【作者】 刘晓军；

【导师】 陈东；

【作者基本信息】 吉林大学 ， 人体解剖与组织胚胎学， 2016， 博士

【摘要】 宫颈癌(Cervical cancer,CC)是女性常见恶性肿瘤,发病率在全世界女性肿瘤中位居第二位,仅次于乳腺癌。全世界每年新增病例约50万,死亡病例约30万,而我国CC发病率位居世界第二位,且发病年龄日渐年轻化,严重威胁全球女性生命健康。尽管目前对CC的诊治取得了较大进步,但CC患者的预后和死亡率并没有得到明显的改善。早期的CC患者通过手术治疗可以达到较好的预后,而晚期CC患者5年的生存率只有50%左右,治疗效果不佳。其它治疗手段包括化疗和放疗,但均缺乏特异性,同时具有严重的毒副作用。因此深入探讨CC发生发展的病理机制,寻求更好、更有效的治疗方案对CC的临床防治具有重大意义。e IF5A2基因是e IF5A家族的同源基因之一,定位在染色体3q26.2区,在细胞生长、分化、增殖、凋亡等过程中扮演重要角色。既往研究表明,在卵巢癌、肝癌、膀胱癌及食管鳞状细胞癌等多种实体瘤组织中存在e IF5A2异常表达,并作为肿瘤恶性程度高的标志物之一。而e IF5A2沉默能够有效抑制肿瘤细胞恶性增殖、促进细胞凋亡,降低其侵袭转移能力。近年研究发现,e IF5A2在宫颈癌组织中的表达显著升高,且与FIGO分期、淋巴血管侵犯、盆腔淋巴结转移等临床病理参数高度相关,但其在宫颈癌中的具体分子调控机制尚不明确,因此e IF5A2对宫颈癌细胞生物学功能的影响仍需进一步探索。越来越多的研究发现,Rho A/ROCK信号通路在多种肿瘤中持续性激活,且与肿瘤细胞生长、凋亡、侵袭迁移及恶性进展密切相关。另有研究证实,e IF5A2基因通过调控Rho A/ROCK信号通路相关蛋白表达介导胰腺癌侵袭转移。此外,e IF5A2过表达促进肝癌细胞增殖和迁移的同时显著激活Rho A/Rac1信号通路。表明e IF5A2可以通过调控Rho A/ROCK信号通路活性介导肿瘤细胞的增殖和迁移。但e IF5A2在宫颈癌中是否通过Rho A/ROCK信号通路影响肿瘤细胞的生物学活性尚不清楚。本研究采用real-time PCR和western blot分析了e IF5A2表达与CC的相关性;并应用RNA干扰技术降低e IF5A2在宫颈癌Hela细胞中的表达,结合MTT、流式细胞术、克隆形成、划痕实验及Transwell等实验观察e IF5A2基因干扰对CC细胞增殖周期和迁移的影响;为探讨相关的分子机制,本研究检测了Rho A及ROCK在CC组织中的表达,并通过构建Rho A过表达载体及干扰片段研究Rho A异常表达对CC细胞增殖和迁移的影响;最后在e IF5A2基因干扰基础上转染Rho A过表达载体,深入探讨e IF5A2基因对Rho A/ROCK信号通路的调控作用,明确e IF5A2基因在CC发病、进展中的作用和具体分子机制。本研究结果旨在进一步阐明CC的发病机理,并为宫颈癌临床防治提供新型分子靶点和更多理论依据。1.e IF5A2在宫颈癌组织中的表达分析目的:分析e IF5A2在CC组织和癌旁正常组织中是否存在差异表达,论证e IF5A2基因同CC发病存在相关性。方法:采集15对CC和对应的癌旁正常组织样本,real-time PCR技术检测各样本中e IF5A2 m RNA表达,western blot技术检测各样本中e IF5A2蛋白表达,分别从m RNA和蛋白水平分析e IF5A2与CC发病的关系。结果:同癌旁正常组织样本比较,e IF5A2 m RNA和蛋白在CC组织样本中的表达均显著升高,且差异具有统计学意义(p<0.05)。结论:成功证实e IF5A2在CC组织中异常高表达,进一步明确了e IF5A2可能成为人类肿瘤病理诊断的重要分子标志和临床治疗的潜在分子靶点。并为应用Real-time PCR或免疫组化技术分析e IF5A2在宫颈活检组织中的表达进行CC病理诊断奠定了一定的理论基础。2.e IF5A2基因干扰对宫颈癌细胞增殖、迁移的影响目的:明确e IF5A2基因干扰对Hela细胞增殖和迁移的影响。方法:通过构建e IF5A2干扰载体,转染Hela细胞并采用G418筛选e IF5A2稳定干扰细胞。实验设计:空白对照组(Control)、阴性对照组(NC)和e IF5A2-sh RNA干扰组(she IF5A2)。通过MTT实验检测e IF5A2干扰对Hela细胞增殖的影响;PI单染流式细胞术分析各组细胞周期变化;克隆形成实验观察各组细胞增殖能力;划痕实验及Transwell实验检测各组细胞迁移能力。结果:e IF5A2基因干扰后,MTT检测结果显示细胞增殖能力显著下降(p<0.05);流式细胞术检测细胞周期结果显示G0期细胞比例增加,S期和G2期细胞比例明显减少(p<0.05或p<0.01);细胞克隆形成实验结果显示细胞克隆形成率显著降低(p<0.05);划痕实验及Transwell实验结果显示细胞迁移能力显著减弱(p<0.05)。结论:e IF5A2基因干扰能将Hela细胞阻滞于G0期,有效抑制细胞增殖和细胞迁移。3.e IF5A2基因影响宫颈癌细胞增殖、迁移的分子机制目的:(1)分析Rho A表达与CC临床病理参数的相关性,明确Rho A过表达或干扰对CC细胞增殖和迁移的影响。(2)明确e IF5A2基因干扰对CC细胞中Rho A/ROCK信号通路活性的影响以及e IF5A2基因是否通过调控Rho A/ROCK通路介导Hela细胞增殖和迁移。方法:(1)采集46例CC和34例宫颈炎组织样本,Western blot技术检测Rho A及ROCK在各组织样本中的表达,分析Rho A同患者年龄、肿瘤体积、HPV感染、FIGO分期、血管浸润、淋巴结转移、雌激素受体及孕激素受体的相关性;构建Rho A过表达及干扰片段,分别转染Hela细胞。实验设计:A空白对照组(Hela)、B空载对照组(Hela-Con)、C Rho A过表达组(Hela-Rho A+)、D阴性对照组(si RNA-Con)、E Rho A干扰组(si RNA-Rho A)。通过CCK-8实验检测Rho A异常表达对CC细胞增殖的影响,划痕实验检测细胞迁移能力变化。(2)western blot检测Control组、NC组和she IF5A2组中Rho A、ROCKⅠ和ROCKⅡ的表达,验证e IF5A2基因干扰对Rho A/ROCK通路具有调控作用后,构建Rho A过表达载体并转染e IF5A2基因稳定干扰的Hela细胞,实验设计:NC组、she IF5A2组、she IF5A2+空载转染组(Vector)和she IF5A2+Rho A过表达组(Rho A)。通过MTT实验检测Rho A过表达对e IF5A2干扰细胞增殖能力的影响,划痕实验分析各组细胞迁移能力变化。结果:(1)与对照组比较,Rho A、ROCKⅠ和ROCKⅡ在CC组织中的表达显著增加(p<0.05或p<0.01),且同FIGO、血管浸润及淋巴结转移高度相关;Rho A过表达后,CCK-8实验结果显示细胞增殖能力显著增加(p<0.05或p<0.01),划痕实验结果显示Hela细胞迁移能力显著增强(p<0.01),而干扰Rho A能有效抑制细胞增殖和迁移(p<0.05)。(2)western blot结果显示e IF5A2基因干扰后Rho A、ROCKⅠ、ROCKⅡ的表达显著降低(p<0.05);在e IF5A2基因干扰基础上转染Rho A过表达载体后,同she IF5A2组比较,MTT和划痕实验检测结果显示Rho A过表达部分逆转了e IF5A2干扰对Hela细胞的调控作用,Hela细胞的增殖和迁移能力均显著上升,且差异均具有统计学意义(p<0.05)。结论:(1)Rho A在宫颈癌组织中异常高表达,且同肿瘤临床分期和侵袭转移密切相关,Rho A过表达显著促进宫颈癌细胞增殖和迁移。(2)e IF5A2基因通过激活Rho A/ROCK信号通路,增加Rho A、ROCKⅠ、ROCKII的表达诱导Hela细胞增殖,促进细胞迁移。更多还原

