miR-20b靶向STAT3负向调控H22细胞VEGF的表达

发布时间：2018-07-12 17:01

本文选题：VEGF + miR-20b　；参考：《蚌埠医学院》2017年硕士论文

【摘要】：研究背景:血管内皮生长因子(vascular endothelial growth factor,VEGF)是由两条相同多肽链通过二硫键构成的同源二聚体糖蛋白。VEGF可以由上皮细胞、平滑肌细胞、内皮细胞、巨噬细胞和肿瘤细胞等多种细胞产生。VEGF是内皮细胞的有丝分裂原,可以促进内皮细胞的存活和迁移。VEGF是肿瘤生长中最重要的血管生成因子,可以作为潜在的肿瘤治疗靶点。VEGF组成性及诱导性的表达是一个复杂的事件,涉及多条胞内信号通路及多种转录因子,目前VEGF表达确切的机制仍然不清。micro RNAs(miRNAs)是一群非编码RNA,长度为23bp左右,其主要通过抑制靶基因的翻译或者直接引起m RNA降解来调控其表达。miRNAs在细胞代谢、增生、分化、凋亡等多种生物学过程中均有重要作用。近来有报道表明,miRNAs可以参与血管的生成和VEGF表达的调控。miR-20b是miR-106a-363基因簇家族成员之一,与来自于miR-17-92基因簇的miR-20a和来自于miR-106b-25基因簇的miR-93具有高度同源性。最近,miR-20a和miR-93相继被报道可以调控VEGF的产生,但是目前关于miR-20b调控VEGF产生的研究报道较少。目的:探讨miR-20b对H22细胞VEGF产生的影响及其相关机制。方法:TGF-β1刺激小鼠肝癌细胞株H22细胞后,荧光定量PCR检测miR-20b、VEGF基因表达情况,免疫印迹检测VEGF蛋白表达情况。H22细胞转染miR-20b模拟物(mimics)或其阴性对照(scramble)24h后,在有或无TGF-β1刺激的情况下,荧光定量PCR及免疫印迹检VGEF基因和蛋白的表达,同时对参与VEGF表达的转录因子STAT3的表达情况进行了检测。miRanda算法预测STAT3 3’-UTR区是否存在miR-20b的结合位点;构建含有STAT3 3’-UTR的双荧光素酶报告基因重组质粒载体,然后使用重组质粒与miR-20b mimics/scramble共转染Hela细胞,通过双荧光素酶活性检测试剂盒检测报告基因海肾荧光素酶的活性;H22细胞使用si RNA干扰STAT3基因表达后,荧光定量PCR检测VEGF基因表达情况,免疫印迹检测VEGF的蛋白表达情况;miR-20b mimics转染H22细胞,然后使用STAT3 si RNA干扰,免疫印迹检测VEGF的蛋白表达情况。结果:TGF-β1刺激H22细胞6h、12h、24h,miR-20b相对表达下调(P0.05),TGF-β1刺激H22细胞6h、12h VEGF m RNA的相对表达量上调(P0.05),刺激H22细胞12h、24h,VEGF蛋白相对表达量上调(P0.05)。相对于TGF-β1单刺激组,转染miR-20b mimics的TGF-β1刺激组VEGF基因和蛋白相对表达量均显著下调(P0.01)。H22细胞转染miR-20b mimics或miR-20b scramble 24h后,与空白对照(control)组相比,miR-20b mimics组VEGF m RNA的相对表达无明显变化,但VEGF蛋白水平相对表达明显下调(P0.05)。利用miRanda算法可以预测到STAT3 3’-UTR区存在miR-20b的结合位点(GCACUUU序列);H22细胞转染miR-20b mimics后STAT3蛋白水平相对表达下调(P0.05)。电泳法和测序法证明成功构建pmiR-RB-ReportTM-STAT3 3'-UTR双荧光素酶报告载体;重组质粒+miR-20b mimics共转染组荧光素酶活性明显低于空质粒+miR-20b mimics及空质粒+miR-20b scramble共转染组(P0.05)。H22细胞STAT3基因干扰后,VEGF的m RNA和蛋白相对表达量明显下调(P0.05)。H22细胞转染miR-20b mimics或miR-20b scramble,STAT3基因干扰24h后,相对于control组,si-STAT3+miR-20b mimics组、si-STAT3对照物(si-NC)+miR-20b mimics组的VEGF蛋白相对表达均有下调,而miR-20b mimics+si-NC组与miR-20b mimics+si-STAT3相比,VEGF蛋白相对表达下降的更为明显(P0.05)。结论:TGF-β1诱导H22细胞中VEGF表达上调,miR-20b表达下调;miR-20b负向调控H22细胞VEGF的表达;miR-20b直接靶向STAT3 3’-UTR而负性调节其表达;STAT3正向调控H22细胞VEGF的表达;STAT3参与miR-20b负向调控H22细胞VEGF表达的过程。
[Abstract]:Background: vascular endothelial growth factor (vascular endothelial growth factor, VEGF) is a homologous two polyglycoprotein.VEGF composed of two same polypeptide chains through two sulfur bonds, which can produce mitogen from epithelial cells, smooth muscle cells, endothelial cells, macrophages, and tumor cells, and.VEGF is the mitogen of endothelial cells. To promote the survival and migration of endothelial cells (.VEGF) is the most important angiogenic factor in tumor growth. It is a complex event, involving multiple intracellular signaling pathways and multiple transcription factors, which can be used as a potential target for tumor therapy, which involves multiple intracellular signaling pathways and multiple transcription factors. The exact mechanism of VEGF expression is still unclear in.Micro RNAs (.Micro RNAs). MiRNAs) is a group of non coded RNA with a length of about 23bp, which mainly regulates the expression of.MiRNAs in many biological processes, such as cell metabolism, proliferation, differentiation and apoptosis by inhibiting the translation of the target gene or directly causing m RNA degradation. Recently, it has been reported that miRNAs can be involved in the formation of blood vessels and the modulation of VEGF expression. Controlled.MiR-20b