishment miR-200a over-expression EOC cells model Objective:
Establishment of miR-200a over-expression EOC cells via a lentiviral expression vector in both2D and3D culture, for further exploring the biological role and mechanism of miR-200a in EOC.
Methods:
1. Identification of miR-200a over-expression lentiviral vector and control empty vector.
Over-expression of miR-200a lentivirus vector pLV-miR-200a and control empty vector pLV-control were purchased from Shenzhen, China Anping Kang Biotechnology Co., Ltd., through plasmid transformation, plasmid amplification, plasmid extraction, PCR amplification, DNA electrophoresis and DNA sequencing, identification miR-200a expression lentiviral vector and control empty vector.
2. Packaging miR-200a over-expression and control lentivirus.
Lentiviral packaging components psPAX2and pMD2.G were kept by the Southern Medical University Cancer Institute. Lentiviral packaging systems was composed by three plasmids pLV-miR-200a or pLV-control, psPAX2and pMD2.G.293T cells transfected with lentiviral packaging system by Lipofectamine2000, were utilized to produce lentivirus carrying the vector over-expression of miR-200a and control empty vector.
3. Infection EOC cells by miR-200a over-expression and control lentivirus.
Infection EOC cell line OVCAR-3by miR-200a over-expression and control lentivirus, then monitor the infection efficiency by green fluorescent protein (GFP) expression under inverted fluorescence microscope, and sorting GFP+cells by flow cytometry.
4. Verify miR-200a expression level in EOC cells after infection.
Extraction total RNA from infected EOC cells, then detection the level of miR-200a by qRT-PCR. Frozen and expanded miR-200a over-expression ovarian cancer cells and control cells for subsequent experiments.
5. Establish suspended growth model of EOC cells over-expression of miR-200a.
Collecting miR-200a over-expression EOC cells and control cells, washing twice with PBS, resuspending in tumor sphere medium (containing EGF20ng/ml, bFGF lOng/ml,0.4%BSA, insulin5μg/ml,100U/ml penicillin and100U/ml streptomycin DMEM/F12), inoculated at ultra-low petri dishes with conventional conditions, adding appropriate amount of tumor sphere medium72h later, collecting spheroid cells7days later. The collected cells were digested with0.02%EDTA-2Na and a1:1mixture of0.25%trypsin at37℃at about lmin to form single cells, re-suspend in tumor sphere medium and continue culture with the above method, then obtaining suspended growth of EOC cells. Detected the level of miR-200a in EOC cells grown in suspension by qRT-PCR.
6. Statistical Analysis. Statistical analyses were performed using SPSS version16.0, one-sample t-test was utilized to analyze the differential expression of miR-200a in adherent and suspension growth of ovarian cancer cells, the definition of significant level α=0.05, P<0.05was considered statistically significant, all data are presented as "mean values±SD".
Results:
1. DNA sequencing confirmed that the sequence of miR-200a over-expression lentiviral vector and control empty vector were correct.
2. Successfully produced miR-200a over-expression and control lentivirus for infection EOC cell line OVCAR-3. The expression of GFP was approximately20%-30%under inverted fluorescence microscope, after sorting GFP+cells by FACS, the infection efficiency was approximately95%.
3. Obtained miR-200a over-expression adhesive cells model of EOC. qRT-PCR validation the expression level of miR-200a increased about (3.67±0.45) times compared with the control, and the difference between the two group was statistically significant(t=10.187, P=0.009).
4. Obtained miR-200a over-expression suspended spheroid cells model of EOC. qRT-PCR validation the expression level of miR-200a increased about (2.05±0.39) times compared with the control, and the difference between the two group was statistically significant (t=4.666, P=0.043).
Conclusion:
1. Successfully integrated miR-200a into EOC cells, and achieved its high expression. Establishing a stable miR-200a over-expression EOC cells in the presence of adherent and suspended growth.