【Abstract】 Cervical carcinoma( CC) is the common tumor occurring in women worldwide,and ranking the second in mortality rate of various malignancies for women, just after breast cancer. There are approximately 500,000 new cervical cancer cases in the world each year, and more than 300,000 of these cases die annually. The Chinese incidence of cervical cancer ranks second in the world and the age tends to be younger, which seriously damages female health. At present, although the diagnosis and treatment for cervical cancer has made great progress, its prognosis and mortality have not been significantly improved. The early-stage patients treated by surgery could achieve good prognosis, however those patients with advanced cervical cancer 5 years of survival rate is only 50 %, the treatment is however not acceptable reasonably. Other treatments including chemotherapy and radiotherapy, have serious side effects as well as lacking specificity. Therefore it contains a great significance for cervical cancer to probe its origin and development pathological mechanism and seek its better and more effective treatments.EIF5A2 gene is one of the homologous genes e IF5 A family and locates in human chromosomes 3q26.2, which plays an importantly regulatory role in the process of cell growth, differentiation, proliferation and apoptosis. Abnormal expression of e IF5A2 was found in ovarian cancer, liver cancer, bladder cancer and esophageal squamous cell carcinoma, which was considered as a tumor marker with high malignant degree. Silencing e IF5A2 could effectively inhibit proliferation, invasion, metastasis and induce apoptosis of tumor cells. Recent studies found that expression of e IF5A2 in cervical cancer tissue was significantly increased, and was correlated with higher FIGO stage, lymphovascular space involvement and pelvic lymph node metastasis. However, the regulation mechanism of e IF5A2 in cervical cancer and the VI effects on biological function of cervical cancer cell are still not identified clearly, which needs further research.More and more studies during recent years discovered that Rho A/ROCK signal pathway was continuously activated in a wide variety of tumor and closely related to proliferation, apoptosis, invasion and metastasis of tumor cell. Another study confirms that the e IF5A2 gene mediated invasion and metastasis of pancreatic cancer through regulating the signaling pathway of the Rho A/ROCK. In addition, overexpression of e IF5A2 promoted the proliferation and migration of hepatocellular carcinoma, meanwhile significantly activating Rho A/Rac1 signaling pathway. These studies indicated that e IF5A2 gene regulated tumor cell proliferation and migration through Rho A/ROCK signaling pathway, but how its mechanism works is still not identified clearly in cervical cancer.Our study firstly, we analyzed the correlation between e IF5A2 expression and cervical cancer by real-time PCR and western blot, and applicated RNA interference technology to reduce e IF5A2 expression in Hela