本文選題：長鏈非編碼RNA + H19�。� 參考：《重慶醫(yī)科大學》2017年碩士論文

【摘要】：目的探索lnc RNA H19通過miR-29作為ce RNA調(diào)控miR-29靶基因表達促進對膀胱癌EMT和侵襲轉(zhuǎn)移的作用,獲得lnc RNA H19作為miR-29的ce RNA調(diào)控靶基因及相關(guān)信號傳導通路的可靠證據(jù),旨在闡明lnc RNA H19的功能,揭示膀胱癌發(fā)生EMT及侵襲轉(zhuǎn)移新的分子機制,為尋找新的膀胱癌診斷和治療的靶基因和藥物提供原創(chuàng)性的科學依據(jù)。方法通過芯片篩選出差異表達的lnc RNAs,并確定H19為研究對象,利用生物信息學繪制ce RNA網(wǎng)絡(luò),并通過組織qRT-PCR實驗驗證H19與DNMT3B在人膀胱癌與正常膀胱中的的表達量。接著在體外水平驗證H19對細胞增殖、遷移、侵襲、骨架以及EMT的影響,在體內(nèi)驗證H19對膀胱癌的增殖、侵襲、血管生成及EMT的影響。接著通過生物信息學網(wǎng)站預測H19和DNMT3B與miR-29b-3p的結(jié)合位點。通過雙熒光素酶報告實驗驗證H19/DNMT3B與miR-29具有結(jié)合位點,且影響相互結(jié)合。然后在人膀胱癌和癌旁組織以及膀胱癌細胞進行H19與miR-29b的共定位,接著利用RIP、RNA pull-down實驗進一步驗證H19與miR-29b的結(jié)合關(guān)系,并利用qRT-PCR檢測H19、DNMT3B與miR-29b的相互調(diào)控關(guān)系。最后我們通過WB測定H19可以體外調(diào)節(jié)DNMT3B來影響EMT標記蛋白的水平。結(jié)果芯片結(jié)果顯示并用RT-q PCR驗證了H19與DNMT3B在人膀胱癌中均高表達,接著利用CCK8,Ed U,平板克隆實驗驗證H19可以影響膀胱癌細胞的增殖;利用劃痕及transwell實驗證明H19能夠影響膀胱癌細胞的遷移與侵襲;通過骨架實驗和穩(wěn)轉(zhuǎn)細胞的形態(tài)觀察發(fā)現(xiàn)H19能夠影響微絲重排與細胞運動;干擾H19表達起相反作用。我們利用動物實驗驗證H19促進體內(nèi)膀胱細胞的腫瘤發(fā)生,轉(zhuǎn)移和血管發(fā)生。在體內(nèi)觀察H19調(diào)節(jié)DNMT3B蛋白水平及EMT相關(guān)的蛋白質(zhì)的表達。利用生物信息學預測H19/DNMT3B與miR-29b具有結(jié)合位點,并用雙熒光素酶報告實驗驗證了兩者分別與miR-29b具有結(jié)合關(guān)系,且H19通過吸附miR-29b-3p而作為ce RNA起作用,因此在功能上消除了miR-29b對靶基因DNMT3B的內(nèi)源抑制作用。結(jié)合利用FISH實驗驗證了H19與miR-29b共定位于細胞質(zhì)及核中。我們進一步利用RIP及RNA pull down實驗驗證H19與miR-29b能夠相互結(jié)合。接著在膀胱癌細胞中通過RT-q PCR驗證三者之間的調(diào)控關(guān)系,過表達miR-29b mimics能夠降低靶基因DNMT3B的表達且H19與DNMT3B表達協(xié)同。最后通過WB實驗驗證H19通過與miR-29b相互影響而調(diào)控DNMT3B的表達進而影響EMT。結(jié)論1.H19和DNMT3B在膀胱癌組織中高表達且兩者表達存在正相關(guān)。2.在體內(nèi)外過表達H19調(diào)控膀胱癌細胞增殖,遷移,侵襲、血管形成及EMT。3.H19通過結(jié)合miR-29b-3p調(diào)節(jié)其靶基因及EMT蛋白的表達。
[Abstract]:Objective to investigate the effect of lnc RNA H19 on EMT and invasion and metastasis of bladder cancer by using miR-29 as ce RNA to regulate the expression of miR-29 target gene, and to obtain reliable evidence that lnc RNA H19 is the ce RNA target gene and related signal transduction pathway of miR-29. The purpose of this study is to elucidate the function of lnc RNA H19, to reveal the new molecular mechanism of EMT and invasion and metastasis of bladder cancer, and to provide scientific basis for finding new target genes and drugs for the diagnosis and treatment of bladder cancer. Methods the differentially expressed lnc RNAss were screened by microarray, and H19 was selected as the research object. Ce RNA network was drawn by bioinformatics, and the expression of H19 and DNMT3B in human bladder cancer and normal bladder was verified by tissue qRT-PCR experiment. Then the effects of H19 on cell proliferation, migration, invasion, cytoskeleton and EMT were examined in vitro. In vivo, the effects of H19 on proliferation, invasion, angiogenesis and EMT of bladder cancer were tested. Then the binding sites of H 19 and DNMT3B to miR-29b-3p were predicted by bioinformatics websites. The double luciferase report showed that H19/DNMT3B and miR-29 had binding sites and influenced their binding. Then, the co-localization of H19 and miR-29b was performed in human bladder cancer and adjacent tissues and bladder cancer cells. The binding relationship between H19 and miR-29b was further verified by rifampicin pull-down assay, and the interregulatory relationship between H19 DNMT3B and miR-29b was detected by qRT-PCR. Finally, we can regulate DNMT3B in vitro by WB determination of H 19 to influence the level of EMT labeled protein. Results the microarray results showed that both H19 and DNMT3B were overexpressed in human bladder cancer by RT-q PCR. The results of scratch and transwell showed that H19 could affect the migration and invasion of bladder cancer cells, the cytoskeleton test and morphological observation of stable cells showed that H19 could affect the microfilament rearrangement and cell movement, and interfere with the expression of H19. We used animal experiments to demonstrate that H 19 promotes tumorigenesis, metastasis and angiogenesis of bladder cells in vivo. To observe the regulation of DNMT3B protein level and EMT related protein expression by H 19 in vivo. Bioinformatics was used to predict the binding sites between H19/DNMT3B and miR-29b, and double luciferase reports were used to verify the binding relationship between miR-29b and H19, and H19 acted as ce RNA by adsorbing miR-29b-3p. Therefore, the endogenous inhibitory effect of miR-29b on target gene DNMT3B was eliminated functionally. The co-localization of H19 and miR-29b in cytoplasm and nucleus was verified by FISH assay. We further use RIP and RNA pull down experiments to verify that H19 and miR-29b can be combined with each other. In bladder cancer cells, RT-q PCR was used to verify the regulatory relationship between them. Overexpression of miR-29b mimics could reduce the expression of target gene DNMT3B, and the expression of H19 and DNMT3B was synergistic. Finally, it was verified by WB experiment that H19 regulated the expression of DNMT3B by interacting with miR-29b and then affected the expression of DNMT3B. Conclusion 1.H19 and DNMT3B are highly expressed in bladder cancer and there is a positive correlation between them. Overexpression of H19 in vitro and in vivo regulates the proliferation, migration, invasion, angiogenesis and EMT.3.H19 regulation of the expression of target gene and EMT protein by binding to miR-29b-3p.
【學位授予單位】：重慶醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R737.14

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