URI通过调控Vimentin表达增强宫颈癌细胞迁移与侵袭能力的研究

发布时间：2018-03-17 06:00

本文选题：URI　切入点：宫颈癌细胞　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：研究背景宫颈癌是最常见的女性生殖系统恶性肿瘤,是威胁广大发展中国家女性生命健康的重要疾病。宫颈癌一旦发生远端转移,将严重影响患者的生存率和治愈效果。因此,宫颈癌转移机制的研究被持续重点关注。RNA聚合酶2第五亚基(RPB5)作用蛋白RMP,即非经典前折叠素RPB5作用因子URI具有癌蛋白的特性,本课题组的前期研究显示在体外,URI能够促进宫颈癌细胞的迁移,但具体机制不明,推测可能与细胞骨架波形蛋白(Vimentin)有关。在前期研究工作的基础上,本研究以宫颈癌细胞株He La和C33A为研究对象,通过干预URI和Vimentin的表达,进一步探讨URI对宫颈癌细胞迁移和侵袭的作用及机制,并探讨URI与Vimentin之间的关系。研究目的本研究主要有三个目的:(1)探究URI对两株宫颈癌细胞He La和C33A迁移和侵袭的生物学行为的作用。(2)揭示URI对宫颈癌细胞运动能力的影响是否与Vimentin有关。(3)探索URI对Vimentin调控的潜在机制。研究方法(1)采用q RT-PCR和western blot检测两株宫颈癌细胞中URI的基础表达水平,然后在He La和C33A细胞中,运用化学转染试剂分别转染URI特异性si RNA、URI表达质粒PCMV6-URI以构建URI干扰和过表达细胞株。(2)运用Transwell小室实验检测URI敲低或过表达后细胞的迁移和侵袭能力。(3)在He La和C33A细胞中,分别敲低或过表达URI后运用q RT-PCR和western blot检测分析Vimentin的表达情况。(4)在URI干预的宫颈癌细胞中,分别用TGF-β、Vimentin特异性si RNA诱导或敲低Vimentin表达后观察细胞的运动能力。(5)将Vimentin基因上游启动子序列克隆到带有荧光素酶报告基因的p GL3/Basic载体中,通过与URI表达质粒共转染293T细胞,荧光素酶报告实验检测URI对Vimentin启动子活性的影响。(6)使用染色质免疫共沉淀(Ch IP)实验分析URI在Vimentin基因启动子区域的可能结合情况。研究结果(1)两株宫颈癌细胞URI基础表达水平和细胞转染研究:q RT-PCR和western blot检测结果显示,He La细胞URI的基础表达水平比C33A细胞高。转染URI特异性si RNA后,He La细胞URI的m RNA和蛋白表达水平明显低于对照组,而转染URI表达质粒PCMV6-URI后,C33A细胞URI的m RNA和蛋白表达水平明显高于对照组。(2)干预URI可影响细胞迁移和侵袭能力:Transwell小室迁移实验显示,在He La细胞中,URI干扰组比对照组细胞迁移穿过小室的数目明显减少:在C33A细胞中,URI过表达组比对照组细胞迁移穿过小室的数目明显增多。Transwell小室侵袭实验显示,同对照组相比,URI干扰后He La细胞侵袭穿过小室的数目明显减少;而过表达URI后C33A细胞侵袭穿透小室的数目明显增多。(3)干预URI可调控Vimentin的表达:q RT-PCR和western blot检测表明,同对照组相比,URI干扰后,He La细胞内Vimentin的m RNA和蛋白表达水平表达都下降;URI过表达后,C33A细胞内Vimentin的m RNA和蛋白表达水平都增加。(4)URI对细胞迁移和侵袭能力的影响同Vimentin有关:Transwell小室迁移实验显示,在URI干扰的He La细胞中,经10ng/ml TGF-β处理后,细胞迁移穿过小室的数目比未处理组多;在URI过表达的C33A细胞中,经Vimentin特异性si RNA转染后的,细胞迁移穿过小室的数目比对照组少。Transwell小室侵袭实验显示,在URI干扰的He La细胞中,10ng/ml TGF-β处理组细胞侵袭穿过小室的数目明显比未处理组多;在URI过表达的C33A细胞中,Vimentin特异性si RNA转染组细胞侵袭穿过小室的数目明显比对照组少。(5)URI可间接活化Vimentin启动子活性:在293T细胞中,荧光素酶报告实验结果显示URI过表达组的荧光素酶活性比对照组增强了约1.57倍,但在He La和C33A两株宫颈癌细胞中,运用染色质免疫共沉淀实验分析,q PCR结果显示URI蛋白在Vimentin启动子各区域结合情况同阴性对照组相比没有统计学差异。结论(1)URI可促进宫颈癌细胞迁移和侵袭。(2)URI可增强Vimentin的m RNA和蛋白表达。(3)Vimentin有助于URI增强宫颈癌细胞的运动能力。(4)URI可增强Vimentin启动子的活性,但尚无证据表明其可以直接结合到Vimentin启动子上。
[Abstract]:Backgroundcervical cancer is the most common malignant tumors of the female reproductive system, is an important disease threat to the life and health of the women in developing countries. Once the occurrence of distant metastasis of cervical cancer, will seriously affect the survival rate of the patients and the cure effect. Therefore, research the mechanism of cervical cancer metastasis by continued focus on 2.RNA polymerase subunit fifth (RPB5 the role of protein RMP, namely non) characteristics of the classical RPB5 before folding factor URI protein with cancer, ourprevious studies showed that in vitro, URI can promote the migration of cervical cancer cells, but the mechanism is not clear, presumably with vimentin (Vimentin). On the basis of previous study in this study, La and C33A in cervical cancer cell line He as the research object, through the intervention of URI and Vimentin, and further explore the mechanism of URI on migration and invasion of cervical cancer cells, And to investigate the relationship between URI and Vimentin. The purpose of this study has three main purposes: (1) to explore the biological behavior of URI in two cervical carcinoma cell lines He La and C33A migration and invasion effect. (2) reveal the effect of URI on exercise ability of cervical cancer cells and whether Vimentin (3. URI on the regulation of Vimentin) to explore the potential mechanism. Methods (1) by Q RT-PCR and Western blot based detection of two cervical carcinoma cell lines in the expression of URI in He, then La and C33A cells, the use of chemical transfection reagents were transfected with URI specific Si RNA URI expression plasmid PCMV6-URI to construct the URI interference and over expression cell lines. (2) using Transwell assay URI knockdown or overexpression of migration and invasion of cells. (3) He in La and C33A