and observed the effect of e IF5A2 interference on proliferation, cycle and migration of Hela cell by MTT, flow cytometry and wound healing assay. Finally,we tested the expression of Rho A and ROCK in cervical cancer tissue, and study goes further to the effect of abnormal expression of Rho A on the proliferation and migration of cervical cancer cells through by upregulating or downregulating the Rho A expression,we transfected Rho A expression vector into Hela cell with e IF5A2-sh RNA to explore the effect of e IF5A2 gene on the regulation of Rho A/ROCK signaling pathway and clarify its mechanism and effect in the progress of cervical cancer. This study aims to further clarify the pathogenesis of cervical cancer and provide a new molecular target and more theoretical analyses for the clinical prevention. Part 1. Analysis the expression of e IF5A2 in cervical cancer tissueObjective: To analyze whether there remains a difference expression of e IF5A2 in cervical cancer and adjacent normal clinical tissue, and argument whether the expression of e IF5A2 is in correlation with cervical cancer.Method: 15 pairs cervical cancer and corresponding adjacent normal clinical tissue samples were collected and used to detect the m RNA and protein expression of e IF5A2, by real-time PCR and western blot. The relationship between e IF5A2 and cervical cancer was analyzed from the level of molecules and protein respectively.Result: The expression of e IF5A2 were significantly higher in cervical cancer tissues in comparison with adjacent normal clinical tissues(p<0.05).Conclusion: We successfully confirmed that the expression of e IF5A2 was increased in cervical cancer tissue, and confirmed the potential of e IF5A2 as an important bio-marker for the prognosis and a potential molecular target for clinical treatment. Meanwhile, in cervical cancer pathological diagnosis, we successfully provided the theoretical analysis of the real-time PCR or immunohistochemical technology for the expression of e IF5A2 in cervical biopsy samples. Part 2 The effect of silencing e IF5A2 gene on proliferation and migration of cervical cancer cellsObjective: To explore the effect of silencing e IF5A2 gene on proliferation and migration of cervical cancer cellsMethod: constructing e IF5A2 interference carrier, transfecting Hela cells and screening stability cell by G418. The experiment was divided into three groups: Control group(Cont), negative control group(NC) and e IF5A2-sh RNA group(she IF5A2). The effect of e IF5A2 gene silencing on cell proliferation was detected by MTT assay. The effect of e IF5A2 gene silencing on cell cycle was examined by flow cytometry. The cell cloning efficiency in vitro was measured by colony formation assay. The cell migration ability was tested by transwell and wound healing assay.Result: The sh RNA-mediated e IF5A2 gene significantly affects the cell-growth inhibition in she IF5A2 group(p<0.05) in MTT. There came an accumulation in cells, in the G0 phase; and there also came a reduction, in S and G2 in she IF5A2 group, compared with NC group(p<0.05 or p<0.01). The cell cloning efficiency in vitro and cell migration ability was both significantly inhibited after e IF5A2 