本文選題：YTHDF2 + 增殖�。� 參考：《江蘇大學》2017年碩士論文

【摘要】：目的:本研究旨在探討YTH結(jié)構(gòu)域m6A RNA結(jié)合蛋白2(YTHDF2)在胰腺癌組織和三種胰腺癌細胞株的表達,并分析其對胰腺癌細胞增殖、遷移、侵襲等生物學行為的影響,初步探討其影響上皮間質(zhì)轉(zhuǎn)化(epithelial-mesenchymal transition,EMT)作用的分子機制,為胰腺癌的臨床診斷、治療提供新的策略。方法:(1)運用公共數(shù)據(jù)庫資料比較正常胰腺組織和胰腺癌組織YTHDF2 mRNA及蛋白表達水平;(2)Western blot,熒光定量PCR檢測人胰腺癌細胞株(PaTu8988、SW1990、BxPC3)中YTHDF2 mRNA及蛋白質(zhì)的表達水平;利用慢病毒介導的RNA干擾技術,構(gòu)建YTHDF2 shRNA干擾質(zhì)粒sh-YTHDF2,并進行慢病毒包裝,感染胰腺癌細胞,嘌呤霉素篩選得到能穩(wěn)定低表達YTHDF2的胰腺癌細胞株,Western blot、熒光定量PCR檢測干擾效率;(3)用平板克隆形成實驗和CCK-8法檢測YTHDF2對胰腺癌細胞增殖能力的影響,Western blot檢測增殖相關指標CyclinD1及其上游蛋白;(4)Transwell遷移實驗和劃痕實驗檢測YTHDF2對胰腺癌細胞遷移能力的影響,Transwell侵襲實驗檢測YTHDF2對胰腺癌細胞侵襲能力的影響,熒光定量PCR、Western blot檢測侵襲相關指標MMP2和MMP9 mRNA及蛋白表達量的影響;(5)Western blot檢測上皮間質(zhì)轉(zhuǎn)化相關指標E-cadherin、Vimentin和Snail的蛋白水平的變化,以及影響上皮間質(zhì)轉(zhuǎn)化的關鍵蛋白YAP,初步探明YTHDF2影響EMT的分子機制。結(jié)果:(1)數(shù)據(jù)庫顯示正常胰腺組織的YTHDF2 mRNA及蛋白表達水平均低于胰腺癌組織,腫瘤分期級別越高的患者其YTHDF2的表達量也越高,并且病理分期T3和T4的患者YTHDF2的表達高于T1和T2的患者;(2)胰腺癌細胞株SW1990、BxPC3中YTHDF2表達量相對較高;靶向干擾YTHDF2 shRNA慢病毒載體構(gòu)建成功,慢病毒包裝并感染SW1990、Bx PC3細胞株,Western blot、熒光定量PCR檢測結(jié)果表明YTHDF2 shRNA在胰腺癌細胞中能有效的干擾YTHDF2的蛋白和mRNA表達水平;(3)SW1990和Bx PC3中分別下調(diào)YTHDF2的表達,細胞增殖率、克隆形成能力降低,p-Akt、p-GSK3β和CyclinD1的表達水平下降;(4)SW1990和Bx PC3中分別下調(diào)YTHDF2的表達,胰腺癌細胞的遷移能力、侵襲能力增強,MMP2和MMP9 mRNA及蛋白表達水平升高;(5)靶向沉默YTHDF2的表達后,細胞上皮表型標志物E-cadherin表達水平下降,間質(zhì)表型標志物Vimentin和Snail表達水平明顯升高,促進了上皮間質(zhì)轉(zhuǎn)化的途徑不是經(jīng)典的TGF-β/Smad通路,而是通過上調(diào)了YAP的表達。結(jié)論:(1)YTHDF2表達水平在胰腺癌組織升高;(2)YTHDF2協(xié)調(diào)了胰腺癌細胞的增殖和上皮間質(zhì)轉(zhuǎn)化;(3)下調(diào)YTHDF2促進上皮間質(zhì)轉(zhuǎn)化的途徑不是經(jīng)典的TGF-β/Smad通路,而是通過上調(diào)了YAP的表達。
[Abstract]:Objective: to investigate the expression of YTH domain m6A RNA binding protein 2 (YTHDF2) in pancreatic carcinoma tissues and three pancreatic cancer cell lines, and to analyze the effects of m6A RNA binding protein 2 on the proliferation, migration and invasion of pancreatic cancer cells.To explore the molecular mechanism of EMTs affecting epithelial-mesenchymal transition, and to provide a new strategy for clinical diagnosis and treatment of pancreatic cancer.Methods the expression levels of YTHDF2 mRNA and protein in normal pancreatic tissues and pancreatic carcinoma tissues were compared by using the common database data. The expression levels of YTHDF2 mRNA and protein in human pancreatic cancer cell line PaTu8988 / SW1990 (BxPC3) were detected by fluorescence quantitative PCR.YTHDF2 shRNA interference plasmid sh-YTHDF2 was constructed by using lentivirus-mediated RNA interference technique. The plasmid sh-YTHDF2 was packaged with lentivirus to infect pancreatic cancer cells.Western blot of pancreatic cancer cell line with stable and low expression of YTHDF2 was obtained