低剂量放疗诱导宫颈癌上皮—间质转化分子机制的研究
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摘要
背景与目的：
恶性肿瘤严重危害着人类的健康,其中宫颈癌是女性生殖系统常见的恶性肿瘤,其发病率在女性生殖系统肿瘤发病率位居第二,死亡率位居第五。全世界每年约有50万新发病例,其中约30万的患者死于宫颈癌,在我国宫颈癌发病率居世界第二位,严重危害女性健康。目前,治疗宫颈癌的方法主要有手术治疗、放射治疗和化学治疗,根据2010NCCN宫颈癌临床实践指南,放疗是治疗宫颈癌的重要手段之一,其适应症广泛,除严重的肝肾功能,造血功能障碍外,Ⅰ～Ⅳ期患者均可进行放射治疗,尤其对于晚期、复发或者无法手术的患者,放射治疗是治疗的重要方法。然而由于个体体质的差异以及肿瘤的异质性,不同患者对放射治疗的反应不同,存在着放疗敏感性和放疗耐受性的差异,放疗后达到治疗效果也不一样,放疗耐受是宫颈癌治疗失败而出现复发转移的重要原因之一。因此,阐明宫颈癌放疗耐受性的机制将有望为设计宫颈癌的个体化治疗提供理论基础,也将为开发逆转宫颈癌放疗耐受的治疗手段提供新的思路。
在肿瘤微环境中,许多因素影响肿瘤细胞对放射治疗的反应。离子放射激活与细胞凋亡,DNA损伤修复,细胞粘附和血管新生信号通路相关的一系列基因,反过来,这些基因又调节细胞内的放疗反应,影响放疗效果。上皮-间质转化(epithelial-mesenchymal transition, EMT)是上皮细胞丢失细胞间的连接,获得间质特性的过程。包括细胞上皮特异性连接蛋白E-cadherin的重新分布或丢失,间质marker N-cadherin和vimentin的发动,细胞的形态改变为狭长的纺锤状,迁移能力增强。EMT是上皮细胞在特定的生理和病理情况下转化为具有间质表型细胞的生物学过程,该过程也发生在肿瘤进展过程中。近年来上皮间质转化在肿瘤中的作用逐渐被重视,研究报道EMT在肿瘤的发生发展、浸润转移等方面发挥着重要的作用。最近研究报道一些microRNA可以调控这一重要的生物学过程。
MicroRNA(miRNA)是内源基因编码的、长度约20-24个核许酸的非编码单链小RNA分子。microRNA通过调节关键的靶基因mRNA,影响分化、增殖、代谢和凋亡多种细胞过程。miRNA在肿瘤里的图谱和在正常组织的表达图谱不同,这类图谱可以作为疾病分类的新的方法。miRNA对EMT有很重要的调节作用,miRNA在细胞增殖、分化、凋亡、基因调控及疾病的发生中扮演重要的角色。miRNA-200c直接作用于转录抑制因子ZEBl及ZEB2的mRNA,上调肿瘤细胞系中E-cadherin的表达,从而减少EMT的发生。miR-200家族的成员(miR-200a, miR-200b, miR-200c, miR-141, miR-429)和miR-205表达在发生EMT的MDCK细胞中显著下调。microRNA的下调是EMT过程中的一个必要成分,已经证实TGFβ敏感的miR-200和ZEB之间通过双向负反馈环(double-negative feedback loop)影响EMT。EMT在肺癌中影响miRNA的表达,并且通过EMT调节肺癌细胞的侵袭和转移。因而阐明调控肿瘤细胞发生EMT过程的分子机制,明确其在恶性肿瘤的发生、进展的作用,并探索基于EMT的诊断方法具有重要的临床意义。
NF-κB是在调节细胞凋亡和肿瘤发生中起重要作用的转录因子,而且是在诱导EMT和维持EMT中是必不可少的。NF-κB信号的强度和持续时间由多个负反馈机制控制,从而使NF-κB信号在癌细胞中保持其致癌性。microRNA对NF-κB有调节作用来调控NF-κB调节的基因表达,影响这些基因的功能。在神经胶质瘤miR-182过表达,直接抑制CYLD(肿瘤抑制基因)(NF-κB的负性调节子)的表达,促进泛素结合NF-κB信号通路成分诱导胶质细胞农现侵袭性表型。并且TGF-β诱导的miR-182表达可以延长NF-KB的活化时间。miR-146a在大部分胃癌表达上调,抑制GPCR(G蛋白偶联受体)介导的NF-κB的激活,因而减少NF-κB调节的肿瘤促进激酶表达和生长因子的表达。通过作用于NF-κB活化通路的一些成分,miR-146a在调节NF-κB活性中起关键作用。miRNA与p53, NF-κ B, β-catenin通路相互作用在调节EMT和保持肿瘤干细胞中起重要作用。乳腺癌中抑制miR-448通过直接作用于特异的AT-富集序列结合蛋白-1(SATB1)的mRNA,导致双调蛋白水平升高,表皮生长因子(EGFR)受体介导的Twistl表达升高,NF-κ B激活,从而可以诱导EMT。基于这些报道,我们猜想miRNA调节NF-κ B在宫颈癌放疗耐受相关EMT中起作用。
本研究旨在阐明宫颈癌放疗耐受的机制,为设计宫颈癌的个体化治疗提供理论基础。本课题进行了三部分的研究：(1)建立宫颈癌放疗耐受细胞株；(2)宫颈癌放疗耐受细胞株EMT的发生及机制研究；(3)宫颈癌放疗耐受相关miRNA的筛选及功能分析。
方法：
根据文献报道及2010NCCN宫颈癌临床实践指南自行建立放疗耐受(FIR)的细胞模型进行实验研究。采用低剂量持续分级放疗的方法,给予2Gy/day的剂量照射宫颈癌细胞株SiHa, C-33A,5days/week,至总剂量达到75Gy,细胞休息2周后实验。进行克隆形成实验、细胞周期及凋亡的检测验证FIR细胞是否对放疗耐受。
在建立FIR细胞进行放疗的过程中,进行形态学观察,发现与亲本细胞相比,FIR细胞株发生了显著的形态学改变,此改变符合EMT。为了证实FIR细胞株是否真正发生了EMT,我们进行western blot实验检测了EMT的标志蛋白E-cadherin和N-cadherin的变化。确定FIR细胞株发生EMT后,进行定量PCR实验在mRNA水平,western blot实验在蛋白水平检测FIR细胞与亲本细胞(N细胞)其他EMT marker的变化,并进行分子机制研究。使用siRNA干扰FIR细胞中NF-κ B p65表达后,检测EMT marker表达,研究NF-κ B p65是否参与调控低剂量放疗诱导的宫颈癌EMT。
miRNA表达谱芯片是研究miRNA的有力工具,通过对宫颈癌放疗耐受细胞株与亲本细胞的miRNA表达谱的分析,筛选出放疗耐受相关的miRNA,为今后开发逆转宫颈癌患者放疗耐受的新治疗手段提供新的靶标。我们使用microRNA芯片检测宫颈癌N细胞及FIR细胞,筛选与放疗耐受相关的microRNA。合成miRNA的引物,定量PCR验证芯片数据结果,并对显著差异的miRNA进行功能研究,筛选放疗耐受相关miRNA。合成FIR细胞表达降低的miRNA的模拟物mimic miRNA,导入FIR细胞后,定量PCR和western blot检测EMT marker表达变化。同时western blot检测宫颈癌FIR细胞表达NF-κB p65的变化；siRNA干扰FIR细胞表达的NF-κB p65后定量PCR实验检测miRNA表达的变化。
结果：
克隆形成实验显示亲木细胞和FIR细胞接受2Gy的X-ray照射后,FIR细胞克隆形成的能力比亲本细胞增强；FIR细胞倍增的时间比亲本细胞长；接受不同剂量的X-ray照射后,FIR细胞凋亡率低于亲本细胞,说明FIR细胞耐受放疗。这些结果表明宫颈癌FIR细胞具有宫颈癌放疗耐受的特点,宫颈癌放疗耐受细胞系建立成功。
在建立宫颈癌FIR细胞,对宫颈癌N细胞进行放疗的过程中,通过形态学观察,发现与亲本细胞相比,FIR细胞株发生了显著的形态学改变,细胞由铺路石样的上皮表型变成细长的间质表型,此改变符合上皮-间质转化,同时细胞迁移和侵袭的能力增强。
