本文選題：URI　切入點：宮頸癌細胞　出處：《江蘇大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：研究背景宮頸癌是最常見的女性生殖系統(tǒng)惡性腫瘤,是威脅廣大發(fā)展中國家女性生命健康的重要疾病。宮頸癌一旦發(fā)生遠端轉(zhuǎn)移,將嚴重影響患者的生存率和治愈效果。因此,宮頸癌轉(zhuǎn)移機制的研究被持續(xù)重點關(guān)注。RNA聚合酶2第五亞基(RPB5)作用蛋白RMP,即非經(jīng)典前折疊素RPB5作用因子URI具有癌蛋白的特性,本課題組的前期研究顯示在體外,URI能夠促進宮頸癌細胞的遷移,但具體機制不明,推測可能與細胞骨架波形蛋白(Vimentin)有關(guān)。在前期研究工作的基礎(chǔ)上,本研究以宮頸癌細胞株He La和C33A為研究對象,通過干預(yù)URI和Vimentin的表達,進一步探討URI對宮頸癌細胞遷移和侵襲的作用及機制,并探討URI與Vimentin之間的關(guān)系。研究目的本研究主要有三個目的:(1)探究URI對兩株宮頸癌細胞He La和C33A遷移和侵襲的生物學(xué)行為的作用。(2)揭示URI對宮頸癌細胞運動能力的影響是否與Vimentin有關(guān)。(3)探索URI對Vimentin調(diào)控的潛在機制。研究方法(1)采用q RT-PCR和western blot檢測兩株宮頸癌細胞中URI的基礎(chǔ)表達水平,然后在He La和C33A細胞中,運用化學(xué)轉(zhuǎn)染試劑分別轉(zhuǎn)染URI特異性si RNA、URI表達質(zhì)粒PCMV6-URI以構(gòu)建URI干擾和過表達細胞株。(2)運用Transwell小室實驗檢測URI敲低或過表達后細胞的遷移和侵襲能力。(3)在He La和C33A細胞中,分別敲低或過表達URI后運用q RT-PCR和western blot檢測分析Vimentin的表達情況。(4)在URI干預(yù)的宮頸癌細胞中,分別用TGF-β、Vimentin特異性si RNA誘導(dǎo)或敲低Vimentin表達后觀察細胞的運動能力。(5)將Vimentin基因上游啟動子序列克隆到帶有熒光素酶報告基因的p GL3/Basic載體中,通過與URI表達質(zhì)粒共轉(zhuǎn)染293T細胞,熒光素酶報告實驗檢測URI對Vimentin啟動子活性的影響。(6)使用染色質(zhì)免疫共沉淀(Ch IP)實驗分析URI在Vimentin基因啟動子區(qū)域的可能結(jié)合情況。研究結(jié)果(1)兩株宮頸癌細胞URI基礎(chǔ)表達水平和細胞轉(zhuǎn)染研究:q RT-PCR和western blot檢測結(jié)果顯示,He La細胞URI的基礎(chǔ)表達水平比C33A細胞高。轉(zhuǎn)染URI特異性si RNA后,He La細胞URI的m RNA和蛋白表達水平明顯低于對照組,而轉(zhuǎn)染URI表達質(zhì)粒PCMV6-URI后,C33A細胞URI的m RNA和蛋白表達水平明顯高于對照組。(2)干預(yù)URI可影響細胞遷移和侵襲能力:Transwell小室遷移實驗顯示,在He La細胞中,URI干擾組比對照組細胞遷移穿過小室的數(shù)目明顯減少:在C33A細胞中,URI過表達組比對照組細胞遷移穿過小室的數(shù)目明顯增多。Transwell小室侵襲實驗顯示,同對照組相比,URI干擾后He La細胞侵襲穿過小室的數(shù)目明顯減少;而過表達URI后C33A細胞侵襲穿透小室的數(shù)目明顯增多。(3)干預(yù)URI可調(diào)控Vimentin的表達:q RT-PCR和western blot檢測表明,同對照組相比,URI干擾后,He La細胞內(nèi)Vimentin的m RNA和蛋白表達水平表達都下降;URI過表達后,C33A細胞內(nèi)Vimentin的m RNA和蛋白表達水平都增加。(4)URI對細胞遷移和侵襲能力的影響同Vimentin有關(guān):Transwell小室遷移實驗顯示,在URI干擾的He La細胞中,經(jīng)10ng/ml TGF-β處理后,細胞遷移穿過小室的數(shù)目比未處理組多;在URI過表達的C33A細胞中,經(jīng)Vimentin特異性si RNA轉(zhuǎn)染后的,細胞遷移穿過小室的數(shù)目比對照組少。Transwell小室侵襲實驗顯示,在URI干擾的He La細胞中,10ng/ml TGF-β處理組細胞侵襲穿過小室的數(shù)目明顯比未處理組多;在URI過表達的C33A細胞中,Vimentin特異性si RNA轉(zhuǎn)染組細胞侵襲穿過小室的數(shù)目明顯比對照組少。(5)URI可間接活化Vimentin啟動子活性:在293T細胞中,熒光素酶報告實驗結(jié)果顯示URI過表達組的熒光素酶活性比對照組增強了約1.57倍,但在He La和C33A兩株宮頸癌細胞中,運用染色質(zhì)免疫共沉淀實驗分析,q PCR結(jié)果顯示URI蛋白在Vimentin啟動子各區(qū)域結(jié)合情況同陰性對照組相比沒有統(tǒng)計學(xué)差異。結(jié)論(1)URI可促進宮頸癌細胞遷移和侵襲。(2)URI可增強Vimentin的m RNA和蛋白表達。(3)Vimentin有助于URI增強宮頸癌細胞的運動能力。(4)URI可增強Vimentin啟動子的活性,但尚無證據(jù)表明其可以直接結(jié)合到Vimentin啟動子上。
[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.

【學(xué)位授予單位】：江蘇大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R737.33

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本文編號：1623440