摘要
恶性肿瘤的发病率和病死率都呈逐年增加趋势,对人类的健康已造成严重威胁。侵袭与转移是恶性肿瘤共同的生物学特性,也是影响肿瘤患者治疗及预后的重要因素。恶性肿瘤发生侵袭转移是目前肿瘤临床治疗的难点,对其发生机制的探讨是当今国内外学者研究的热点。食管癌是我国最常见的恶性肿瘤之一,河南省是高发省份,因此,探讨食管癌浸润、转移的机制具有重要意义。
肿瘤发生浸润转移是一个复杂过程,包括肿瘤细胞的粘附、运动、侵袭和血管新生等。S100A4属于钙离子结合蛋白家族成员,是由肿瘤细胞和肿痛活化的间质细胞分泌的活性肿瘤转移调节因子。已被归类为转移相关蛋白。已经证实多种恶性肿瘤存在S100A4高表达。它不仅以非共价同型二聚体的形式存在于细胞内,而且还被分泌出细胞外,以共价二聚体的形式存在于细胞间质而有细胞外作用。存在于细胞间质中的S100A4蛋白是细胞之间信号传导、细胞与细胞基质相互联系、相互作用的基础,也是肿瘤细胞侵袭和转移的病理基础。是一种有很高预后意义的肿瘤转移潜能分子标记物。现认为,S100A4蛋白通过和多种靶蛋白结合发挥细胞内和细胞外作用,可以调节细胞周期的进展,改变细胞的黏附性和运动能力,并增加肿瘤细胞的存活能力,调节肿瘤细胞的侵袭和转移能力。应用RNA干扰或反义寡核苷酸技术抑制S100A4蛋白的表达可使肿瘤细胞的活动能力下降2倍以上。S100A4蛋白尚可影响基质金属蛋白酶(MMPs)及基质金属蛋白酶组织抑制剂(TIMPs)的表达和活性,破坏基底膜的完整性,起到使肿瘤细胞转移的作用。E—钙粘蛋白(E-cadherin)通过维持正常细胞间的黏附力抑制细胞的侵袭,其功能丧失是细胞获得高侵袭性的关键。是公认的肿瘤转移抑制基因。有文献报道在非小细胞性肺癌、宫颈癌等肿瘤中S100A4与E-cadherin呈负相关。使S100A4成为旨在抑制肿瘤转移力的基因治疗的理想选靶目标,为肿瘤的防治提供了一条新的途径。
国外已有学者应用免疫组织化学法证实食管鳞癌中存在S100A4过表达,并和食管鳞癌的浸润转移有关,S100A4阳性的患者预后不良。但S100A4在食管鳞癌浸润转移中的分子机制及作用途径尚不清楚。本课题进行了以下研究:1.利用免疫组化技术、Western Blotting及RT-PCR等方法检测S100A4在食管鳞癌组织和食管鳞癌细胞系EC-1、Eca109、EC9706、TE-1中的表达。观察食管鳞癌组织中S100A4表达与MMP-2、E-cadherin的表达的关系及在浸润转移中的作用;采用Boyden侵袭小室检测EC-1、Eca109、EC9706、TE-1 4种食管鳞癌细胞系的体外侵袭力,并分析其与S100A4表达的关系。2.利用RNA干扰技术沉默筛选出来的具有高侵袭性的EC-1细胞中S100A4基因的活性,观察其对EC-1细胞体外侵袭力以及对MMP-2、E-cadherin表达的影响;MTT实验观察S100A4siRNA对食管鳞癌细胞增殖的影响。3.构建裸鼠食管癌移植瘤动物模型,观察移植瘤生长情况。本实验分为以下三部分。
第一部分S100A4在食管鳞癌中的表达和MMP-2、E-cadherin表达的相关性及与侵袭潜能的关系
方法
1.采用免疫组化法对100例食管鳞癌组织及其相应的癌旁正常食管黏膜组织中S100A4、MMP-2及E-cadherin蛋白的表达进行检测。分析S100A4与MMP-2、E-cadherin蛋白表达的关系。
2.采用Western Blotting和免疫细胞化学法检测4种食管鳞癌细胞系(EC-1、EC9706、Eca109、TE-1)中S100A4蛋白的表达。采用RT-PCR技术检测4种食管鳞癌细胞系中S100A4 mRNA的表达。
3.采用Boyden小室法测定4种食管鳞癌细胞系EC-1、EC9706、Eca109、TE-1的体外侵袭力和运动能力,并分析与S100A4表达的关系。
4.统计学处理:利用SPSS11.0软件处理数据,计数资料统计阳性率,采用x~2检验(chi-square);计量资料采用(?)±S表示;两组均数的比较用t检验(t-test);多组均数的比较用方差分析(ANVOA);相关性分析采用Pearson′s相关分析法。检验水准取α=0.05。
结果
1.S100A4在食管鳞癌组织中的表达及与MMP-2及E-cadherin表达的关系以及与临床病理学特征的关系
1.1 S100A4蛋白在食管鳞癌组织中的表达
S100A4在食管鳞癌细胞中为胞浆着色,食管正常黏膜上皮中胞浆、胞核均着色,食管鳞癌组织中大多数阳性着色细胞散在分布,少数阳性细胞呈灶状分布。间质中可见血管平滑肌细胞、内皮细胞着色;间质淋巴细胞、纤维细胞的胞浆着色较强。在食管鳞癌组织中的阳性表达率为52.00%,明显高于食管正常黏膜上皮中的表达率26.00%,两者相比具有显著性差异(P=0.001)。S100A4蛋白的表达与食管鳞癌的分化程度有关,高、中、低分化鳞癌中S100A4的阳性率分别为36.00%、51.56%、90.91%,差异具有显著性(P=0.040);S100A4蛋白的表达与食管鳞癌的浸润深度有关,浸润至深层(深肌层和外膜)的阳性表达率(59.38%)明显高于浸润至浅层(黏膜和浅肌层)的阳性率(38.89%)(P=0.049)。S100A4蛋白的表达与食管鳞癌的淋巴结转移有关,在淋巴结转移组S100A4蛋白的阳性表达率(69.50%)高于无淋巴结转移组(40.00%),差异具有统计学意义(P=0.004)。
1.2 MMP-2蛋白在食管鳞癌组织中的表达