Chapter2The roles and mechanisms of miR-200a regulation on chemosensitivity, proliferation and CSCs in EOC
Section1The role of miR-200a regulation on chemosensitivity in EOC Objective:
Verifying the relationship between miR-200a and the chemo-sensitivity of paclitaxel and cisplatin in adherent and suspension growth of EOC cells model.
Method:
1. MTT detection of miR-200a regulation on chemosensitivity in adherent growth of EOC cells.
MiR-200a over-expressing or control adherent growth of EOC cells were collected and resuspended in RPMI1640containing10%FBS medium.5×103miR-200a over-expressing or control adherent growth of EOC cells were seeded in normal96-well plates, respectively, routine cultured12-16h later, paclitaxel (final concentration:2nM,4nM,8nM,16nM,32nM) or cisplatin (final concentration:1ug/mL,2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL), were added in the cells, each concentration was repeated four holes,48h after drug treatment, added20νL of cell viability assay MTT (5mg/mL) to each well, continue regular culture4h, the supernatant was carefully discarded and added100uL DMSO in each well, after mixing in dark, absorbance values (OD) of each well was measured by enzyme-linked detector, detection wavelength was490nm. Cell viability (SR) according to the following formula:(the average OD of dosing wells/the average OD of control wells) x100%. Draw viability curves with SR value in each group. This experiment was repeated three times.
2. MTT detection of miR-200a regulation on chemosensitivity in suspension growth of EOC cells.
MiR-200a over-expressing or control suspension growth of EOC cells were collected and resuspended in tumour spheroid medium,1×104miR-200a over-expressing or control suspension growth of EOC cells were seeded in ultra-low attach96-well plates, respectively, routine cultured24h later, paclitaxel (final concentration:2nM,8nM,32nM,128nM,512nM) or cisplatin (final concentration:2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL), were added in the cells, each concentration was repeated four holes.72h after drug treatment, added20μL of cell viability assay MTT (5mg/mL) to each well, continue regular culture4h, box-type centrifuge3000rpm,15min, then visible tumor cells aggregates in the side plates, using1mL syringe suction supernatant carefully, added100uL DMSO in each well, after mixing in dark, absorbance values (OD) of each well was measured by enzyme-linked detector, detection wavelength was490nm. Cell viability (SR) according to the following formula:(the average OD of dosing wells/the average OD of control wells) x100%. Draw viability curves with SR value in each group. This experiment was repeated three times.
3. Statistical Analysis. Statistical analyses were performed using SPSS version16.0, factorial design analysis of variance and two independent samples t-test were utilized to analyze the differences of cell viability in cytotoxicity tests. P<0.05was considered statistically significant, data are presented as "mean values±SD".
Results:
1. The cell survival rate (%) of miR-200a over-expressing adherent growth of EOC cells and control cells, after treated with a final concentration of paclitaxel:2nM,4nM,8nM,16nM,32nM, were (85.86±4.16vs.87.80±2.22),(74.66±3.19vs.80.62±2.60),(65.85±4.24vs.76.15±4.46),(54.91±4.43vs.69.53±3.31),(54.62±1.76vs.66.91±3.41), respectively. These results showed that, the difference of paclitaxel cytotoxicity between miR-200a over-expressing adherent growth of EOC cells and control cells, was statistically significant (F=63.464, P<0.001).
The cell survival rate (%) of miR-200a over-expressing adherent growth of EOC cells and control cells, after treated with a final concentration of cisplatin (final concentration:1ug/mL,2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL), were (84.26±5.50vs.90.73±5.19),(71.95±6.68vs.66.96±3.57),(66.46±2.89vs.60.52±4.93),(57.48±3.70vs.54.45±2.63),(48.91±2.40vs.51.48±1.52),(43.54±4.34vs.45.87±4.19), respectively. These results showed that, the difference of cisplatin cytotoxicity between miR-200a over-expressing adherent growth of EOC cells and control cells, was not statistically significant (F=0.247, P=0.622).