为了证实FIR细胞株是否真正发生了EMT,我们进行western blot检测了EMT的标志蛋白E-cadherin和N-cadherin的变化,结果显示FIR细胞表达的E-cadherin降低,N-cadherin表达升高,从而初步证实了FIR细胞有EMT的改变。进一步进行定量PCR实验在mRNA水平,western blot实验在蛋白水平检测FIR细胞与亲本细胞其他EMT marker的变化,发现上皮性marker CK-18表达降低,间质marker vimentin表达升高。使用siRNA干扰FIR细胞中,p65农达后,E-cadherin表达升高,N-cadherin农达降低,说明NF-κB p65参与调控低剂量分级放疗诱导的宫颈癌放疗耐受相关EMT。
mieroRNA芯片检测宫颈癌亲本细胞及FIR细胞,筛选与放疗耐受相关的miRNA。结果显示,在1198个mieroRNA探针中有20个mieroRNA有差异,定量PCR验证了世片数据结果。合成FIR细胞表达降低的miRNA的模拟物mimic miRNA,导入FIR细胞后,定量PCR和western blot检测EMT marker表达变化,其中导入mimic miR-1236后FIR细胞表达的E-cadherin升高和N-cadherin表达降低,差异有显著性,说明miR-1236参与调节低剂量分级放疗引起的宫颈癌放疗耐受相关EMT。
进一步研究发现FIR细胞导入miR-1236后NF-κ B p65的表达降低,同时使用siRNA干扰NF-κ B P65后FIR细胞表达的miR-1236比对照组显著升高,说明miR-1236和NF-κ B p65相互调节在低剂量分级放疗诱导的宫颈癌EMT导致的放疗耐受中起调控作用。
结论：
1.低剂量分级持续放疗可以诱导宫颈癌细胞发生上皮-间质转化而发生放疗耐受。
2. NF-κ B p65参与调控宫颈癌放疗耐受相关的EMT。
3. miR-1236参与调控宫颈癌放疗耐受相关的EMT。
4. miR-1236和NF-κ B p65相互调节在低剂量分级放疗诱导的宫颈癌EMT导致的放疗耐受中起调控作用。意义：
1.阐明宫颈癌放疗耐受的机制将有望为设计宫颈癌的个体化治疗提供理论基础。
2. NF-kB p65及筛选出来的的microRNA也将为开发逆转宫颈癌放疗耐受的治疗手段提供新的靶标。
3.本研究建立的宫颈癌放疗耐受细胞株可以用于放疗耐受相关的其他分子生物学研究。
BACKGROUND AND OBJECT
Cervical cancer is one of the most common genital cancers in women worldwide. It is the second most prevalent and the fifth most deadly malignancy. There are approximately500,000new cervical cancer cases in the world each year, and more than60%of these cases die annually. And incidence of cervical cancer in our country ranks second in the world. Currently, many therapy methods are available for cervical cancer, such as surgery, radiotherapy and chemotherapy. According to the NCCN Clinical Practice Guidelines in Oncology—Gervical Cancer Guideline2010, radiotherapy is one of the important means for the treatment of cervical cancer. Radiotherapy is effective for almost all the stages of cervical cancer, particularly for patients with advanced cervical cancers or who can not be cured surgically. However the response to radiotherapy varies among patients because of the heterogeneity of individual conditions and tumors. Radiation resistance has become one of the most reasons of failure of cure and recurrence and metastasis. Therefore to clarify the mechanisms of radiation resistance may provide theoretical basis for individual treatment and also provide new therapeutic targets to reverse the radiation resistance of cervical cancer.
The response of tumor cells to radiotherapy was affected by many factors in tumor microenvironment. Radiation ionizing activates genes which are related to cell apoptosis, DNA damage and repair, cell adhesion and angiogenesis. In return these genes regulate cell response to radiation. Epithelial-mesenchymal transition (EMT) is a process that epithelial cells lose their cell-cell adhesion and obtain mesenchymal features. Including redistribution or lost of specific epithelial connecting protein, E-cadherin, and mesenchymal markers, including N-cadherin and vimentin, were activated. Morphology of cell changes to cambiform phenotype and the ability of migration is enhanced. EMT is a biological process that epithelial cells transited to cells with mesenchymal characters in some physiological and pathological conditions. And it also occurs in the development of tumors. In recent years, the functions of EMT have been paid more attention. It has been reported that EMT played important role in development and progression, invasion and migration of tumor. And many factors regulated EMT.