MMP-2在癌细胞中的阳性表达主要定位于胞浆,部分间质细胞也有表达。MMP-2在食管鳞癌组织中的阳性率(67.00%)明显高于食管正常黏膜上皮的阳性率(31.00%)(P=0.001)。并且与食管鳞癌的分化程度有关(P=0.027),高、中、低分化鳞癌MMP-2蛋白表达阳性率为48.00%、70.31%、90.91%。MMP-2的表达还与食管鳞癌的浸润转移有关,浸润至深层的食管鳞癌组织中MMP-2蛋白阳性表达率(78.13%)明显高于浸润至浅层的食管鳞癌组织中的阳性表达率(47.22%),二者相比差异显著(P=0.002),在淋巴结转移组中MMP-2蛋白的阳性表达率(86.96%)高于无淋巴结转移组(47.00%)(P=0.020)。
1.3 E-cadherin蛋白在食管鳞癌组织中的表达
E-cadherin在正常黏膜组织主要表现为胞膜的强着色,在癌组织中则胞膜、胞浆均有着色,以胞浆着色为主。E-cadherin在食管鳞癌组织中的表达(44.00%)明显低于食管正常黏膜中的表达(86.00%)(P=0.001);随癌细胞分化程度的降低E-cadherin蛋白表达也逐渐降低,在高、中、低分化鳞癌中E-cadherin蛋白表达阳性率分别为64.00%、40.63%、18.18%(P=0.026);随癌细胞浸润深度增加E-cadherin蛋白表达呈降低趋势,但不存在统计学意义,浸润至深层的阳性表达率为39.06%,浸润至浅层的阳性率52.78%(P=0.185)。E-cadherin的表达与淋巴结转移有关,在淋巴结转移组中E-cadherin蛋白的阳性表达率(26.87%)低于无淋巴结转移组(49.35%),差异具有统计学意义(P=0.049)。
1.4 S100A4、MMP-2和E-cadherin蛋白表达的相关性分析
食管鳞癌组织中S100A4蛋白表达和MMP-2蛋白表达呈正相关关系(r=0.262,P=0.009);S100A4蛋白表达和E-cadherin蛋白表达呈负相关关系(r=-0.237,P=0.018);MMP-2蛋白表达和E-cadherin蛋白表达的相关系数为-0.363(P=0.001),两者也呈负相关关系。
2.S100A4在4种食管鳞癌细胞系(EC-1、Eca109、EC9706、TE-1)中的表达
2.1 S100A4蛋白在4种食管鳞癌细胞系中的表达
免疫细胞化学结果显示:S100A4蛋白在4株食管鳞癌细胞系EC-1、Eca109、EC9706、TE-1中均有表达,阳性表达主要定位于细胞浆,在Eca109细胞中偶见胞核着色。采用HPIAS-1000高清晰图像处理系统进行图像分析,所测得平均光密度(OD值)为EC-1(0358±0.016)、Eca109(0326±0.025)、EC9706(0.310±0.019)、TE-1(0.285±0.017),四组相比具有统计学意义(P=0.001);组间两两比较:Eca109和EC9706之间无统计学意义(P=0.134),其余各组间均有显著性差异(P＜0.05)。
Western Blotting结果表明:在4株食管鳞癌细胞系EC-1、Eca109、EC9706、TE-1中均有S100A4蛋白的表达,在EC-1中表达最高(0.897±0.053),依次为Eca109(0.806±0.058)、EC9706(0.720±0.068)、TE-1(0.645±0.089),差异具有统计学意义(P=0.001),组间两两比较:EC9706和TE-1之间无统计学意义(P=0.073),其余各组间均有显著性差异(P＜0.05)。
2.2 S100A4 mRNA在4种食管鳞癌细胞系中的表达
RT-PCR结果显示:S100A4 mRNA在4株食管鳞癌细胞系EC-1、Eca109、EC9706、TE-1中均有目的条带出现,其相对表达量为:EC-1(0.894±0.021)、Eca109(0.890±0.022)、EC9706(0.842±0.028)、TE-1(0.812±0.040),具有显著性差异(P=0.001),组间两两比较:EC1和Eca109之间无统计学意义(P=0.723),其余各组间均有显著性差异(P＜0.05)。
3.4种食管鳞癌细胞体外侵袭力和运动能力测定
在Boyden侵袭小室实验中,以穿过滤膜的细胞数表示细胞的侵袭力。4种食管鳞癌细胞系中,EC-1过膜细胞数最多(91.00+17.44),依次Eca109(83.60±12.93)、EC9706(79.00±11.29)、TE-1(61.80±11.10),4组间差异具有显著性(P=0.001),组间两两比较:EC-1和Eca109之间无统计学意义(P=0.575),其余各组间均有显著性差异(P＜0.05);4种食管鳞癌细胞系的体外运动能力依次为:EC-1(274.28±33.24)、Eca109(251.95±32.71)、EC9706(223.32±56.81)、TE-1(200.12±18.15),4组数据比较,差异具有显著性(P=0.016)。
4.S100A4表达与食管鳞癌细胞体外侵袭力的关系
食管鳞癌细胞中S100A4表达与侵袭力的相关性分析显示:S100A4蛋白表达与细胞体外侵袭力的相关系数为0.573(P=0.03),S100A4mRNA表达与细胞体外侵袭力的相关系数为0.832(P=0.001)。S100A4表达与细胞的侵袭力呈正相关关系,提示细胞的高侵袭性与S100A4的高表达有关。
第二部分S100A4siRNA对食管鳞癌细胞体外侵袭力和增殖的影响
方法
1.将化学合成的50nM的S100A4siRNA用转染试剂转入EC-1细胞,以单纯转染试剂转染的细胞作为对照组。
2.RT-PCR及Western Blotting法检测转染S100A4siRNA后0,24,48及72h S100A4 mRNA和蛋白的表达水平。
3.Boyden小室法检测转染S100A4 siRNA后0,24,48及72h,对EC-1细胞体外侵袭力及运动能力的影响。
4.RT-PCR及Western Blotting法检测转染S100A4siRNA后0,24,48及72h MMP-2和E-cadherin的mRNA和蛋白表达水平。
