LncRNAH19通过miR-29b-3p作为ceRNA调控膀胱癌EMT的机制研究

发布时间：2018-05-14 12:01

本文选题：长链非编码RNA + H19　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的探索lnc RNA H19通过miR-29作为ce RNA调控miR-29靶基因表达促进对膀胱癌EMT和侵袭转移的作用,获得lnc RNA H19作为miR-29的ce RNA调控靶基因及相关信号传导通路的可靠证据,旨在阐明lnc RNA H19的功能,揭示膀胱癌发生EMT及侵袭转移新的分子机制,为寻找新的膀胱癌诊断和治疗的靶基因和药物提供原创性的科学依据。方法通过芯片筛选出差异表达的lnc RNAs,并确定H19为研究对象,利用生物信息学绘制ce RNA网络,并通过组织qRT-PCR实验验证H19与DNMT3B在人膀胱癌与正常膀胱中的的表达量。接着在体外水平验证H19对细胞增殖、迁移、侵袭、骨架以及EMT的影响,在体内验证H19对膀胱癌的增殖、侵袭、血管生成及EMT的影响。接着通过生物信息学网站预测H19和DNMT3B与miR-29b-3p的结合位点。通过双荧光素酶报告实验验证H19/DNMT3B与miR-29具有结合位点,且影响相互结合。然后在人膀胱癌和癌旁组织以及膀胱癌细胞进行H19与miR-29b的共定位,接着利用RIP、RNA pull-down实验进一步验证H19与miR-29b的结合关系,并利用qRT-PCR检测H19、DNMT3B与miR-29b的相互调控关系。最后我们通过WB测定H19可以体外调节DNMT3B来影响EMT标记蛋白的水平。结果芯片结果显示并用RT-q PCR验证了H19与DNMT3B在人膀胱癌中均高表达,接着利用CCK8,Ed U,平板克隆实验验证H19可以影响膀胱癌细胞的增殖;利用划痕及transwell实验证明H19能够影响膀胱癌细胞的迁移与侵袭;通过骨架实验和稳转细胞的形态观察发现H19能够影响微丝重排与细胞运动;干扰H19表达起相反作用。我们利用动物实验验证H19促进体内膀胱细胞的肿瘤发生,转移和血管发生。在体内观察H19调节DNMT3B蛋白水平及EMT相关的蛋白质的表达。利用生物信息学预测H19/DNMT3B与miR-29b具有结合位点,并用双荧光素酶报告实验验证了两者分别与miR-29b具有结合关系,且H19通过吸附miR-29b-3p而作为ce RNA起作用,因此在功能上消除了miR-29b对靶基因DNMT3B的内源抑制作用。结合利用FISH实验验证了H19与miR-29b共定位于细胞质及核中。我们进一步利用RIP及RNA pull down实验验证H19与miR-29b能够相互结合。接着在膀胱癌细胞中通过RT-q PCR验证三者之间的调控关系,过表达miR-29b mimics能够降低靶基因DNMT3B的表达且H19与DNMT3B表达协同。最后通过WB实验验证H19通过与miR-29b相互影响而调控DNMT3B的表达进而影响EMT。结论1.H19和DNMT3B在膀胱癌组织中高表达且两者表达存在正相关。2.在体内外过表达H19调控膀胱癌细胞增殖,迁移,侵袭、血管形成及EMT。3.H19通过结合miR-29b-3p调节其靶基因及EMT蛋白的表达。
[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.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.14

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