2. The cell survival rate (%) of miR-200a over-expressing suspension growth of EOC cells and control cells, after treated with a final concentration of paclitaxel:2nM,8nM,32nM,128nM,512nM, were(69.27±3.63vs.83.43±4.01),(57.03±2.88vs.78.91±0.86),(57.47±1.31vs.80.89±1.29),(57.69±1.35vs.80.1±1.80),(55.17±0.60vs.77.96±3.75), respectively. These results showed that, the difference of paclitaxel cytotoxicity between miR-200a over-expressing suspension growth of EOC cells and control cells, was statistically significant (F=721.813, P<0.001).
The cell survival rate (%) of miR-200a over-expressing suspension growth of EOC cells and control cells, after treated with a final concentration of cisplatin (final concentration:2ug/mL,4ug/mL,8ug/mL,16ug/mL,32ug/mL), were (50.80±2.35vs.54.37±1.86),(42.64±2.80vs.41.71±11.62),(49.66±2.03vs.46.92±6.37),(46.84±7.72vs.51.57±5.25),(42.66±2.98vs.45.54±5.36), respectively. These results showed that, the difference of cisplatin cytotoxicity between miR-200a over-expressing suspension growth of EOC cells and control cells, was not statistically significant (F=1.191, P=0.284).
Conclusion:
Over-expression of miR-200a increased the sensitivity of adherent and suspension growth of EOC cells to paclitaxel, without affecting the sensitivity of adherent and suspension growth of EOC cells to cisplatin.
Section2The role of miR-200a regulation on proliferation in EOC
Objective:
Given miR-200a enhanced paclitaxel sensitivity in adherent and suspension growth of EOC cells, but no such effect to cisplatin. Considering paclitaxel is a drug targeting cell cycle, while cisplatin is not a cell cycle dependent drug, we intended to further explore the effects of miR-200a on proliferation in EOC through a series in vitro and in vivo assays.
Methods:
1. Colony forming assay was utilized to detect the impact of miR-200a on the growth state of adherent EOC cells.
MiR-200a over-expressing or control adherent growth of EOC cells were collected and resuspended in RPMI1640containing10%FBS medium.1.5×102over-expression miR-200a or control ovarian cancer cells, were seeded in normal6-well plates, respectively, three wells in each group, routine cultured10d, observed the morphology of cell clones, calculating the number of clone more than50cells, cloning efficiency=(clone number/seeded cells)×100%. Discard cells medium, carefully rinsed cells with PBS three times, fixed with methanol lOmin, hematoxylin5-10min, carefully rinsed cells with water, photographed cloning appearance.
2. Growth curve (CCK8) assay was utilized to detect the impact of miR-200a on the growth state of adherent and suspended EOC cells.
MiR-200a over-expressing or control adherent growth of EOC cells were collected and resuspended in RPMI1640containing10%FBS medium.2.0×103over-expression miR-200a or control adherent growth of EOC cells were seeded in normal96-well culture plates, respectively, six wells in each group, when cultured6h,1,2,3,4,5,6d, adding CCK8with culture medium mixture110μL (ratio of1:10),37℃cultured1.5h, absorbance values (OD) of each well was measured by enzyme-linked detector, detection wavelength was450nm, blank control wells as zero, draw growth curves with the average OD value from6wells in each group.
MiR-200a over-expressing or control suspension growth of EOC cells were collected and resuspended in tumour spheroid medium.4.0×103over-expression miR-200a or control suspended growth of ovarian cancer cells were seeded in ultra-low attach96-well culture plates, respectively, six wells in each group, when cultured1,2,3,4,5,6,7d, adding10μL CCK8,37℃cultured4h, absorbance values (OD) of each well was measured by enzyme-linked detector, detection wavelength was450nm, blank control wells as zero, draw growth curves with the average OD value from6wells in each group.