5.MTT实验检测转染S100A4siRNA后对EC-1细胞增殖的影响。
6.统计学处理:利用SPSS11.0软件处理数据,计量资料采用(?)±S表示;两组均数的比较用t检验(t-test);多组均数的比较用方差分析(ANVOA);显著性水准为α=0.05。
结果
1.转染前后细胞生长情况
用S100A4siRNA转染食管鳞癌细胞系EC-1后,倒置显微镜下观察发现:转染后细胞融合度变差,细胞中颗粒增多,细胞周围碎片增多,有部分变圆浮起,培养基中出现悬浮细胞。增殖速度明显减慢。未转染细胞贴壁生长,细胞轮廓清楚,细胞间结构紧密,生长旺盛。
2.S100A4siRNA有效沉默EC-1细胞中S100A4基因的表达
Western Blotting结果表明,S100A4siRNA转染EC-1细胞后,S100A4蛋白的表达量逐渐降低。S100A4/β-actin蛋白半定量结果为:转染0h(0.658±0.017)、24h(0.508±0.010)、48h(0.436±0.008)、72h(0.247±0.006),统计学分析,转染后24h、48h、72h与各时间点对照组及转染组转染0h相比均有显著性差异(P＜0.05),且转染后72h抑制效果最佳,S100A4蛋白表达量下降62.46%。RT-PCR显示,转染组S100A4 mRNA半定量结果为:转染0h(0.775±0.014)、24h(0.634±0.006)、48h(0.498±0.008)、72h(0.233±0.012)。经统计分析,转染后24h、48h、72h与各时间点对照组比较均有显著性差异(P＜0.05);转染组各时间点组间比较均有显著性差异(P＜0.05),转染72h后S100A4 mRNA的抑制率为69.94%,效果最佳。说明S100A4 siRNA对S100A4表达的抑制作用具有高度特异性和时间依赖性。
3.S100A4siRNA抑制食管鳞癌细胞体外侵袭力和运动能力
EC-1细胞转染S100A4siRNA 48h后,细胞的体外侵袭力和运动力明显下降,且随转染时间延长下降更加突出,转染72h细胞体外侵袭力下降60.66%,体外侵袭力:转染0h(85.46±15.10)、24h(76.86±18.64)、48h(59.39±7.47)、72h(33.62±7.82)(P＜0.05);转染72h细胞体外运动力下降60.80%,体外运动能力:转染0h(270.33±27.11)、24h(264.55±34.24)、48h(163.13±34.15)、72h(105.96±6.64)(P＜0.05)。
4.S100A4siRNA抑制EC-1细胞中MMP-2的表达
RT-PCR及Western Blotting结果表明,EC-1细胞转染S100A4siRNA后,MMP-2蛋白和MMP-2 mRNA的表达水平逐渐降低。MMP-2/β-actin mRNA半定量结果为:转染0h(1.004±0.058)、24h(0.969±0.077)、48h(0.622±0.035)、72h(0.275±0.078)。转染后48h、72h与相应对照组相比有明显差异(P＜0.05);MMP-2/β-actin蛋白半定量结果为:转染0h(0.864±0.054)、24h(0.710±0.041)、48h(0.549±0.041)、72h(0.288±0.050)。转染后24h、48h、72h与相应对照组相比,均有统计学意义(P＜0.05)。由此可见,S100A 4siRNA下调MMP-2蛋白的表达出现相对较早,表明S100A4除影响MMP-2 mRNA转录活性外,也可能对MMP-2 mRNA转录后蛋白合成有直接调节作用。
5.S100A4 siRNA对E-cadherin表达的影响
EC-1细胞转染S100A4siRNA后,RT-PCR及Western Blotting结果表明:E-cadherin蛋白和mRNA的表达水平逐渐增强。转染0h与24h无明显变化,转染后48h、72h与对照组相比均有显著性差异(P＜0.05)。E-cadherin/β-actin mRNA半定量结果为:转染后0h(0.318±0.013)、24h(0.334±0.023)、48h(0.476±0.022)、72h(0.586±0.013);E-cadherin/β-actin蛋白半定量结果为:转染后0h(0.289±0.012)、24h(0.294±0.020)、48h(0.479±0.014)、72h(0.625±0.022)。
6.S100A4 siRNA体外抑制食管鳞癌细胞增殖
MTT结果显示,转染S100A4 siRNA后细胞增殖能力明显受到抑制,转染后48h,转染组和对照组的吸光度值分别为(0.487±0.019)、(0.571±0.019)(P＜0.05);转染后72h,转染组和对照组的吸光度值分别为(0.488±0.008)、(0.681±0.013)(P＜0.05)。转染后48h和72h对细胞生长的抑制率分别为14.71%、28.34%,说明S100A4 siRNA有抑制食管鳞癌细胞增殖的作用。
第三部分S100A4siRNA对裸鼠体内食管癌移植瘤生长及基因表达的影响
方法
1.裸鼠食管癌异种移植瘤动物模型的建立:取对数生长期EC-1细胞,调整细胞浓度为(1×10~7/ml),在每只裸鼠前肢右腋背部皮下接种0.2ml,构建裸鼠移植瘤模型。
2.将成功构建的食管癌裸鼠异种移植瘤动物模型随机分成3组:S100A4siRNA组(A)、阴性对照siRNA(B)、空白对照组(C)。每组5只。分别给予50nM S100A4siRNA、50nM无关序列siRNA和PBS,每只200μl瘤旁注射,隔日治疗1次,共14次。
3.每日观察裸鼠肿瘤体积变化(采用游标卡尺进行测量),绘制肿瘤生长曲线。实验结束时统一处死小鼠,称取瘤重。计算肿瘤抑制率,公式为:肿瘤抑制率=(对照组瘤体重量-实验组瘤体重量)/对照组瘤体重量×100%。