MicroRNAs (miRNAs) are small endogenous single-stranded,22-24nucleotide non-coding RNAs. Maps of miRNAs are different between tumors and normal tissues. Therefore these maps can be used as new methods of disease classification. MiRNAs influence differentiation, proliferation, metabolism, apoptosis of cells and development of diseases by regulating key target mRNAs. And in regulation of EMT, miRNAs also play important role. As a result it is very important to clarify the molecular mechanisms of occurrence of EMT and the functions of EMT in development of tumors. And it is also very meaningful to explore the diagnostic methods which are based on EMT.
NF-κB is a key transcription factor in regulation of cell apoptosis and tumor progression. It is essential in induction and maintain of EMT. MiRNAs can regulate NF-κB and consequently the expression of its downstream genes were affected. The signal strength and lasting time of NF-κB are controlled by multiple negative feedback mechanisms. Thus NF-κB can keep its functions of oncogene in tumor cells.
This study aimed to clarify the mechanisms of radiation resistance of cervical cancer and provide theoretic basis for individual treatment of cervical cancer. In this project three parts were studied. Part1was to establish radiation resistance cervical cancer cells. Part2was to study the molecular mechanisms of low-dose radiation induced EMT. And Part3was the screening of miRNAs which are resistant to radiation of cervical cancer and analysis of their functions.
METHODS
Fractionated X-ray irradiation was utilized to generate radioresistant (FIR) cell model according to methods reported and NCCN Clinical Practice Guidelines in Oncology—Cervical Cancer Guideline2010. Cervical cancer cells, SiHa and C-33A, were irradiated with2GY of low-dose fractionated X-ray irradiation per day on five consecutive days per week, until the total concentration reached75GY. There was a two-week interval between the last fractionated irradiation and the experiments. Clonogenic assay, cell cycle and apoptotic assays were conducted to test the radiresistance of cells.
During this process, morphological changes of FIR cells, which corresponded with EMT, were observed comparing of parent cells. To confirm the existence of EMT, we detected the expression of E-cadherin and N-cadherin, the marker proteins of EMT. After that, other EMT markers and detailed molecular signaling pathways were detected by real-time PCR on mRNA level and by western blot on protein level. SiRNA was used to interfere the expression of NF-κB p65to study the role of NF-κB p65in low-dose radiation induced EMT involved in cervical cancer radioresistance.
Recently,microRNAs (miRNAs) were reported to play important role in regulation of EMT, cell proliferation, cell differentiation, apoptosis and the development of diseases. For this reason, we used miRNA microarray analysis to detect the FIR cells and parent cells to find the miRNAs which were related to radioresistance. The primers of relative miRNAs were synthetized and real-time PCR was conducted to further confirm the results of microarray and the functions of miRNAs which work in radioresistance of cervical cancers were studied. Mimics of miRNAs which were down-regulated in FIR cells were synthetized and transfected to detect the change of EMT markers. At the same time, the expressions of NF-κB p65 were detected by western blot. SiRNA was used to silence the expression of NF-κB p65to make sure the expression change of miRNAs by real-time PCR.
RESULTS
Clonogenic assays revealed that the ability of FIR cells to form clones were enhanced comparing with parent cells after2GY of X-ray irradiation. When accepting different dose of X-ray irradiation, the apoptosis rate of FIR cells decreased obviously. This indicated that FIR cells were resistant to radiation and cervical cancer cell model of radioresistance were generated successfully.
During the processes of generating radioresistant cervical cancer cells, FIR cells were observed obvious morphological changes which corresponded with EMT. And the abilities of migration and invasion of FIR cells also enhanced.
To clarify the existence of EMT, we detected the expression of E-cadherin and N-cadherin which were marker proteins of EMT and found that E-cadherin had low expression and N-cadherin had high expression in FIR cells. This indicated that FIR cells developed EMT. Further studies, including real-time PCR on mRNA level and western blot on protein level, revealed that another epithelial marker, CK-18, was down-regulated and mesenchymal marker, vimentin, was up-regulated. After silencing of p65by siRNA in FIR cells, E-cadherin was up-regulated and N-cadherin was down-regulated. These results showed that NF-κB p65worked in regulation of low-dose radiation induced EMT in cervical cancer.
MiRNAs expression profile is a powerful tool to study miRNAs. After analyzing the miRNAs expression profiles of radiation resistance cervical cancer cells and parent cells, miRNAs which are related to radiation resistance can be screened out. And this will provide new therapeutic targets to reverse the radiation resistance of cervical cancer patients. The results of miRNA microassay analysis showed that20in total1198miRNAs varied in FIR cells compared with parent cells. And real-time PCR confirmed these results. Mimics of miRNAs which were down-regulated in FIR cells were synthesized and transfected into FIR cells to detect the changes of EMT markers. Results revealed that after transfection of mimic miR-1236, the expression of E-cadherin was enhanced and N-cadherin was decreased obviously in FIR cells. It indicated that miR-1236play role in low-dose radiation induced EMT involved in cervical cancer radioresistance.
Further detailed studies found that after transfection of miR-1236, the expression of NF-κB p65were up-regulated. And when using siRNA to interfere the expression of NF-κB p65, miR-1236was elevated compared with control group. This illustrated that the mutual regulation between miR-1236and NF-κB p65regulated the low-dose radiation induced EMT in cervical cancer.
CONCLUSION
1. low-dose radiation could induce EMT in cervical cancer to develop the radioresistance.
2. NF-κB p65played role in regulation of low-dose radiation induced EMT in cervical cancer radioresistance.
3. MiR-1236worked in regulation of low-dose radiation induced EMT in cervical cancer radioresistance.
4. Mutual regulation between miR-1236and NF-κB p65regulated the low-dose radiation induced EMT involved in cervical cancer radioresistance.