is one of the members of the miR-106a-363 gene cluster family, highly homologous with the miR-20a from the miR-17-92 gene cluster and the miR-93 from the miR-106b-25 gene cluster. Recently, miR-20a and miR-93 have been reported to regulate the production of VEGF. But there are few reports on VEGF production of miR-20b modulation control. The effect of R-20b on the production of VEGF in H22 cells and its related mechanisms. Methods: TGF- beta 1 stimulated the H22 cells of the mouse liver cancer cell line. The fluorescence quantitative PCR was used to detect miR-20b, the expression of VEGF gene, and the expression of VEGF protein was detected by Western blot, and.H22 cells were transfected into miR-20b analogue (mimics) or its negative control. In the case of excitation, the expression of VGEF gene and protein in PCR and immunoblotting, and the expression of the transcription factor STAT3 involved in the expression of VEGF, the.MiRanda algorithm was used to predict the binding site of miR-20b in STAT3 3 '-UTR region, and the construction of a recombinant plasmid vector containing the double luciferase reporter gene containing STAT3 3' -UTR. The recombinant plasmid was then co transfected with miR-20b mimics/scramble to transfect Hela cells, and the activity of luciferase was detected by the double luciferase activity detection kit. H22 cells used Si RNA to interfere with STAT3 gene expression, and the fluorescence quantitative PCR detected the expression of the VEGF gene, and the protein expression of VEGF was detected by immunoblotting; miR-. 20b mimics transfected H22 cells, then using STAT3 Si RNA interference and immunoblotting to detect the protein expression of VEGF. Results: TGF- beta 1 stimulates H22 cell 6h, 12h, 24h, relative expression level. (P0.05). Relative to the TGF- beta 1 single stimulation group, the VEGF gene and protein relative expression of the TGF- beta 1 stimulation group transfected with miR-20b mimics decreased significantly (P0.01).H22 cells transfected to miR-20b mimics or miR-20b scramble, but compared with the blank control group, the relative expression was not significantly changed. MiRanda algorithm could predict the presence of miR-20b binding site (GCACUUU sequence) in STAT3 3 '-UTR region, and H22 cells were down regulated (P0.05) of STAT3 protein level after miR-20b mimics transfected into H22 cells (P0.05). The luciferase activity of the recombinant plasmid +miR-20b mimics co transfection group was significantly lower than that of the empty plasmid +miR-20b mimics and the empty plasmid +miR-20b scramble co transfection group (P0.05).H22 cell STAT3 gene interference, and the m RNA and protein relative expression of VEGF decreased significantly. After 4h, the relative expression of VEGF protein in the si-STAT3 control group (si-NC) +miR-20b mimics group decreased compared to the control group and the si-STAT3 control group (si-NC) +miR-20b mimics group. Expression downregulation; miR-20b negatively regulated the expression of VEGF in H22 cells; miR-20b directly targets STAT3 3 '-UTR to regulate its expression; STAT3 regulates the expression of VEGF in H22 cells; STAT3 participates in the process of miR-20b negative direction regulating the expression of H22 cells.
【学位授予单位】：蚌埠医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R363

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