4.各组裸鼠肿瘤组织进行常规病理切片及HE染色;免疫组化SP法检测上述各组瘤体组织中S100A4、MMP-2和E-cadherin蛋白的表达情况。
5.RT-PCR检测各组瘤体组织中S100A4、MMP-2和E-cadherin mRNA的表达情况。
6.统计学处理:利用SPSS11.0软件处理数据,资料采用(?)±S表示;两组均数的比较用t检验(t-test);多组均数的比较用方差分析(ANVOA)。以α=0.05为显著性水准。
结果
1.15只裸鼠皮下全部成瘤,EC-1细胞株异种移植致瘤率为100%,成瘤时间均在接种后1周左右。
2.不同方法处理后各组裸鼠肿瘤体积和瘤体重量的比较
各组裸鼠治疗前肿瘤体积经统计学分析无显著性差异。不同方法处理后,A组肿瘤体积(997.74±82.54mm3)与B组(1405.10±182.71mm3)和C组(1459.75±138.43mm3)相比明显较小,具有统计学差异(P＜0.05),B组与C组肿瘤体积无明显差异(P＞0.05)。处死动物后,分离肿瘤并称重。A组移植瘤重量较轻(0.493±0.021g),和B组(0.680±0.031g)、C组(0.701±0.046g)相比差异均有显著性意义(P＜0.01)。B组与C组相比差异无统计学意义(P＞0.05)。
3.不同方法处理后对食管癌异种移植瘤细胞形态的影响
光镜下观察发现,A组肿瘤细胞体积较小,核染色质稀疏,少见痛巨细胞,瘤组织间可见点、片状坏死灶。B组和C组肿瘤细胞大小不一,核染色质多,核大不规则,瘤巨细胞常见,异型性明显。
4.不同方法处理后食管癌异种移植瘤中S100A4、MMP-2和E-cadherin蛋白的表达
免疫组化结果以随机计数5个高倍视野,每视野记录200个细胞内阳性细胞数的平均数表示,S100A4蛋白在抑制瘤中的表达量为:A组(68±4)、B组(123±14)、C组(135±18),S100A4蛋白在A组瘤体内的表达低于在B组和C组的表达(P＜0.05),B组和C组相比无明显统计学差异(P＞0.05),说明瘤旁注射S100A4siRNA可以有效抑制移植瘤内S100A4蛋白的表达;MMP-2蛋白在A组瘤体内的表达(76±8)低于B组(134±9)和C组(142±7)。E-cadherin蛋白在A组瘤体内的表达(68±6)均高于在B组(41±2)和C组(36±4)的表达(P＜0.05)。与体外实验结果相一致。
5.3组食管癌异种移植瘤中S100A4、MMP-2和E-cadherin mRNA的表达
RT-PCR半定量结果显示:S100A4 mRNA在三组中的表达量分别为:A组(0.334±0.027)、B组(0.689±0.035)、C组(0.702±0.047);MMP-2 mRNA表达量为:A组(0.572±0.084)、B组(0.804±0.173)和C组(0.922±0.451)。S100A4和MMP-2mRNA在A组瘤体内的表达均低于在B组和C组的表达(P＜0.05),B组和C组相比无明显统计学差异(P＞0.05)。E-cadherin mRNA在A组瘤体内的表达(0.516±0.274)均高于在B组(0.327±0.033)和C组(0.284±0.057)的表达(P＜0.05)。
结论
1.食管鳞癌组织及食管鳞癌细胞系中S100A4高表达并与食管鳞癌的浸润转移有关。
2.体外实验提示,4种食管鳞癌细胞系中EC—1细胞具有高侵袭性。食管鳞癌细胞的高侵袭性与S100A4的高表达有关。
3.S100A4siRNA能有效地阻断食管鳞癌细胞中S100A4的表达,并对细胞的侵袭力及运动能力具有抑制作用。
4.S100A4与MMP—2的正相关关系及与E-cadherin的负相关关系,表明S100A4对食管鳞癌浸润转移力的影响与MMP—2及E-cadherin具有协同作用;S100A4 siRNA沉默S100A4活性可导致MMP—2的表达受到抑制,而E-cadherin的表达增加,提示S100A4可能对MMP—2及E-cadherin基因存在某种调节机制。
5.S100A4 siRNA具有抑制食管鳞癌细胞增殖的能力。
6.裸鼠移植瘤模型体内研究显示,S100A4siRNA能有效降低移植瘤中S100A4的表达,抑制裸鼠移植瘤生长,表明靶向S100A4的RNA干扰技术有可能成为食管癌基因治疗的新策略。
The incidence and fatality of Malignant tumors increase year by year,which have seriously threaten human health.The invasion and the metastasis are common bionomics of the malignant tumor which play an important role in the treatment and the prognosis of patients.They are the hot topics that scholars are studying nowadays and the nodus of tumor clinical treatment,esophageal carcinoma is one of the most common malignant tumors in our country especially in Henan Province.Therefore,it is significant to have a discussion about the mechanism of invasion and metastasis of esophageal carcinoma.