by purine mycin screening. The interference efficiency was detected by fluorescence quantitative PCR. The effect of YTHDF2 on proliferation of pancreatic cancer cells was detected by plate clone formation assay and CCK-8 assay. Western blot was used to detect the proliferation of pancreatic cancer cells.The influence of YTHDF2 on the migration ability of pancreatic cancer cells was detected by CyclinD1 and its upstream protein, transwell migration assay and scratch test. The influence of YTHDF2 on the invasion ability of pancreatic cancer cells was detected by Transwell invasion assay.Detection of invasive markers MMP2 and MMP9 mRNA and protein expression by fluorescent quantitative PCR Western blot was used to detect the protein levels of E-cadherin Vimentin and Snail in epithelial mesenchymal transformation.And the key protein Yap, which is the key protein affecting epithelial mesenchymal transformation, has been preliminarily proved to be the molecular mechanism of YTHDF2 affecting EMT.Results the YTHDF2 mRNA and protein expression levels in normal pancreatic tissues were lower than those in pancreatic cancer tissues, and the YTHDF2 expression levels in patients with higher tumor stages were higher than those in normal pancreatic tissues.The expression of YTHDF2 in T 3 and T 4 patients was higher than that in T 1 and T 2 (P < 0.05). The expression of YTHDF2 in SW1990 BxPC3 cell line was higher than that in T 1 and T 2 cell line, and the target interfering YTHDF2 shRNA lentivirus vector was successfully constructed.Lentivirus was packaged and infected with SW1990 Bx PC3 cell line. The results of fluorescence quantitative PCR analysis showed that YTHDF2 shRNA could effectively interfere with the expression of YTHDF2 protein and mRNA in pancreatic cancer cells and down-regulate the expression of YTHDF2 and the proliferation rate of BX PC3, respectively.The down-regulation of YTHDF2 expression in SW1990 and BX PC3, the enhancement of migration ability and invasion ability of pancreatic cancer cells, the enhancement of MMP2 and MMP9 mRNA, and the increase of protein expression level of YTHDF2 were also observed after the expression of YTHDF2 was silenced.The expression level of E-cadherin decreased, and the expression of Vimentin and Snail increased significantly. The pathway of promoting epithelial interstitial transformation was not the classical TGF- 尾 / Smad pathway, but the up-regulation of YAP expression.Conclusion the expression of TGF- 尾 / Smad is not the classical TGF- 尾 / Smad pathway, but by up-regulation of YAP expression.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.9

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本文編號：1749358