YB-1通过调控GSK-3β表达促进胰腺癌增殖的机制研究

发布时间：2018-03-24 07:37

本文选题：胰腺癌　切入点：增殖　出处：《华中科技大学》2016年博士论文

【摘要】：研究目的：在多种恶性肿瘤中,YB-1表达水平的增高可促进肿瘤的发生发展。而在胰腺癌中YB-1的表达情况,是否影响胰腺癌进展,特别是其作用机制,并没有明确的报道。本研究旨在探讨YB-1在胰腺癌中的表达及与胰腺癌进展的关系,并深入探讨YB-1发挥生物学效应的分子机制。实验方法：本研究通过免疫组织化学(IHC)检测90对胰腺癌和癌旁组织中YB-1的表达,并分析YB-1的阳性表达与胰腺癌分化程度的相关性。分析通过western blot法检测胰腺癌细胞系中YB-1的表达。使用慢病毒沉默技术抑制胰腺癌细胞系中YB-1的表达,通过CCK-8法和流式细胞术检测YB-1沉默对于胰腺癌细胞系增殖能力和对细胞周期的影响。通过western blot检测其对细胞周期相关蛋白表达的影响。通过western blot法和qPCR法检测YB-1沉默的胰腺癌细胞中GSK-3β蛋白表达和mRNA表达的变化。使用慢病毒技术过表达胰腺癌细胞系中的YB-1,通过western blot法检测YB-1过表达的胰腺癌细胞系中GSK-3β表达的变化。应用GSK-3β抑制剂TWS119,通过CCK-8法和流式细胞术检测其过表达以及加用GSK-3β抑制剂对胰腺癌细胞增殖能力及细胞周期的影响,是否能够逆转过表达YB-1的作用。应用PI3K/AKT、 MEK/ERK信号通路抑制剂,通过Western blot技术检测在胰腺癌细胞系中,细胞信号通路对YB-1的调控的机制。实验结果：YB-1在胰腺癌中显著高于癌旁组织,且与胰腺癌临床分化程度呈正相关。YB-1在胰腺癌细胞系中高表达。使用慢病毒沉默胰腺癌细胞中YB-1的表达后,可以显著抑制胰腺癌的增殖,并使胰腺癌细胞出现G1期阻滞,慢病毒过表达的胰腺癌细胞系中,GSK-3β表达明显上调,增殖能力显著增强,G1期细胞比例明显减少。抑制YB-1后,胰腺癌细胞中GSK-3β mRNA水平明显降低,且蛋白表达水平变化与mRNA水平变化一致；相反,过表达YB-1后,GSK-3β mRNA与蛋白表达均表达明显上调。使用GSK-3β特异性抑制剂处理YB-1过表达的胰腺癌细胞系后,YB-1过表达所致的细胞增殖能力增强被抑制,且CyclinD1、E1蛋白表达水平与细胞增殖水平变化一致。应用PI3K/AKT及MEK/ERK信号通路的抑制剂发现,YB-1同时受这两条信号通路调控,其中PI3K/AKT在转录水平调控,MEK/ERK在翻译水平调控。研究结论：YB-1过表达是胰腺癌具有高度恶性生物学行为的重要机制,其通过调控GSK-3β的表达促进胰腺癌细胞的增殖与肿瘤进展。YB-1和GSK-3β具有成为成为胰腺癌治疗的靶点的潜力。
[Abstract]:Objective: to study whether the expression of YB-1 in pancreatic cancer affects the progression of pancreatic cancer, especially its mechanism. The purpose of this study was to investigate the expression of YB-1 in pancreatic cancer and its relationship with the progression of pancreatic cancer. In this study, the expression of YB-1 in pancreatic cancer and adjacent tissues was detected by immunohistochemistry. The expression of YB-1 in pancreatic cancer cell line was detected by western blot method. Lentivirus silencing technique was used to inhibit the expression of YB-1 in pancreatic cancer cell line. The effect of YB-1 silencing on the proliferation and cell cycle of pancreatic cancer cell line was detected by CCK-8 and flow cytometry. The expression of cell cycle related protein was detected by western blot. YB-1 was detected by western blot and qPCR. The changes of GSK-3 尾 protein and mRNA expression in silent pancreatic cancer cells. YB-1 in pancreatic cancer cell line was overexpressed by lentivirus technique, and GSK-3 尾 expression was detected by western blot assay. GSK-3 尾 was used to detect the expression of GSK-3 尾 in pancreatic cancer cell line with YB-1 overexpression. TWS119 was used to detect the overexpression of TWS119 by CCK-8 and flow cytometry, and the effect of GSK-3 尾 inhibitor on the proliferation and cell cycle of pancreatic cancer cells. Whether it can reverse the effect of overexpression of YB-1. Using PI3K / AKT, MEK/ERK signaling pathway inhibitor and Western blot technique to detect the regulation mechanism of cell signal pathway to YB-1 in pancreatic cancer cell line. The results showed that the ratio of yb-1 in pancreatic cancer was significantly higher than that in adjacent tissues. There was a positive correlation between YB-1 and clinical differentiation degree of pancreatic carcinoma. After using lentivirus to silence the expression of YB-1 in pancreatic cancer cell line, the proliferation of pancreatic carcinoma was significantly inhibited and G1 phase arrest appeared. The expression of GSK-3 尾 was upregulated in pancreatic cancer cell lines with lentivirus overexpression, and the proportion of cells in G 1 phase was significantly increased. After inhibiting YB-1, the level of GSK-3 尾 mRNA in pancreatic cancer cells decreased significantly. The change of protein expression level was consistent with that of mRNA. The expression of GSK-3 尾 mRNA and protein was up-regulated after overexpression of YB-1. The proliferation of pancreatic cancer cell line YB-1 overexpression was inhibited after GSK-3 尾 specific inhibitor was used to treat the overexpression of YB-1. The expression level of Cyclin D1 F1E 1 protein was consistent with that of cell proliferation. Using the inhibitors of PI3K/AKT and MEK/ERK signaling pathway, it was found that YB-1 was regulated by these two signaling pathways at the same time. Among them, PI3K/AKT is regulated at the transcription level and MEK / ERK is regulated at the translation level. Conclusion the overexpression of PI3K/AKT is an important mechanism for the highly malignant biological behavior of pancreatic cancer. By regulating the expression of GSK-3 尾, it promotes the proliferation and progression of pancreatic cancer cells. YB-1 and GSK-3 尾 have the potential to become targets for the treatment of pancreatic cancer.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.9

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