The process of invasion and metastasis of tumor is a complex course which involves many complex mechanisms,including the adherency,movement,invasion of tumor cells and the angiogenesis.S100A4 belongs to the family of calcium ion bond protein which is an active tumor metastasis regulatory factor secreted by the tumor cell and the tumor interstitial cells.It has been confirmed that there are S100A4 high expression in many kinds of malignant tumors.S100A4 not only exists in the intracellular in the form of non-covalence same type dimer,but also is secreted to the extracellular and exists in the intercellular substance in the form of covalence dimer with extracellular function.The S100A4 existing in the intercellular substance is the foundation of the signal conduction between cells,the mutual relation and the interaction between the cell and the cell matrix.In addition,it is also the pathological foundation of the invasion and metastasis of tumor cells.Thus,S100A4 which has been classified as the metastasis related protein is an very important biomarker to predict the prognsis of the tumors.Nowadays,it is believed that S100A4 protein united with many kinds of target proteins can exert its intracellular and extracellular function to regulate the progress of cell cycle,change the cell adhesivity and the movement ability,increase the survival ability of tumor cells,and can adjust the ability of infiltration and metastasis of tumor cells.Suppressing the expression of the S100A4 protein with siRNA or the antisense oligonucleotides technology can drop the locomotor activity of tumor cells about 2 times.In addition,the S100A4 can influence the expression and activity of matrix metalloproteinase and the inhibitor of matrix metalloproteinase,destroys the integrity of basilar membrane and causes metastasis of tumor cells.E-cadherin is important in keep the adhesion between the normal cells and inhibition of the invasion.Its functional incapacitation is critial to the invasion of tumor.E-cadherin as anti-metastasis gene is well-known.It was reported that there was negative correlation between the expression of S100A4 and E-cadherin in non-minicelllung cancer and uterine cervix cancer.This provided a new way to prevent the metastasis of tumor through suppressing the expression of the S100A4.
It has been confirmed by the foreign scholars that there are over expression of S100A4 in the esophageal squamous cell carcinoma(ESCC) detected by immunohistochemical method which concerned with the infiltration and metastasis of tumor,and these studies suggest that patients with the over expression of S100A4 protein have bad prognosis.But it is unclear the role of S100A4 in the infiltration and metastasis,whether which concerned with the expression of MMP-2 and E-cadherin? We well study this in the following reasearch.In our research,we have studied the molecular mechanism further and the role of S100A4 in the process of metastasis of esophageal scale cancer:1.We applied the immunohistochemistry technology, Western Blotting and the RT-PCR methods to examine the expression of S100A4, MMP-2 and the E-cadherin simultaneously and analyzed their relevance in ESCC organization and the ESCC cell lines EC-1,Eca109,EC9706,TE-1.The Boyden chamber was used to observe the invasiveness for 4 kinds of ESCC cell EC-1,Eca109, EC9706,TE-1 and their relation with S100A4 in vitro.2.Using RNAi technology to silence the S100A4 gene in the ESCC cell and observing the influence to invasiveness of the ESCC cell and its regulative function to MMP-2,and the expression of E-cadherin in vitro.MTT methods was used to observe the influence of S100A4siRNA to the multiplication of ESCC cells.3.By constructing of the athymic mouse model,we observed the growth of transplantation tumor with transfection cells..This experiment was divided into three parts as follows.
Part one The expression of S100A4 in ESCC,its relationship with the expression of MMP-2 and E-cadherin and the relationship with the potential invassiveness of cell line of ESCC
Methods
1.Immunohistochemical method was used to detect the expression of S100A4 protein, MMP-2 protein and E-cadherin protein in the tissues of ESCC and the relatively normal tissue besides the tumor in 100 samples.Analyzing the relationship between the expression of S100A4 protein and MMP-2 as well as the E-cadherin protein.
2.Western Blotting and immunocytochemical methods were applied to examine the expression of S100A4 protein in 4 kinds of ESCC cell(EC-1,EC9706,Eca109,TE-1). RT-PCR technology was used to examine the expression of S100A4 mRNA in 4 kinds of ESCC cell.
3.the Boyden cab was used to determine the ability of invasion of 4 kind of ESCC cells(EC-1,EC9706,Eca109,the TE-1) in vitro,examining the relationship between the expression of S100A4 and them.
4.statistical treatment:SPSS13.0 software was used,positive ratio of the enumeration data was statisticed with x~2 tests(chi-square);The measurement data was manifested with X±S;The comparison between two groups of mean values with T-test(t-test) and multi-group mean values with variance analysis(ANVOA);The relevant analysis used the Pearson's related analytic method.size of testα=0.05.
Results
1.The results of S100A4,MMP-2 and E-cadherin protein expression in relation to clinicopathological parameters in esophageal squamous cell carcinoma
1.1.The expression of S100A4 protein in esophageal squamous cell carcinoma
S100A4 is cytoblaslema coloration in the ESCC cells,but it is coloured in cytoblaslema and nucleus in epithelium mucosae of esophagus,almost all the positive cells scatter in the tissues of ESCC,only a few assemble in some positions.Vascular smooth muscle cells and endothelial cells are also found to be coloured in interstitial substance,and lymphocyte and fibrocellular was coloured more strongly in cytoblaslema.The positive rate is 52.00%in the ESCC which is obviously higher than the expression rate 26.00%(P=0.001) in the esophagus normal mucous membrane organization.There is significant difference between two groups.The positive rate of S100A4 in the high,middle and low differentiation ESCC is 36.00%,51.56%, 90.91%,respectively and there is diffference,too(P=0.04);In the ESCC infiltrates to in-depth(deep myo-level and external coat periplast),the positive rate is 59.38% which is obviously higher than 38.89%(P=0.049) in the shallow layer.(mucous membrane and shallow myo-level).The positive rate of the expression of S100A4 protein(69.50%) in the lymph node is higher than that in the non-lymph node (40.00%),and the difference has statistics significance(P=0.004).