SIGNIFICANCE
1. Clarifying the mechanisms of radioresistance in cervical cancer could provide the theoretical basis for individual treatment of cervical cancer patients.
2. NF-kB p65and miRNAs which were found relative with cervical cancer could provide new therapeutic targets for cervical cancer to reverse the radioresistance.
3. The.radioresistance cervical cancer cells which we established in this study could be used in other molecular research related to radioresistance.
恶性肿瘤严重危害着人类的健康,其中宫颈癌是女性生殖系统常见的恶性肿瘤,其发病率在女性生殖系统肿瘤发病率位居第二,死亡率位居第五。全世界每年约有50万新发病例,其中约30万的患者死于宫颈癌,在我国宫颈癌发病率居世界第二位,严重危害女性健康。目前,治疗宫颈癌的方法主要有手术治疗、放射治疗和化学治疗,根据2010NCCN宫颈癌临床实践指南,放疗是治疗宫颈癌的重要手段之一,其适应症广泛,除严重的肝肾功能,造血功能障碍外,Ⅰ～Ⅳ期患者均可进行放射治疗,尤其对于晚期、复发或者无法手术的患者,放射治疗是治疗的重要方法。然而由于个体体质的差异以及肿瘤的异质性,不同患者对放射治疗的反应不同,存在着放疗敏感性和放疗耐受性的差异,放疗后达到治疗效果也不一样,放疗耐受是宫颈癌治疗失败而出现复发转移的重要原因之一。因此,阐明宫颈癌放疗耐受性的机制将有望为设计宫颈癌的个体化治疗提供理论基础,也将为开发逆转宫颈癌放疗耐受的治疗手段提供新的思路。
在肿瘤微环境中,许多因素影响肿瘤细胞对放射治疗的反应。离子放射激活与细胞凋亡,DNA损伤修复,细胞粘附和血管新生信号通路相关的一系列基因,反过来,这些基因又调节细胞内的放疗反应,影响放疗效果。上皮-间质转化(epithelial-mesenchymal transition, EMT)是上皮细胞丢失细胞间的连接,获得间质特性的过程。包括细胞上皮特异性连接蛋白E-cadherin的重新分布或丢失,间质marker N-cadherin和vimentin的发动,细胞的形态改变为狭长的纺锤状,迁移能力增强。EMT是上皮细胞在特定的生理和病理情况下转化为具有间质表型细胞的生物学过程,该过程也发生在肿瘤进展过程中。近年来上皮间质转化在肿瘤中的作用逐渐被重视,研究报道EMT在肿瘤的发生发展、浸润转移等方面发挥着重要的作用。最近研究报道一些microRNA可以调控这一重要的生物学过程。
MicroRNA(miRNA)是内源基因编码的、长度约20-24个核许酸的非编码单链小RNA分子。microRNA通过调节关键的靶基因mRNA,影响分化、增殖、代谢和凋亡多种细胞过程。miRNA在肿瘤里的图谱和在正常组织的表达图谱不同,这类图谱可以作为疾病分类的新的方法。miRNA对EMT有很重要的调节作用,miRNA在细胞增殖、分化、凋亡、基因调控及疾病的发生中扮演重要的角色。miRNA-200c直接作用于转录抑制因子ZEBl及ZEB2的mRNA,上调肿瘤细胞系中E-cadherin的表达,从而减少EMT的发生。miR-200家族的成员(miR-200a, miR-200b, miR-200c, miR-141, miR-429)和miR-205表达在发生EMT的MDCK细胞中显著下调。microRNA的下调是EMT过程中的一个必要成分,已经证实TGFβ敏感的miR-200和ZEB之间通过双向负反馈环(double-negative feedback loop)影响EMT。EMT在肺癌中影响miRNA的表达,并且通过EMT调节肺癌细胞的侵袭和转移。因而阐明调控肿瘤细胞发生EMT过程的分子机制,明确其在恶性肿瘤的发生、进展的作用,并探索基于EMT的诊断方法具有重要的临床意义。
NF-κB是在调节细胞凋亡和肿瘤发生中起重要作用的转录因子,而且是在诱导EMT和维持EMT中是必不可少的。NF-κB信号的强度和持续时间由多个负反馈机制控制,从而使NF-κB信号在癌细胞中保持其致癌性。microRNA对NF-κB有调节作用来调控NF-κB调节的基因表达,影响这些基因的功能。在神经胶质瘤miR-182过表达,直接抑制CYLD(肿瘤抑制基因)(NF-κB的负性调节子)的表达,促进泛素结合NF-κB信号通路成分诱导胶质细胞农现侵袭性表型。并且TGF-β诱导的miR-182表达可以延长NF-KB的活化时间。miR-146a在大部分胃癌表达上调,抑制GPCR(G蛋白偶联受体)介导的NF-κB的激活,因而减少NF-κB调节的肿瘤促进激酶表达和生长因子的表达。通过作用于NF-κB活化通路的一些成分,miR-146a在调节NF-κB活性中起关键作用。miRNA与p53, NF-κ B, β-catenin通路相互作用在调节EMT和保持肿瘤干细胞中起重要作用。乳腺癌中抑制miR-448通过直接作用于特异的AT-富集序列结合蛋白-1(SATB1)的mRNA,导致双调蛋白水平升高,表皮生长因子(EGFR)受体介导的Twistl表达升高,NF-κ B激活,从而可以诱导EMT。基于这些报道,我们猜想miRNA调节NF-κ B在宫颈癌放疗耐受相关EMT中起作用。
本研究旨在阐明宫颈癌放疗耐受的机制,为设计宫颈癌的个体化治疗提供理论基础。本课题进行了三部分的研究：(1)建立宫颈癌放疗耐受细胞株；(2)宫颈癌放疗耐受细胞株EMT的发生及机制研究；(3)宫颈癌放疗耐受相关miRNA的筛选及功能分析。
方法：
根据文献报道及2010NCCN宫颈癌临床实践指南自行建立放疗耐受(FIR)的细胞模型进行实验研究。采用低剂量持续分级放疗的方法,给予2Gy/day的剂量照射宫颈癌细胞株SiHa, C-33A,5days/week,至总剂量达到75Gy,细胞休息2周后实验。进行克隆形成实验、细胞周期及凋亡的检测验证FIR细胞是否对放疗耐受。
在建立FIR细胞进行放疗的过程中,进行形态学观察,发现与亲本细胞相比,FIR细胞株发生了显著的形态学改变,此改变符合EMT。为了证实FIR细胞株是否真正发生了EMT,我们进行western blot实验检测了EMT的标志蛋白E-cadherin和N-cadherin的变化。确定FIR细胞株发生EMT后,进行定量PCR实验在mRNA水平,western blot实验在蛋白水平检测FIR细胞与亲本细胞(N细胞)其他EMT marker的变化,并进行分子机制研究。使用siRNA干扰FIR细胞中NF-κ B p65表达后,检测EMT marker表达,研究NF-κ B p65是否参与调控低剂量放疗诱导的宫颈癌EMT。
