YTHDF2协调胰腺癌细胞的增殖和上皮间质转化

发布时间：2018-04-14 12:35

本文选题：YTHDF2 + 增殖　；参考：《江苏大学》2017年硕士论文

【摘要】：目的:本研究旨在探讨YTH结构域m6A RNA结合蛋白2(YTHDF2)在胰腺癌组织和三种胰腺癌细胞株的表达,并分析其对胰腺癌细胞增殖、迁移、侵袭等生物学行为的影响,初步探讨其影响上皮间质转化(epithelial-mesenchymal transition,EMT)作用的分子机制,为胰腺癌的临床诊断、治疗提供新的策略。方法:(1)运用公共数据库资料比较正常胰腺组织和胰腺癌组织YTHDF2 mRNA及蛋白表达水平;(2)Western blot,荧光定量PCR检测人胰腺癌细胞株(PaTu8988、SW1990、BxPC3)中YTHDF2 mRNA及蛋白质的表达水平;利用慢病毒介导的RNA干扰技术,构建YTHDF2 shRNA干扰质粒sh-YTHDF2,并进行慢病毒包装,感染胰腺癌细胞,嘌呤霉素筛选得到能稳定低表达YTHDF2的胰腺癌细胞株,Western blot、荧光定量PCR检测干扰效率;(3)用平板克隆形成实验和CCK-8法检测YTHDF2对胰腺癌细胞增殖能力的影响,Western blot检测增殖相关指标CyclinD1及其上游蛋白;(4)Transwell迁移实验和划痕实验检测YTHDF2对胰腺癌细胞迁移能力的影响,Transwell侵袭实验检测YTHDF2对胰腺癌细胞侵袭能力的影响,荧光定量PCR、Western blot检测侵袭相关指标MMP2和MMP9 mRNA及蛋白表达量的影响;(5)Western blot检测上皮间质转化相关指标E-cadherin、Vimentin和Snail的蛋白水平的变化,以及影响上皮间质转化的关键蛋白YAP,初步探明YTHDF2影响EMT的分子机制。结果:(1)数据库显示正常胰腺组织的YTHDF2 mRNA及蛋白表达水平均低于胰腺癌组织,肿瘤分期级别越高的患者其YTHDF2的表达量也越高,并且病理分期T3和T4的患者YTHDF2的表达高于T1和T2的患者;(2)胰腺癌细胞株SW1990、BxPC3中YTHDF2表达量相对较高;靶向干扰YTHDF2 shRNA慢病毒载体构建成功,慢病毒包装并感染SW1990、Bx PC3细胞株,Western blot、荧光定量PCR检测结果表明YTHDF2 shRNA在胰腺癌细胞中能有效的干扰YTHDF2的蛋白和mRNA表达水平;(3)SW1990和Bx PC3中分别下调YTHDF2的表达,细胞增殖率、克隆形成能力降低,p-Akt、p-GSK3β和CyclinD1的表达水平下降;(4)SW1990和Bx PC3中分别下调YTHDF2的表达,胰腺癌细胞的迁移能力、侵袭能力增强,MMP2和MMP9 mRNA及蛋白表达水平升高;(5)靶向沉默YTHDF2的表达后,细胞上皮表型标志物E-cadherin表达水平下降,间质表型标志物Vimentin和Snail表达水平明显升高,促进了上皮间质转化的途径不是经典的TGF-β/Smad通路,而是通过上调了YAP的表达。结论:(1)YTHDF2表达水平在胰腺癌组织升高;(2)YTHDF2协调了胰腺癌细胞的增殖和上皮间质转化;(3)下调YTHDF2促进上皮间质转化的途径不是经典的TGF-β/Smad通路,而是通过上调了YAP的表达。
[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.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.9

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