3. Flow cytometry was utilized to detect the impact of miR-200a on the cell cycle progression of adherent and suspended EOC cells.
Collecting over-expression miR-200a and control EOC cells growth in adherent and suspension, respectively, rinsed twice with cold PBS, adding cold70%ethanol,4℃fixed overnight or-20℃long-term fixed, cells were rinsed once with PBS before detection, incubated with a solution containing50μg/mL ethidium bromide (PI),100μg/mL RNase A,0.2%Triton X-100in500μL PBS for30min at room temperature in the dark, using a standard procedure by flow cytometry, counting from10,000to20,000cells, the results were intended by the cell cycle software ModFit analysis.
4. In vivo xenograft experiment was utilized to detect the impact of miR-200a on the proliferation of EOC cells.
Selected4-5week-old female BALB/c-nu/nu mice as transplant objects, subcutaneously transplanted nude mice back at right and left side with over-expression miR-200a and control EOC cells growth in adherent, respectively. Each point transplantation1.5x106cells, the total volume of the cell suspension was1100μL. The number of nude mice used in this experiment was eight. Observed the general status, measured the growth of mass in vaccination site, and record the weight of nude mice every three days. Timely drawn and correlation analysis based on tumor growth.
5. Immunohistochemistry assay was utilized to detect the expression of Ki67in transplanted tumor tissue, photographed under an optical microscope, randomly selected three high-power fields, calculate the proportion of positive cells per field. The percentage of positive cells per field=the number of positive cells/total cells per field×100%.
6. Statistical Analysis. Statistical analyses were performed using SPSS version16.0, two independent samples t-test was utilized to analyze the differences of colony formation rate, Ki67-positive rate and cell cycle distribution, and factorial design analysis of variance was utilized to analyze the differences of growth curves (CCK8) and xenograft in nude mice. P<0.05was considered statistically significant, all data are presented as "mean values±SD".
Results:
1. Colony forming assay shown, over-expression miR-200a adherent growth of EOC cells became smaller in size, arranged closer, more inclined epithelial phenotype compared with control group. The colony formation rate(%) of over-expression miR-200a adherent growth of ovarian cancer cells and control cells was (92.52±5.60vs.79.12±4.41). Statistical analysis showed that the difference of colony formation rate between miR-200a over-expressing adherent growth of EOC cells and control cells, was statistically significant (t=-4.461, P=0.001).
2. Growth curves (CCK8) experiment shown, the OD value of miR-200a over-expressing adherent growth of EOC cells and control cells, cultured in8h,1,2,3,4,5,6d, were (0.278±0.008vs.0.275±0.002),(0.500±0.096vs.0.433±0.035), (0.624±0.058vs.0.528±0.046),(1.028±0.115vs.0.710±0.048),(1.530±0.160vs.1.000±0.171),(1.993±0.206vs.1.239±0.159),(2.450±0.233vs.1.428±0.161), respectively. Statistical analysis showed that, the difference of cell growth between miR-200a over-expressing adherent growth of EOC cells and control cells, was statistically significant (F=199.507, P<0.001).
The OD value of miR-200a over-expressing suspended growth of EOC cells and control cells, cultured in1,2,3,4,5,6,7d, were (0.218±0.015vs.0.216±0.013),(0.333±0.013vs.0.323±0.014),(0.462±0.028vs.0.437±0.021),(0.720±0.078vs.0.565±0.050),(0.952±0.081vs.0.688±0.062),(1.149±0.116vs.0.803±0.054),(1.434±0.135vs.0.957±0.080), respectively. Statistical analysis showed that, the difference of cell growth between miR-200a over-expressing suspended growth of EOC cells and control cells, was statistically significant (F=159.036, P<0.001).