miRNA表达谱芯片是研究miRNA的有力工具,通过对宫颈癌放疗耐受细胞株与亲本细胞的miRNA表达谱的分析,筛选出放疗耐受相关的miRNA,为今后开发逆转宫颈癌患者放疗耐受的新治疗手段提供新的靶标。我们使用microRNA芯片检测宫颈癌N细胞及FIR细胞,筛选与放疗耐受相关的microRNA。合成miRNA的引物,定量PCR验证芯片数据结果,并对显著差异的miRNA进行功能研究,筛选放疗耐受相关miRNA。合成FIR细胞表达降低的miRNA的模拟物mimic miRNA,导入FIR细胞后,定量PCR和western blot检测EMT marker表达变化。同时western blot检测宫颈癌FIR细胞表达NF-κB p65的变化；siRNA干扰FIR细胞表达的NF-κB p65后定量PCR实验检测miRNA表达的变化。
结果：
克隆形成实验显示亲木细胞和FIR细胞接受2Gy的X-ray照射后,FIR细胞克隆形成的能力比亲本细胞增强；FIR细胞倍增的时间比亲本细胞长；接受不同剂量的X-ray照射后,FIR细胞凋亡率低于亲本细胞,说明FIR细胞耐受放疗。这些结果表明宫颈癌FIR细胞具有宫颈癌放疗耐受的特点,宫颈癌放疗耐受细胞系建立成功。
在建立宫颈癌FIR细胞,对宫颈癌N细胞进行放疗的过程中,通过形态学观察,发现与亲本细胞相比,FIR细胞株发生了显著的形态学改变,细胞由铺路石样的上皮表型变成细长的间质表型,此改变符合上皮-间质转化,同时细胞迁移和侵袭的能力增强。
为了证实FIR细胞株是否真正发生了EMT,我们进行western blot检测了EMT的标志蛋白E-cadherin和N-cadherin的变化,结果显示FIR细胞表达的E-cadherin降低,N-cadherin表达升高,从而初步证实了FIR细胞有EMT的改变。进一步进行定量PCR实验在mRNA水平,western blot实验在蛋白水平检测FIR细胞与亲本细胞其他EMT marker的变化,发现上皮性marker CK-18表达降低,间质marker vimentin表达升高。使用siRNA干扰FIR细胞中,p65农达后,E-cadherin表达升高,N-cadherin农达降低,说明NF-κB p65参与调控低剂量分级放疗诱导的宫颈癌放疗耐受相关EMT。
mieroRNA芯片检测宫颈癌亲本细胞及FIR细胞,筛选与放疗耐受相关的miRNA。结果显示,在1198个mieroRNA探针中有20个mieroRNA有差异,定量PCR验证了世片数据结果。合成FIR细胞表达降低的miRNA的模拟物mimic miRNA,导入FIR细胞后,定量PCR和western blot检测EMT marker表达变化,其中导入mimic miR-1236后FIR细胞表达的E-cadherin升高和N-cadherin表达降低,差异有显著性,说明miR-1236参与调节低剂量分级放疗引起的宫颈癌放疗耐受相关EMT。
进一步研究发现FIR细胞导入miR-1236后NF-κ B p65的表达降低,同时使用siRNA干扰NF-κ B P65后FIR细胞表达的miR-1236比对照组显著升高,说明miR-1236和NF-κ B p65相互调节在低剂量分级放疗诱导的宫颈癌EMT导致的放疗耐受中起调控作用。
结论：
1.低剂量分级持续放疗可以诱导宫颈癌细胞发生上皮-间质转化而发生放疗耐受。
2. NF-κ B p65参与调控宫颈癌放疗耐受相关的EMT。
3. miR-1236参与调控宫颈癌放疗耐受相关的EMT。
4. miR-1236和NF-κ B p65相互调节在低剂量分级放疗诱导的宫颈癌EMT导致的放疗耐受中起调控作用。意义：
1.阐明宫颈癌放疗耐受的机制将有望为设计宫颈癌的个体化治疗提供理论基础。
2. NF-kB p65及筛选出来的的microRNA也将为开发逆转宫颈癌放疗耐受的治疗手段提供新的靶标。
3.本研究建立的宫颈癌放疗耐受细胞株可以用于放疗耐受相关的其他分子生物学研究。
BACKGROUND AND OBJECT
Cervical cancer is one of the most common genital cancers in women worldwide. It is the second most prevalent and the fifth most deadly malignancy. There are approximately500,000new cervical cancer cases in the world each year, and more than60%of these cases die annually. And incidence of cervical cancer in our country ranks second in the world. Currently, many therapy methods are available for cervical cancer, such as surgery, radiotherapy and chemotherapy. According to the NCCN Clinical Practice Guidelines in Oncology—Gervical Cancer Guideline2010, radiotherapy is one of the important means for the treatment of cervical cancer. Radiotherapy is effective for almost all the stages of cervical cancer, particularly for patients with advanced cervical cancers or who can not be cured surgically. However the response to radiotherapy varies among patients because of the heterogeneity of individual conditions and tumors. Radiation resistance has become one of the most reasons of failure of cure and recurrence and metastasis. Therefore to clarify the mechanisms of radiation resistance may provide theoretical basis for individual treatment and also provide new therapeutic targets to reverse the radiation resistance of cervical cancer.
