本文選題：VEGF + miR-20b��； 參考：《蚌埠醫(yī)學(xué)院》2017年碩士論文

【摘要】：研究背景:血管內(nèi)皮生長因子(vascular endothelial growth factor,VEGF)是由兩條相同多肽鏈通過二硫鍵構(gòu)成的同源二聚體糖蛋白。VEGF可以由上皮細(xì)胞、平滑肌細(xì)胞、內(nèi)皮細(xì)胞、巨噬細(xì)胞和腫瘤細(xì)胞等多種細(xì)胞產(chǎn)生。VEGF是內(nèi)皮細(xì)胞的有絲分裂原,可以促進(jìn)內(nèi)皮細(xì)胞的存活和遷移。VEGF是腫瘤生長中最重要的血管生成因子,可以作為潛在的腫瘤治療靶點。VEGF組成性及誘導(dǎo)性的表達(dá)是一個復(fù)雜的事件,涉及多條胞內(nèi)信號通路及多種轉(zhuǎn)錄因子,目前VEGF表達(dá)確切的機(jī)制仍然不清。micro RNAs(miRNAs)是一群非編碼RNA,長度為23bp左右,其主要通過抑制靶基因的翻譯或者直接引起m RNA降解來調(diào)控其表達(dá)。miRNAs在細(xì)胞代謝、增生、分化、凋亡等多種生物學(xué)過程中均有重要作用。近來有報道表明,miRNAs可以參與血管的生成和VEGF表達(dá)的調(diào)控。miR-20b是miR-106a-363基因簇家族成員之一,與來自于miR-17-92基因簇的miR-20a和來自于miR-106b-25基因簇的miR-93具有高度同源性。最近,miR-20a和miR-93相繼被報道可以調(diào)控VEGF的產(chǎn)生,但是目前關(guān)于miR-20b調(diào)控VEGF產(chǎn)生的研究報道較少。目的:探討miR-20b對H22細(xì)胞VEGF產(chǎn)生的影響及其相關(guān)機(jī)制。方法:TGF-β1刺激小鼠肝癌細(xì)胞株H22細(xì)胞后,熒光定量PCR檢測miR-20b、VEGF基因表達(dá)情況,免疫印跡檢測VEGF蛋白表達(dá)情況。H22細(xì)胞轉(zhuǎn)染miR-20b模擬物(mimics)或其陰性對照(scramble)24h后,在有或無TGF-β1刺激的情況下,熒光定量PCR及免疫印跡檢VGEF基因和蛋白的表達(dá),同時對參與VEGF表達(dá)的轉(zhuǎn)錄因子STAT3的表達(dá)情況進(jìn)行了檢測。miRanda算法預(yù)測STAT3 3’-UTR區(qū)是否存在miR-20b的結(jié)合位點;構(gòu)建含有STAT3 3’-UTR的雙熒光素酶報告基因重組質(zhì)粒載體,然后使用重組質(zhì)粒與miR-20b mimics/scramble共轉(zhuǎn)染Hela細(xì)胞,通過雙熒光素酶活性檢測試劑盒檢測報告基因海腎熒光素酶的活性;H22細(xì)胞使用si RNA干擾STAT3基因表達(dá)后,熒光定量PCR檢測VEGF基因表達(dá)情況,免疫印跡檢測VEGF的蛋白表達(dá)情況;miR-20b mimics轉(zhuǎn)染H22細(xì)胞,然后使用STAT3 si RNA干擾,免疫印跡檢測VEGF的蛋白表達(dá)情況。結(jié)果:TGF-β1刺激H22細(xì)胞6h、12h、24h,miR-20b相對表達(dá)下調(diào)(P0.05),TGF-β1刺激H22細(xì)胞6h、12h VEGF m RNA的相對表達(dá)量上調(diào)(P0.05),刺激H22細(xì)胞12h、24h,VEGF蛋白相對表達(dá)量上調(diào)(P0.05)。相對于TGF-β1單刺激組,轉(zhuǎn)染miR-20b mimics的TGF-β1刺激組VEGF基因和蛋白相對表達(dá)量均顯著下調(diào)(P0.01)。H22細(xì)胞轉(zhuǎn)染miR-20b mimics或miR-20b scramble 24h后,與空白對照(control)組相比,miR-20b mimics組VEGF m RNA的相對表達(dá)無明顯變化,但VEGF蛋白水平相對表達(dá)明顯下調(diào)(P0.05)。利用miRanda算法可以預(yù)測到STAT3 3’-UTR區(qū)存在miR-20b的結(jié)合位點(GCACUUU序列);H22細(xì)胞轉(zhuǎn)染miR-20b mimics后STAT3蛋白水平相對表達(dá)下調(diào)(P0.05)。電泳法和測序法證明成功構(gòu)建pmiR-RB-ReportTM-STAT3 3'-UTR雙熒光素酶報告載體;重組質(zhì)粒+miR-20b mimics共轉(zhuǎn)染組熒光素酶活性明顯低于空質(zhì)粒+miR-20b mimics及空質(zhì)粒+miR-20b scramble共轉(zhuǎn)染組(P0.05)。H22細(xì)胞STAT3基因干擾后,VEGF的m RNA和蛋白相對表達(dá)量明顯下調(diào)(P0.05)。H22細(xì)胞轉(zhuǎn)染miR-20b mimics或miR-20b scramble,STAT3基因干擾24h后,相對于control組,si-STAT3+miR-20b mimics組、si-STAT3對照物(si-NC)+miR-20b mimics組的VEGF蛋白相對表達(dá)均有下調(diào),而miR-20b mimics+si-NC組與miR-20b mimics+si-STAT3相比,VEGF蛋白相對表達(dá)下降的更為明顯(P0.05)。結(jié)論:TGF-β1誘導(dǎo)H22細(xì)胞中VEGF表達(dá)上調(diào),miR-20b表達(dá)下調(diào);miR-20b負(fù)向調(diào)控H22細(xì)胞VEGF的表達(dá);miR-20b直接靶向STAT3 3’-UTR而負(fù)性調(diào)節(jié)其表達(dá);STAT3正向調(diào)控H22細(xì)胞VEGF的表達(dá);STAT3參與miR-20b負(fù)向調(diào)控H22細(xì)胞VEGF表達(dá)的過程。
[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.
【學(xué)位授予單位】：蚌埠醫(yī)學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R363

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 Mehmet Coskun;Jacob Tveiten Bjerrum;Jakob Benedict Seidelin;Jesper Thorvald Troelsen;JΦrgen Olsen;Ole Haagen Nielsen;;miR-20b, miR-98, miR-125b-1*, and let-7e* as new potential diagnostic biomarkers in ulcerative colitis[J];World Journal of Gastroenterology;2013年27期

2 王蘋;付濤;王緒銳;祝威;;應(yīng)用微陣列芯片分析喉鱗狀細(xì)胞癌miRNA與正常黏膜表達(dá)差異的初步研究[J];臨床耳鼻咽喉頭頸外科雜志;2010年12期

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