1.2.The expression of MMP-2 protein in esophageal squamous cell carcinoma.
The positive expression of MMP-2 in cancer cell mainly locates in cytoplast and partially in mesenchymal.The positive rate in the ESCC organization is 67.00% which is obviously higher than the rate in normal mucous membrane organization of esophagus which is 31%(P=0.001).There is significant difference among groups different differentiated degree(P=0.027).The positive rate of the expression of MMP-2 protein in high,middle and poorly differentiation ESCC is 48.00%,70.31%, and 90.91%,respectively.The difference of the positive rate of the expression of MMP-2 protein in the in-depth ESCC 78.13%and 47.22%in the shallow layer ESCC is significant which has remarkable statistics significance(P=0.002).The positive rate of the expression of MMP-2 protein(86.96%) in the metabasis lymph node is higher than 47.00%in the non- metabasis lymph node group and there were the statistics significance(P=0.020).
1.3.The expression of E-cadherin protein in esophageal squamous cell carcinoma.
E-cadherin located in the cell membrane in the normal mucous membrane organization detected by immunocytochemical.However,it located in the cytoplast in cancer organization.The expression of E-cadherin in the cancer organization(44.00%) is obviously lower than its level(86.00%) in the normal tissue.The difference of the expression has statistics significance(P=0.001).The positive rate of the expression of E-cadherin protein in the high,middle and poorly differentiated ESCC was 64.00%, 40.63%and 18.18%(P=0.026),respectively.The E-cadherin protein expression rate reduces gradually with the increasing invasion depth of the cancer cell.The positive rate of its expression was 39.06%in the ESCC invaded deeply was lower than the rate for the invaded shallowly which is 52.78%,but there is no statistics significance (P=0.185).The positive rate of E-cadherin protein(26.87%) with the lymph node metabasis is lower than the rate(49.35%) withnot and the difference has statistics significance(P=0.049).
1.4.The relevant analysis about the expression of S100A4 proteins,MMP-2 protein and E-cadherin protein
In the ESCC organizations,the correlation coefficient between the expression of S100A4 protein and the MMP-2 protein is 0.262(P=0.009).The correlation coefficient between the expression of S100A4 protein and the E-cadherin protein is-0.237(P=0.018);The correlation coefficient between the expression of MMP-2 protein and the E-cadherin protein is -0.363(P=0.001).The S100A4 protein and the MMP-2 protein are connected positively obviously.The S100A4 protein and the MMP-2 protein assume the inverse correlation relations with the E-cadherin protein.
2.The expression of S100A4 in 4 kind of esophageal carcinoma cell lines(EC-1, Eca109,EC9706,TE-1)
2.1.Expression of S100A4 proteins in four ESCC cell lines
The results of immunocytochemical method shows that there is expression of the S100A4 protein in 4 cell lines,the positive expression mainly locates in the cytoplasma with faint yellow and the yellowish brown color,and partially concerned with the nucleus coloration in the Eca109 cell line.The image analysis applied by the HPIAS-1000 high clear imagery processing system shows that the average light density(the OD value) is EC-1(0.358±0.016),Eca109(0.326±0.025), EC9706(0.310±0.019),TE-1(0.285±0.017).Comparion of four groups there were significant difference(F=17.589,P=0.001).The comparison between Eca109 and EC9706 show that there is no statistics significance(P=0.134),the other comparisons between two groups has significant difference(P＜0.05).
The results of the Western Blotting indicated that,there were the expression of S100A4 protein in 4 kinds of esophageal carcinoma cell lines.The expression of S100A4 protein in EC-1 is(0.897±0.053) which is higher than(0.806±0.058) in Eca109,(0.720±0.068) in EC9706,and(0.645±0.089) in TE-1.there was significant difference(P=0.001).The comparison between EC9706 and TE-1 shows that there is no statistics significance(P=0.073),the other comparisons between two groups has significant difference(P＜0.05).
2.2.The expression of S100A4 mRNA in four ESCC cell lines.
RT-PCR results showed that there were the goal bandings of S100A4 mRNA in 4 kinds of cell lines.The semi-quantitative analysis showed that the relative expression quantity of S100A4 mRNA compared with EC-1(0.894±0.021),Eca109(0.890±0.022), EC9706(0.842±0.028),TE-1(0.812±0.040).There was significant difference (P=0.001),The comparison between EC1 and Eca109 show that there is non-statistics significance(P＞0.05),the other comparisons between two groups has significant difference(P＜0.05).
3.The examination of the invasiveness for 4 kinds of ESCC cell in vitro.
The number of cells passed through the filter membrane was used to indicate the invasiveness in the Boyden attack cab experiment.In 4 kind of ESCC cell,EC-1 cells had the greatest number(91.00±17.44),followed by Eca109(83.60±12.93),EC9706 (79.00±11.29),TE-1(61.80±11.10).The differences between four groups had significance(P=0.001).The comparison between EC1 and Eca109 showed that there is no statistics significance(P=0.575),the other comparisons between two groups has the significant difference(P＜0.05).The locomotory capacity of 4 cell lines were as the following:EC-1(274.28±33.24),Eca109(251.95±32.71),EC9706 (223.32±56.81),TE-1(200.12±18.15),and there were significant difference between each group(P=0.016).