The response of tumor cells to radiotherapy was affected by many factors in tumor microenvironment. Radiation ionizing activates genes which are related to cell apoptosis, DNA damage and repair, cell adhesion and angiogenesis. In return these genes regulate cell response to radiation. Epithelial-mesenchymal transition (EMT) is a process that epithelial cells lose their cell-cell adhesion and obtain mesenchymal features. Including redistribution or lost of specific epithelial connecting protein, E-cadherin, and mesenchymal markers, including N-cadherin and vimentin, were activated. Morphology of cell changes to cambiform phenotype and the ability of migration is enhanced. EMT is a biological process that epithelial cells transited to cells with mesenchymal characters in some physiological and pathological conditions. And it also occurs in the development of tumors. In recent years, the functions of EMT have been paid more attention. It has been reported that EMT played important role in development and progression, invasion and migration of tumor. And many factors regulated EMT.
MicroRNAs (miRNAs) are small endogenous single-stranded,22-24nucleotide non-coding RNAs. Maps of miRNAs are different between tumors and normal tissues. Therefore these maps can be used as new methods of disease classification. MiRNAs influence differentiation, proliferation, metabolism, apoptosis of cells and development of diseases by regulating key target mRNAs. And in regulation of EMT, miRNAs also play important role. As a result it is very important to clarify the molecular mechanisms of occurrence of EMT and the functions of EMT in development of tumors. And it is also very meaningful to explore the diagnostic methods which are based on EMT.
NF-κB is a key transcription factor in regulation of cell apoptosis and tumor progression. It is essential in induction and maintain of EMT. MiRNAs can regulate NF-κB and consequently the expression of its downstream genes were affected. The signal strength and lasting time of NF-κB are controlled by multiple negative feedback mechanisms. Thus NF-κB can keep its functions of oncogene in tumor cells.
This study aimed to clarify the mechanisms of radiation resistance of cervical cancer and provide theoretic basis for individual treatment of cervical cancer. In this project three parts were studied. Part1was to establish radiation resistance cervical cancer cells. Part2was to study the molecular mechanisms of low-dose radiation induced EMT. And Part3was the screening of miRNAs which are resistant to radiation of cervical cancer and analysis of their functions.
METHODS
Fractionated X-ray irradiation was utilized to generate radioresistant (FIR) cell model according to methods reported and NCCN Clinical Practice Guidelines in Oncology—Cervical Cancer Guideline2010. Cervical cancer cells, SiHa and C-33A, were irradiated with2GY of low-dose fractionated X-ray irradiation per day on five consecutive days per week, until the total concentration reached75GY. There was a two-week interval between the last fractionated irradiation and the experiments. Clonogenic assay, cell cycle and apoptotic assays were conducted to test the radiresistance of cells.
During this process, morphological changes of FIR cells, which corresponded with EMT, were observed comparing of parent cells. To confirm the existence of EMT, we detected the expression of E-cadherin and N-cadherin, the marker proteins of EMT. After that, other EMT markers and detailed molecular signaling pathways were detected by real-time PCR on mRNA level and by western blot on protein level. SiRNA was used to interfere the expression of NF-κB p65to study the role of NF-κB p65in low-dose radiation induced EMT involved in cervical cancer radioresistance.
Recently,microRNAs (miRNAs) were reported to play important role in regulation of EMT, cell proliferation, cell differentiation, apoptosis and the development of diseases. For this reason, we used miRNA microarray analysis to detect the FIR cells and parent cells to find the miRNAs which were related to radioresistance. The primers of relative miRNAs were synthetized and real-time PCR was conducted to further confirm the results of microarray and the functions of miRNAs which work in radioresistance of cervical cancers were studied. Mimics of miRNAs which were down-regulated in FIR cells were synthetized and transfected to detect the change of EMT markers. At the same time, the expressions of NF-κB p65 were detected by western blot. SiRNA was used to silence the expression of NF-κB p65to make sure the expression change of miRNAs by real-time PCR.
RESULTS
Clonogenic assays revealed that the ability of FIR cells to form clones were enhanced comparing with parent cells after2GY of X-ray irradiation. When accepting different dose of X-ray irradiation, the apoptosis rate of FIR cells decreased obviously. This indicated that FIR cells were resistant to radiation and cervical cancer cell model of radioresistance were generated successfully.
During the processes of generating radioresistant cervical cancer cells, FIR cells were observed obvious morphological changes which corresponded with EMT. And the abilities of migration and invasion of FIR cells also enhanced.
To clarify the existence of EMT, we detected the expression of E-cadherin and N-cadherin which were marker proteins of EMT and found that E-cadherin had low expression and N-cadherin had high expression in FIR cells. This indicated that FIR cells developed EMT. Further studies, including real-time PCR on mRNA level and western blot on protein level, revealed that another epithelial marker, CK-18, was down-regulated and mesenchymal marker, vimentin, was up-regulated. After silencing of p65by siRNA in FIR cells, E-cadherin was up-regulated and N-cadherin was down-regulated. These results showed that NF-κB p65worked in regulation of low-dose radiation induced EMT in cervical cancer.
MiRNAs expression profile is a powerful tool to study miRNAs. After analyzing the miRNAs expression profiles of radiation resistance cervical cancer cells and parent cells, miRNAs which are related to radiation resistance can be screened out. And this will provide new therapeutic targets to reverse the radiation resistance of cervical cancer patients. The results of miRNA microassay analysis showed that20in total1198miRNAs varied in FIR cells compared with parent cells. And real-time PCR confirmed these results. Mimics of miRNAs which were down-regulated in FIR cells were synthesized and transfected into FIR cells to detect the changes of EMT markers. Results revealed that after transfection of mimic miR-1236, the expression of E-cadherin was enhanced and N-cadherin was decreased obviously in FIR cells. It indicated that miR-1236play role in low-dose radiation induced EMT involved in cervical cancer radioresistance.
Further detailed studies found that after transfection of miR-1236, the expression of NF-κB p65were up-regulated. And when using siRNA to interfere the expression of NF-κB p65, miR-1236was elevated compared with control group. This illustrated that the mutual regulation between miR-1236and NF-κB p65regulated the low-dose radiation induced EMT in cervical cancer.
CONCLUSION
1. low-dose radiation could induce EMT in cervical cancer to develop the radioresistance.
