LATS1基因去甲基化对人肝癌细胞Hippo-YAP信号通路的影响

发布时间：2018-03-23 15:33

本文选题：肝细胞肝癌　切入点：Hippo信号通路　出处：《重庆医科大学》2017年硕士论文

【摘要】：目的通过去甲基化药物上调LATS1基因在人肝癌细胞中的表达,研究LATS1基因对人肝癌细胞增殖、凋亡、裸鼠成瘤及Hippo信号通路的影响。方法利用蛋白免疫印迹法(Western Blot)检测人肝癌细胞Hep G2与SMMC-7721中大肿瘤抑制基因-1(Large tumor suppressor gene 1,LATS1)和下游癌基因Yes-相关蛋白(Yes-associated protein,YAP)的蛋白表达水平,使用甲基化特异性PCR(Methylation-specific PCR,MSP)与亚硫酸氢盐测序法(Bisulfite sequencing PCR,BSP)检测Hep G2与SMMC-7721细胞中LATS1基因的甲基化水平,利用5-氮杂-2脱氧胞苷(5-Aza-2’-deoxycytidine,DAC)去甲基化处理Hep G2与SMMC-7721细胞后,用MSP和Western Blot分别检测LATS1的甲基化水平与LATS1和YAP的蛋白表达水平,流式细胞术(Flow cytometry,FCM)检测各组细胞凋亡和周期情况,建立细胞裸鼠皮下移植瘤模型,检测各组细胞成瘤情况。结果MSP显示人肝癌细胞系Hep G2与SMMC-7721中LATS1存在高度甲基化;BSP定量显示Hep G2细胞中LATS1甲基化程度(甲基化的CPG岛位点占全部CPG岛位点的比例)为92.4%,SMMC-7721细胞中LATS1甲基化程度为93.1%,LATS1基因启动子区域高度甲基化;WB提示LTAS1低表达,YAP高表达(P0.05)。经DAC去甲基化处理Hep G2与SMMC-7721,MSP显示LATS1的甲基化水平显著降低(P0.05),同时LAST1蛋白表达水平显著升高(P0.05),但YAP蛋白表达水平显著降低(P0.05),细胞周期停滞在G1期(P0.05)、细胞凋亡率显著增加(P0.05)、裸鼠皮下移植瘤大小与体积显著减小(P0.05)。结论LATS1基因在人肝癌细胞中甲基化程度较高,去甲基化后能够上调LATS1基因的蛋白表达水平同时降低YAP蛋白表达水平,抑制并降低Hep G2与SMMC-7721细胞增殖能力、诱导其凋亡,遏制肝癌细胞的成瘤能力。
[Abstract]:Objective to study the effect of LATS1 gene on proliferation and apoptosis of human hepatoma cells by upregulating the expression of LATS1 gene in human hepatoma cells by demethylating drugs. Methods the expression of large tumor suppressor gene 1 (LATS1) and Yes-associated protein (Yes-associated protein) in human hepatoma cell line Hep G2 and SMMC-7721 were detected by Western blot. The methylation level of LATS1 gene in Hep G2 and SMMC-7721 cells was detected by methylation specific PCR(Methylation-specific PCR MSPs and bisulfite sequencing PCR BSPs. Hep G2 and SMMC-7721 cells were treated with 5-aza-2-deoxycytidine demethylation (5-Aza-2-deoxycytidine). The methylation level of LATS1 and the protein expression of LATS1 and YAP were detected by MSP and Western Blot, and apoptosis and cell cycle were detected by flow cytometry. Results MSP showed that there was hypermethylation of LATS1 in Hep G2 and LATS1 in SMMC-7721. The extent of LATS1 methylation in Hep G2 cells was determined quantitatively (the percentage of methylated CPG island loci to all CPG island sites) in Hep G2 cells. The degree of LATS1 methylation in SMMC-7721 cells was 93.1. The high methylation of LTAS1 in the promoter region of LATS1 gene suggested that the low expression of LTAS1 and the high expression of P0.050.The methylation level of Hep G2 and SMMC-7721 showed that the methylation level of LATS1 was significantly lower than that of LAST1 protein surface after DAC demethylation treatment in SMMC-7721 cells. The expression of YAP protein decreased significantly, the cell cycle stagnated in G1 phase, the apoptosis rate increased significantly, and the size and volume of subcutaneous transplanted tumor decreased significantly in nude mice. Conclusion the expression of LATS1 gene in human hepatoma cells is significantly lower than that in human hepatoma cells. Higher levels of methylation, Demethylation can up-regulate the expression of LATS1 gene and decrease the expression of YAP protein, inhibit and decrease the proliferation of Hep G2 and SMMC-7721 cells, induce apoptosis and inhibit the tumorigenic ability of hepatoma cells.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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