4.The relationship between the expression of S100A4 protein,S100A4 mRNA and the invasiveness of ESCC cell in vitro
The relevant analysis demonstrated that the correlation coefficient between the expression of S100A4 protein and the invasion of these cell in vitro was 0.573 (P＜0.05).And the correlation coefficient between S100A4 mRNA expression and the invasiveness of these cells in vitro was 0.832(P=0.001).There was positive correlation between them.Thus,it indicated that there was close correlation between the expression of S100A4 and the invasiveness of ESCC cell in vitro.
Part two The infulence of S100A4siRNA on the Multiplication and Invasiveness of ESCC Cell In Vitro
Methods
1.EC-1 cells transfected with 50nM chemical synthesis S100A4siRNA as Transfected group,EC-1 cells transfected with transfected reagent as control group.
2.After transfection with S100A4siRNA,RT-PCR and Western the Blotting were used to detect the expression of S100A4 in mRNA and protein at 0,24,48 and 72h.
3.The Boyden cab was used to detect the invasion of EC-1 cells after transfection at 0,24,48 and 72h in vitro.
4.After transfected with S100A4siRNA,RT-PCR and Western Blotting were used to detect the expression of MMP-2 and E-cadherin mRNA and protein at 0,24,48 and 72h.
5.MTT experiment was used to detect the influence of S100A4siRNA on the cytostasis of EC-1 cells.
6.statistical treatment:Using the SPSS11.0 software to process data,The measurement data was manifested with(?)±S;The comparison between two group of mean values with T-test(t-test) and multi-group mean values with variance analysis (ANVOA);The relevant analysis was used the Pearson's related analytic method,size of testα=0.05.
Results
1.The comparison of cell growth condition before and after transfected
After transfection with S100A4 siRNA into the EC-1 cells,the morphous of these cells were observed under inverted microscope.The degree of cell fusion goes down;in the butcher the pellet increases;But the control group's cell pastes the wall to grow,the cell outline is clear,the intercellular space structure is close and the growth is exuberant.
2.S100A4siRNA effectively silenced the expression of S100A4 gene in EC-1 cells.
Results of Western Blotting indicated that,afte transfection expression of S100A4 protein reduced.Results of S100A4/β-actin protein were:0 hours (0.658±0.017),24 hours(0.508±0.010),48 hours(0.436±0.008),72 hours (0.247±0.006).After statistics analysis,there were significant difference between 24h、48h、72h after tranfection with control group in each times and 0h after tranfection(P＞0.05).Transfected after 72h,the effectiveness of inhibition was optimization,the expression of S100A4 reduced 62.46%.
Results of semi-quantitative mRNA of S100A4/β-actin after transfection is:0 hours(0.775±0.014),24 hours(0.634±0.006),48 hours(0.498±0.008),72 hours (0.233±0.012).After statistics analysis,there were significant difference between 24h,48h,72h after tranfection with control group in each times and 0h after tranfection (P＞0.05).Transfected after 72h,the effectiveness of inhibition was optimization,the expression of S100A4 reduced 69.94%.Which indicated that there were high degree of specificity and time dependence in the suppression of S100A4 siRNA to S100A4.
3.Influence of S100A4siRNA on the invasiveness and locomotory capacity of ESCC cell in vitro.
After tranfection,the invasiveness and motoricity of EC-1 cells reduced gradually after 48h.After 72h,the invasiveness of EC-1 cells reduced 60.66%,the motoricity of EC-1 cells reduced 60.80%.The results are as the fllowings,invasiveness in vitro:0 hours(85.46±15.10),24 hours(76.86±18.64),48 hours(59.39±7.47),72 hours (33.62±7.82);locomotory capacity in vitro:0 hours(270.33±27.11),24 hours (264.55±34.24),48 hours(163.13±34.15),72 hours(105.96±6.64).
4.S100A4siRNA suppressed the expression of MMP-2 in EC-1 cells
The results of RT-PCR and Western Blotting showed that:after transfected with S100A4siRNA the expression of protein and mRNA of MMP-2 reduced gradually. The results of MMP-2/β-actin mRNA are as the followings:0h(1.004±0.058),24h (0.969±0.077),48h(0.622±0.035),72h(0.275±0.078),there was significant difference between 48h,72h and control group(P<0.05).The results of MMP-2/β-actin:0h(0.864±0.054),24h(0.710±0.041),48h(0.549±0.041),72h (0.288±0.050),there was significant difference between 24,48h,72h and control group(P<0.05).S100A4siRNA reduced the expression of MMP-2 proteion early than MMP-2 mRNA,which indicated that S100A4 not only influence the transcription of MMP-2 mRNA,but also influence the synthesis of protein after transcription.
5.S100A4siRNA suppresses the expression of E-cadherin in EC-1 cells
After transfection with S100A4siRNA,the results of RT-PCR and Western Blotting showed that:the expression of protein and mRNA of E-cadherin increased gradually.there was no significant changes at 0h and 24h,however there was significant difference between 48h,72h and control group.The results of E-cadherin /β-actin mRNA:0h(0.318±0.013),24h(0.334±0.023),48h(0.476±0.022),72h (0.586±0.013);The results of E-cadherin /β-actin:0h(0.289±0.012),24h (0.294±0.020),48h(0.479±0.014),72h(0.625±0.022).
6.Suppression of S100A4siRNA on the multiplication of ESCC cells in vitro
The MTT results showed that:after transfection reproductive activity of these cells were inhibited effectively,after transfected 48h,OD of transfection group,control group was(0.487±0.019),(0.571±0.019)(P＜0.05).after transfected 72h,OD of transfection group,control group was(0.488±0.008),(0.681±0.013) (P＜0.05).Ttransfected after 48h,72h,inhibited ratio to these cells is 14.71%,28.34% respectively.Which indicated that S100A4 siRNA can inhibit the reproductity of ESCC Cell.
part three Research on the effect of S100A4siRNA to the growth of transplants tumor of esophageal carcinoma in the body of athymic mouse.