2. NF-κB p65played role in regulation of low-dose radiation induced EMT in cervical cancer radioresistance.
3. MiR-1236worked in regulation of low-dose radiation induced EMT in cervical cancer radioresistance.
4. Mutual regulation between miR-1236and NF-κB p65regulated the low-dose radiation induced EMT involved in cervical cancer radioresistance.
SIGNIFICANCE
1. Clarifying the mechanisms of radioresistance in cervical cancer could provide the theoretical basis for individual treatment of cervical cancer patients.
2. NF-kB p65and miRNAs which were found relative with cervical cancer could provide new therapeutic targets for cervical cancer to reverse the radioresistance.
3. The.radioresistance cervical cancer cells which we established in this study could be used in other molecular research related to radioresistance.
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36. Dahlberg WK, Azzam El, Yu Y, Little JB. Response of human tumor cells of varying radiosensitivity and radiocurability to fractionated irradiation. Cancer Res 1999; 59:5365-9.
37. Pearce AG, Segura TM, Rintala AC, Rintala-Maki ND, Lee H. The generation and characterization of a radiation-resistant model system to study radioresistance in human breast cancer cells. Radiat Res 2001; 156:739-50.
38. Wazer DE, Joyce M, Jung L, Band V. Alterations in growth phenotype and radiosensitivity after fractionated irradiation of breast carcinoma cells from a single patient. Int J Radiat Oncol Biol Phys 1993; 26:81-8.
39. Fukuda K, Sakakura C, Miyagawa K, Kuriu Y, Kin S, Nakase Y, et al. Differential gene expression profiles of radioresistant oesophageal cancer cell lines established by continuous fractionated irradiation. Br J Cancer 2004; 91:1543-50.
40. Ogawa K, Utsunomiya T, Mimori K, Tanaka F, Haraguchi N, Inoue H, et al. Differential gene expression profiles of radioresistant pancreatic cancer cell lines established by fractionated irradiation. Int J Oncol 2006; 28:705-13.
41. Maity A, Kao GD, Muschel RJ, McKenna WG. Potential molecular targets for manipulating the radiation response. Int J Radiat Oncol Biol Phys 1997; 37:639-53.
42. Pawlik TM, Keyomarsi K. Role of cell cycle in mediating sensitivity to radiotherapy. Int J Radiat Oncol Biol Phys 2004; 59:928-42.
43. McKenna WG, Weiss MC, Endlich B, Ling CC, Bakanauskas VJ, Kelsten ML, ct al. Synergistic effect of the v-myc oncogene with H-ras on radioresistance. Cancer Res 1990; 50:97-102.
44. Kong Z, Xie D, Boike T, Raghavan P, Burma S, Chen DJ, et al. Downregulation of human DAB2IP gene expression in prostate cancer cells results in resistance to ionizing radiation. Cancer Res; 70:2829-39.
45. Iliakis G, Metzger L, Muschel RJ, McKenna WG. Induction and repair of DNA double strand breaks in radiation-resistant cells obtained by transformation of primary rat embryo cells with the oncogencs H-ras and v-myc. Cancer Res 1990; 50:6575-9.
46. O'Connor PM. Mammalian G1 and G2 phase checkpoints. Cancer Surv 1997; 29:151-82.
47. Powell SN, DeFrank JS, Connell P, Eogan M, Preffer F, Dombkowski D, et al. Differential sensitivity of p53(-) and p53(+) cells to caffeine-induced radiosensitization and override of G2 delay. Cancer Res 1995; 55:1643-8.
48. Tsuboi K, Tsuchida Y, Endo K, Yoshii Y, Nose T. Isolation of radiosensitive and radioresistant mutants from a medulloblastoma cell line. Brain Tumor Pathol 1997; 14:19-25.
49. Russell KJ, Wiens LW, Demers GW, Galloway DA, Le T, Rice GC, et al. Preferential radiosensitization of G 1 checkpoint-deficient cells by methylxanthines. Int J Radiat Oncol Biol Phys 1996; 36:1099-106.
50. Haimovitz-Friedman A. Radiation-induced signal transduction and stress response. Radiat Res 1998; 150:S 102-8.
51. Tang JT, Yamazaki H, Inoue T, Koizumi M, Yoshida K, Ozeki S. Mitochondrial DNA influences radiation sensitivity and induction of apoptosis in human fibroblasts. Anticancer Res 1999; 19:4959-64.
52. Thrall DE. Biologic basis of radiation therapy. Vet Clin North Am Small Anim Pract 1997; 27:21-35.
53. Olsen DR. Calculation of the biological effect of fractionated radiotherapy:the importance of radiation-induced apoptosis. Br J Radiol 1995; 68:1230-6.
1. Kuo CJ, LaMontagne KR, Jr., Garcia-Cardena G, Ackley BD, Kalman D, Park S, et al. Oligomerization-dependent regulation of motility and morphogenesis by the collagen XVIII NCl/endostatin domain. J Cell Biol 2001; 152:1233-46.
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1. Sreekumar R, Sayan BS, Mirnezami AH, Sayan AE. MicroRNA Control of Invasion and Metastasis Pathways. Front Genet; 2:58.
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27. Li Y, VandenBoom TG,2nd, Kong D, Wang Z, Ali S, Philip PA, et al. Up-regulation of miR-200 and let-7 by natural agents leads to the reversal of epithelial-to-mesenchymal transition in gemcitabine-resistant pancreatic cancer cells. Cancer Res 2009; 69:6704-12.
28. Howe EN, Cochrane DR, Richer JK. The miR-200 and miR-221/222 microRNA families: opposing effects on epithelial identity. J Mammary Gland Biol Neoplasia; 17:65-77.
29. Ma Y, Li W, Wang H. Roles of miRNA in the initiation and development of colorectal carcinoma. Curr Pharm Des.
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32.汤钊猷.现代肿瘤学.复旦大学出版社,2005.
33. Reducing uncertainties about the effects of chemoradiotherapy for cervical cancer:individual patient data meta-analysis. Cochrane Database Syst Rev:CD008285.
34. Li Z, Xia L, Lee LM, Khaletskiy A, Wang J, Wong JY, et al. Effector genes altered in MCF-7 human breast cancer cells after exposure to fractionated ionizing radiation. Radiat Res 2001; 155:543-53.