3. Flow cytometry cell cycle analysis shown, G0/G1, S, G2/M proportion (%) in over-expression miR-200a adherent growth of EOC cells and control cells, were (55.86±2.55vs.72.41±3.22),(26.15±2.30vs.21.76±2.11),(13.52±1.99vs.6.82±0.76), respectively. Statistical analysis showed that, the difference of G0/G1,S, G2/M ratio between miR-200a over-expressing adherent growth of EOC cells and control cells (t=9.861, P<0.001),(t=-3.445, P=0.006),(t=-7.686, P<0.001) were statistically significant.
The G0/G1, S, G2/M proportion (%) in over-expression miR-200a suspended growth of EOC cells and control cells, were (69.52±3.24vs.90.48±2.36),(21.35±2.10vs.5.49±0.52),(11.31±1.43vs.3.32±0.28), respectively. Statistical analysis showed that, the difference of G0/G1, S, G2/M ratio between over-expression miR-200a suspended growth of EOC cells and control cells (t=11.470, P<0.001),(t=-17.926, P=0.006),(t=-13.386, P<0.001) were statistically significant.
4. In vivo xenograft experiment shown, both miR-200a overexpression EOC cells and control cells could form xenograft in nude mice. The tumor volume (mm3) of over-expression miR-200a and control group after subcutaneous inoculation transplantation50,53,56,59,62d, were (110.27±57.84vs.58.19±19.55),(146.88±73.64vs.67.44±32.58),(167.03±75.68vs.82.25±48.15),(234.01±80.85vs.108.74±42.98),(366.36±99.87vs.137.44±43.13), respectively. Statistical analysis showed that the difference of tumor volume between over-expression miR-200a and control group, was statistically significant (F=63.44, P<0.001).
5. Immunohistochemistry assay shown, the positive expression rate of Ki67(%) between over-expression miR-200a xenograft and control was (70.91±5.18vs.15.21±3.52). Statistical analysis showed that the difference positive expression rate of Ki67between over-expression miR-200a and control group, was statistically significant (t=-21.156, P<0.001).
Conclusion:
Over-expression miR-200a increased EOC cells cloning efficiency, promoted cells growth, induced cells enter into cell cycle, and promoted the subcutaneous xenograft growth in nud mice, suggesting miR-200a faciliated the proliferation of EOC cells, and this effect of promoting proliferation may be one of the mechanisms involved in enhancing the chemosensivitity of paclitaxel regulated by miR-200a.
Section3The roles and mechanisms of miR-200a regulation on CSCs in EOC
Objective:
The above findings showed miR-200a has a certain influence on paclitaxel sensitivity and proliferation in EOC cells. Then we intended to further explore the effects of miR-200a regulation on CSCs in EOC through tumor sphere formation assay, detection the side population (SP) by FACS, and validation stem-related genes expression regulated by miR-200a.
Methods:
1. Tumor sphere formation assay was utilized to detect the impact of miR-200a on the self-renewal capacity in EOC cells.
MiR-200a over-expressing or control suspension growth of EOC cells were collected and resuspended in tumour spheroid medium,1×103over-expression miR-200a or control EOC cells were seeded in ultra-low attach24-well culture plates, respectively, three wells in each group, routine cultured7d, observed the morphology of tumor spheroids, calculating the number of tumor spheroids diameter≥70μm. The experiment was repeated three times.
2. Investigation the proportion of side population (SP) by FACS was utilized to detect the impact of miR-200a on CSCs ratio.
Collecting miR-200a over-expression EOC cells and control cells, resuspended in RPMI1640medium containing2%FBS preheated to37℃, cells'density was adjusted to1×106/mL. Prepare two bottles of cells in each group, Hoeehst33342was added with final concentration of5ug/mL in all four bottles of cells, randomly selected one bottle of cells in each group and verapamil was added with final concentration of50μmol/L, incubated at37℃shaker for90min in the dark, and terminated the reaction on ice.4℃1000rpm centrifuge3min, supernatant was removed, washed with pre-cooling PBS once.40μm filter filtering cells before detection, propionate iodide (PI) was added with final concentration1μg/mL to label dead cells. SP ratio was detected by FACS, Hoeehst33342excitation wavelength350nm,405/30bandpass collected blue,570/20bandpass collect red, PI was excitated by488nm blue,630/30bandpass collected red. Dead cells were directly removed by PI staining, Hoechst Red X-axis, Hoechst Blue Y-axis for the two-dimensional scatter plot. Low Hoechst Red and low Hoechst Blue and verapamil missing region was SP. Comparison the proportion of SP between miR-200a over-expression EOC cells and control cells.