Methods
1.The establishment of the modle of heterogenic transplantation tumor of esophageal carcinoma in the body of athymic mouse:Take the logarithm growth period EC-1 cell, adjust cell density to(1×10~7/ml),hypodermic vaccinates 0.2ml in each bare mouse's foreleg right armpit back,constructs the model of bare mouse transplant tumor.
2.The animals successfully constructed were divided into 3 groups at random: S100A4siRNA group(A),irrelevant sequence siRNA(B),control group(C).There are 5 athymic mice in each group.Drugs was injected with 50nM S100A4siRNA、50nM control siRNA and PBS nearby the tumors to treat 1 time every other day, altogether 14 times.
3.Observe the change of the tumor volume of athymic mice everyday(Vernier caliper was used),and curve the growth of tumor.In the end all athymic mice were executed,and the weight of tumor were measured,the formula is:Tumor suppression rate=(quantity of control group tumor-quantity of experimental group tumor)/ quantity of control group tumor×100%.
4.Tumor organization of each group of athymic mice were carried on the convention pathological slice and dyed with HE;Examine the expression of S100A4,MMP-2 in each group and the expression of E-cadherin protein.
5.Examine the expression S100A4,MMP-2 and E-cadherin mRNA of each group with RT-PCR.
6.statistical treatment:Using the SPSS11.0 software to process data,The measurement data were manifested with(?)±S;The comparison between two groups of mean values with T-test(t-test) and multi-group mean values with variance analysis(ANVOA);The relevant analysis used the Pearson's related analytic method. size of testα=0.05.
Results
1.Tumors were succeeded in the hypodermic of 15 athymic mice,the EC-1 cell line heterotransplantation rate is completely 100%,formed the tumor after 1 week.
2.The comparison of the weight and volume of tumors stripped out of the nude mice in three groups at the end of treament
Before getting treatment,there were no significant difference in the tumor volume(P>0.05).After dealing with different methods,volume of tumors of A group were smaller than B and C groups(P<0.05),and there was no difference between B and C groups.We weighed those tumors after killing these mice,Weight of tumors of A group were smaller than B and C groups(P<0.05),there were siginifant difference between them(P<0.01),But the deffenence between B and C groups had no statistics significance(P>0.05).
3.The influence on morphology of cells after transfected with S100A4siRNA
Observing under the microscope,we found that the tumor cells of A group were smaller and the nuclear chromatins were sparse.In addition the tumor giant cells were rare,and the obvious plot and the laminated necrosis stove were found in the tumor organization.We also find that the size of tumor cells of B group and C group were irregular,and there were a lot of nuclear chromatin,the nucleus were large and irregular,the tumor giant cells were common,and the heterogeneous type was obvious.
4.The expression of S100A4,MMP-2 and E-cadherin protein in three groups
The results of immunocytochemsty were observed under 5 highpower field randomedly,in each field we obersved 200 positive cells.Quantity of the expression of S100A4 protein were as the flowings:A group(68±4),B group(123±14),C group (135±18);The expression of S100A4 in A group were lower than that in B group and C group(P＜0.05),Compared the expression of B group and C group,we found there was no obvious statistics difference(P＞0.05).Which indicated that S100A4siRNA was injected besides the tumor can effectively inhibit the expression of S100A protein;quantity of the expression of MMP-2 protein in A group were (76±8),which was lower than B group(134±9)and C group(142±7).The expression of the E-cadherin protein in the tumor of A group(68±6) was higher than B and C groups(P＜0.05).The results is uniformed with the results of vitro.
5.The expression of S100A4,MMP-2 and E-cadherin mRNA in three groups
The result of RT-PCR showed that quantity of the expression of S100A4 mRNA were as the flowings:A group(0.334±0.027),B group(0.689±0.035),C group (0.702±0.047);quantity of the expression of MMP-2 mRNA were as the flowings:A group(0.572±0.084),B group(0.804±0.173),C group(0.922±0.451).The expression of S100A4 and the MMP-2mRNA in A group was lower than that in B group and C group(P＜0.05).Compared the expression of B group and C group,we found there was no obvious statistics difference(P＞0.05).The expression of the E-cadherin mRNA in the tumor of A group(0.516±0.274) was higher than B group (0.327±0.033) and C group(0.284±0.057)(P＜0.05).
Conclusion
1.There is high expression of S100A4 in the tissue of ESCC and its cell lines,which was positive correlation with the invasiveness of tumor cells.
2.The results of expremient in vitro suggests that EC-1 cell lines have the highist invasion in the 4 different cell lines.The expression of S100A4 concerned with malignant degree and invasiveness of tumor.
3.S100A4siRNA can effectively inhibit the expression of S100A4,invasion and locomotory capacity of tumor cells.
4.There is positive correlation between S100A4 and MMP-2,but there is negative correlation between S100A4 and E-cadherin,which indicated that there were synergistic effect to the influence on the infiltration,metastasis of ESCC between S100A4,MMP-2 and E-cadherin.S100A4siRNA can effectively silence the expression of S100A4 gene,suppresses the expression of MMP-2,but the expression of E-cadherin increased,which indicated that S100A4 may adjust the expression of MMP-2 and E-cadherin through some way.
5.S100A4 has the ability of inhibition of the cell proliferation.
6.The model of heterogenic transplantation tumor of esophageal carcinoma in athymic mice demonstrats that,S100A4siRNA can effectively reduce the expression of S100A4 protein,and suppress the growth transplantation tumor,which indicats S100A4 RNA may become new targat gene for the treatment of esophageal carcinoma.
引文
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