cells, respectively, knockdown or overexpression of URI after RT-PCR and Western using Q blot analysis Vimentin . (4) in the intervention of URI cervical cancer cells, respectively TGF- beta, observe cell motility Vimentin specific Si induced by RNA or knockdown of Vimentin. (5) the upstream promoter sequence of Vimentin gene was cloned into P GL3/Basic vector with luciferase reporter gene, the plasmids were transfected into 293T cells and expression of URI and luciferase reporter assay of URI effect on Vimentin promoter activity. (6) using chromatin immunoprecipitation (Ch IP) experimental analysis of URI promoter region in Vimentin gene may be binding. Results (1) the expression level of research and transfection of two cervical carcinoma cell lines: URI based Q RT-PCR and Western blot showed that the expression level of He La URI cells than C33A cells. Transfection of URI specific Si after RNA, the expression level of M RNA and He La protein in URI cells was significantly lower than the control group, while the transfection of URI The expression plasmid PCMV6-URI, the expression level of M RNA and C33A protein in URI cells was significantly higher than the control group. (2) the intervention of URI can affect the cell migration and invasion: Transwell cell migration assay showed that He in La cells, URI interference group was significantly less than the number of control cells migrate through the cell in C33A cells URI, over expression group than in control group the number of cell migration through the cell significantly increased.Transwell cell invasion assay showed that compared with the control group, the number of URI after He cell invasion through La interference cell significantly decreased; while overexpression of URI after the invasion of C33A cells increased significantly. The number of penetrating chamber (3) intervention on expression of URI regulation Vimentin: Q RT-PCR and Western blot detection showed that, compared with the control group, after URI interference, the expression of M protein and RNA He in La cells Vimentin expression level decreased; overexpression of URI in C33A cells, Vimentin M The expression of RNA and protein increased. (4) the effect of URI on the invasion and migration of cells with Vimentin: Transwell cell migration assay showed that URI interference in He La cells, the 10ng/ml TGF- beta after treatment, cell migration through the cell number than the untreated group; in the URI over expression of C33A in the cell, the Vimentin specific Si RNA after transfection, the number of cell migration through the chamber less than the control group.Transwell chamber invasion assay showed that URI interference in He La cells, the number of 10ng/ml TGF- beta cell invasion through the cell treatment group were significantly higher than the untreated group; in the over expression of URI in C33A cells the number of Vimentin, specific Si RNA cell invasion through the cell transfection group was significantly less than the control group. (5) URI can indirectly activate Vimentin promoter activity in 293T cells, luciferase reporter experiments showed that fluorescence URI overexpression group Luciferase activity than the control group increased about 1.57 times, but in the He La and C33A two cervical carcinoma cell lines, using chromatin immunoprecipitation experiment analysis of co precipitation, Q PCR showed that URI protein in Vimentin promoter regions combined with the negative control group had no significant difference compared. Conclusion (1) URI can promote the migration and invasion of cervical cancer cells. (2) URI can enhance the expression of M protein and RNA Vimentin. (3) Vimentin URI helps enhance athletic ability of cervical cancer cells. (4) URI could enhance Vimentin promoter activity, but there is no evidence that it can be directly binding to the Vimentin promoter.

【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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