35. Russell J, Wheldon TE, Stanton P. A radioresistant variant derived from a human neuroblastoma cell line is less prone to radiation-induced apoptosis. Cancer Res 1995; 55:4915-21.
36. Dahlberg WK, Azzam El, Yu Y, Little JB. Response of human tumor cells of varying radiosensitivity and radiocurability to fractionated irradiation. Cancer Res 1999; 59:5365-9.
37. Pearce AG, Segura TM, Rintala AC, Rintala-Maki ND, Lee H. The generation and characterization of a radiation-resistant model system to study radioresistance in human breast cancer cells. Radiat Res 2001; 156:739-50.
38. Wazer DE, Joyce M, Jung L, Band V. Alterations in growth phenotype and radiosensitivity after fractionated irradiation of breast carcinoma cells from a single patient. Int J Radiat Oncol Biol Phys 1993; 26:81-8.
39. Fukuda K, Sakakura C, Miyagawa K, Kuriu Y, Kin S, Nakase Y, et al. Differential gene expression profiles of radioresistant oesophageal cancer cell lines established by continuous fractionated irradiation. Br J Cancer 2004; 91:1543-50.
40. Ogawa K, Utsunomiya T, Mimori K, Tanaka F, Haraguchi N, Inoue H, et al. Differential gene expression profiles of radioresistant pancreatic cancer cell lines established by fractionated irradiation. Int J Oncol 2006; 28:705-13.
41. Maity A, Kao GD, Muschel RJ, McKenna WG. Potential molecular targets for manipulating the radiation response. Int J Radiat Oncol Biol Phys 1997; 37:639-53.
42. Pawlik TM, Keyomarsi K. Role of cell cycle in mediating sensitivity to radiotherapy. Int J Radiat Oncol Biol Phys 2004; 59:928-42.
43. McKenna WG, Weiss MC, Endlich B, Ling CC, Bakanauskas VJ, Kelsten ML, ct al. Synergistic effect of the v-myc oncogene with H-ras on radioresistance. Cancer Res 1990; 50:97-102.
44. Kong Z, Xie D, Boike T, Raghavan P, Burma S, Chen DJ, et al. Downregulation of human DAB2IP gene expression in prostate cancer cells results in resistance to ionizing radiation. Cancer Res; 70:2829-39.
45. Iliakis G, Metzger L, Muschel RJ, McKenna WG. Induction and repair of DNA double strand breaks in radiation-resistant cells obtained by transformation of primary rat embryo cells with the oncogencs H-ras and v-myc. Cancer Res 1990; 50:6575-9.
46. O'Connor PM. Mammalian G1 and G2 phase checkpoints. Cancer Surv 1997; 29:151-82.
47. Powell SN, DeFrank JS, Connell P, Eogan M, Preffer F, Dombkowski D, et al. Differential sensitivity of p53(-) and p53(+) cells to caffeine-induced radiosensitization and override of G2 delay. Cancer Res 1995; 55:1643-8.
48. Tsuboi K, Tsuchida Y, Endo K, Yoshii Y, Nose T. Isolation of radiosensitive and radioresistant mutants from a medulloblastoma cell line. Brain Tumor Pathol 1997; 14:19-25.
49. Russell KJ, Wiens LW, Demers GW, Galloway DA, Le T, Rice GC, et al. Preferential radiosensitization of G 1 checkpoint-deficient cells by methylxanthines. Int J Radiat Oncol Biol Phys 1996; 36:1099-106.
50. Haimovitz-Friedman A. Radiation-induced signal transduction and stress response. Radiat Res 1998; 150:S 102-8.
51. Tang JT, Yamazaki H, Inoue T, Koizumi M, Yoshida K, Ozeki S. Mitochondrial DNA influences radiation sensitivity and induction of apoptosis in human fibroblasts. Anticancer Res 1999; 19:4959-64.
52. Thrall DE. Biologic basis of radiation therapy. Vet Clin North Am Small Anim Pract 1997; 27:21-35.
53. Olsen DR. Calculation of the biological effect of fractionated radiotherapy:the importance of radiation-induced apoptosis. Br J Radiol 1995; 68:1230-6.
1. Kuo CJ, LaMontagne KR, Jr., Garcia-Cardena G, Ackley BD, Kalman D, Park S, et al. Oligomerization-dependent regulation of motility and morphogenesis by the collagen XVIII NCl/endostatin domain. J Cell Biol 2001; 152:1233-46.
2. Lee MY, Chou CY, Tang MJ, Shen MR. Epithelial-mesenchymal transition in cervical cancer: correlation with tumor progression, epidermal growth factor receptor overexpression, and snail up-regulation. Clin Cancer Res 2008; 14:4743-50.
3. Min C, Eddy SF, Sherr DH, Sonenshein GE. NF-kappaB and epithelial to mesenchymal transition of cancer. J Cell Biochem 2008; 104:733-44.
4. Huber MA, Azoitei N, Baumann B, Grunert S, Sommer A, Pehamberger H, et al. NF-kappaB is essential for epithelial-mesenchymal transition and metastasis in a model of breast cancer progression. J Clin Invest 2004; 114:569-81.
5. Cho KB, Clio MK, Lee WY, Kang K.W. Overexpression of c-myc induces epithelial mesenchymal transition in mammary epithelial cells. Cancer Lett; 293:230-9.
6. Li X, Kong X, Huo Q, Guo H, Yan S, Yuan C, et al. Metadherin enhances the invasiveness of breast cancer cells by inducing epithelial to mesenchymal transition. Cancer Sci; 102:1151-7.
7. Kitahara O, Katagiri T, Tsunoda T, Harima Y, Nakamura Y. Classification of sensitivity or resistance of cervical cancers to ionizing radiation according to expression profiles of 62 genes selected by cDNA microarray analysis. Neoplasia 2002; 4:295-303.
8. Thiery JP. Epithelial-mesenchymal transitions in tumour progression. Nat Rev Cancer 2002; 2:442-54.
9. Schmalhofer O, Brabletz S. Brabletz T. E-cadherin, beta-catenin, and ZEBl in malignant progression of cancer. Cancer Metastasis Rev 2009; 28:151-66.
10. Lee MY, Shen MR. Epithelial-mesenchymal transition in cervical carcinoma. Am J Transl Res; 4:1-13.
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