silencing(p<0.05).Conclusion: EIF5A2 gene silencing induces cervical cancer cell cycle arrest in G0 phase and effectively inhibits cell proliferation and migration. VIII Part 3 The molecular mechanism of e IF5A2 gene silencing on cell proliferation and migration of cervical cancerObjective: i)To analyze the correlation of Rho A with clinical and pathological parameters of cervical cancer and to confirm the effect of abnormal expression of Rho A on the proliferation and migration of cervical cancer cells. ii)To clarify the effect of e IF5A2 gene silencing on Rho A/ROCK signaling pathway in cervical cancer Hela cells, and to clarify the mechanism of Rho A/ROCK on cell proliferation and migration of Hela cells.Method: i)46 specimens of human cervical cancer and 34 cervicitis tissues(as control) were selected and used to detect the expression of Rho A and ROCK by western blot. We analyzed the association of Rho A expression with the age, tumor size, HPV infection, FIGO stage, vascular invasion, lymph node metastasis, expression of estrogen receptor and progesterone receptor. Constructing overexpression vector and interference fragment of Rho A and be transfected into Hela respectively. The experiment was divided into five groups: Hela group, Hela-Con group, Hela-Rho A+ group, si RNA-Con group and si RNA-Rho A group. The effect of abnormal expression of Rho A on cell proliferation was detected by CCK-8 assay, and the cell migration ability was tested by wound healing assay. ii)The effect of e IF5A2 gene silencing on Rho A/ROCK signaling pathway was testified by western blot. Building Rho A overexpression vector which was transfected into she IF5A2 Hela cells. The experiment was divided into four groups: NC group, she IF5A2 group, she IF5A2+Vector group(Vector) and she IF5A2+Rho A group(Rho A).The proliferation of Hela cells was detected by MTT assay. The migration of Hela cells was examined by wound healing assay.Result: i)The expression of Rho A, ROCKⅠand ROCK Ⅱwas significantly higher in cervical cancer tissues in comparison with control group(p<0.05 or p<0.01). And the Rho A was associated with FIGO stage, vascular invasion and lymph node metastasis, whose overexpression resulted in significant cell growth and migration from the results of cell CKK-8 and wound healing assay(p<0.05 or p<0.01). However, interfering Rho A could effectively inhibit proliferation and migration of tumor cells. ii)The protein expression of Rho A, ROCK and ROCK Ⅰ Ⅱ were decreased in Hela cells with e IF5A2 gene silencing, which was reversed after it transfected with Rho A overexpression vector. The ability to proliferate and migrate the Hela cells was significantly improved in Rho A group compared with the she IF5A2 group(p<0.05).Conclusion: i)The expression of Rho A was obviously increased in cervical cancer specimens and was closely related with clinical stage and metastasis of tumor, whose overexpression significantly promoted the proliferation and migration of cervical cancer cells. ii)The e IF5A2 gene mediated proliferation and migration of cervical cancer cells through activating Rho A/ROCK signaling pathway and so as to increase the expression of Rho A, ROCK and ROCKⅠ Ⅱ.更多还原