3. qRT-PCR and Western Blot were utilized to detect the impact of miR-200a on regulation CSCs related genes expression.
qRT-PCR detection method is as follows, total RNA in miR-200a over-expression EOC cells and control cells was extracted, then reversed to cDNA, and qRT-PCR detected (SYBR method) SOX2and OCT4mRNA levels, GAPDH used as an endogenous control.
Western blot methods is as follows:the proteins in over-expression miR-200a or control EOC cells were extracted with RIPA lysis buffer, BCA protein assay kit was employed to assess protein concentrations, protein samples (30μg per lane) were resolved using SDS-PAGE, and then transferred to PVDF membranes, after blocking the nonspecific binding sites with3%BSA, PVDF membranes were incubated with primary antibodies at4℃overnight, specifically, rabbit monoclonal antibody to SOX2and OCT4, followed by incubation with the corresponding secondary antibodies at room temperature for1h, immunoblots were visualized using an enhanced chemiluminescence (ECL) Western Blot Detection System, P-actin used as an endogenous control.
4. Statistical Analysis. Statistical analyses were performed using SPSS version16.0, two independent samples t-test was utilized to analyze the differences of tumor sphere formation and the ratio of SP, one-sample t-test was utilized to analyze the different expression of SOX2and OCT4by qRT-PCR and Western Blot. P<0.05was considered statistically significant, all data are presented as "mean values±SD".
Results:
1. Tumor sphere formation assay showed, over-expression of miR-200a induced the attachment of suspended tumor spheroid cells, even in ultralow attachment dishes, and showed a gradual proliferation state, which could not be seen in control groups. The number of tumor spheroids(/1,000cells) in over-expression miR-200a and control EOC cells was (7.17±1.17vs.17.50±1.87). Statistical analysis showed that, the difference number of tumor spheroids between over-expression miR-200a and control EOC cells was statistically significant (t=11.474, P<0.001).
2. SP ratio by FACS experiments show that two groups of cells, the lower left corner show low fluorescence or negative cell populations, after adding verapamil these cells disappear. SP ratio (%) of over-expression miR-200a and control EOC cells was (0.233±0.076vs.0.850±0.100). Statistical analysis showed that, the differences of SP ratio between over-expression miR-200a and control EOC cells was statistically significant (t=8.488, P=0.001).
3. qRT-PCR showed that SOX2and OCT4mRNA levels in over-expression miR-200a EOC cells, decreased to (0.467±0.085) and (0.361±0.064) times, respectively, compared with control. Statistical analysis showed that, the difference mRNA levels of SOX2and OCT4between over-expression miR-200a EOC cells and control cells (t=10.932, P<0.001),(t=17.416, P=0.003) were statistically significant.
Western Blot results Show that SOX2and OCT4levels in over-expression miR-200a EOC cells, decreased to (0.546±0.077) and (0.518±0.086) times, respectively, compared with control. Statistical analysis showed that, the difference levels of SOX2and OCT4between over-expression miR-200a EOC cells and control cells (t=10.191, P=0.001),(t=9.659, P=0.011) were statistically significant.
Conclusion:
miR-200a weakened the stem characteristics of CSCs in EOC by blocking the formation of tumour spheroids, inducing the differentiation process in3D culture, reducing the proportion of CSCs and down-regulating the CSCs related genes SOX2and OCT4.
引文
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