LATS1基因去甲基化对人肾癌细胞生物学功能及其Hippo-YAP信号通路的影响

发布时间：2018-08-03 13:07

【摘要】：目的探讨LATS1基因去甲基化对人肾癌细胞生物学功能及其Hippo-YAP信号通路的影响。方法利用RT-PCR检测人肾透明细胞癌细胞系786-O和人胚肾细胞系HEK-293中Hippo-YAP信号通路大肿瘤抑制基因1(large tumor suppressor gene 1,LATS1)mRNA和下游癌基因Yes-相关蛋白(yes-associated protein,YAP)mRNA的表达水平,用亚硫酸氢盐测序法(bisulfite sequence-PCR,BSP)对LATS1低表达细胞786-O进行甲基化分析,5-氮杂-2脱氧胞苷(5-aza-2'-deoxycytidine,DAC)处理786-O和HEK-293后,用RT-PCR、Western blot检测各组细胞LATS1和YAP的mRNA和蛋白表达水平,流式细胞术检测各组细胞凋亡和周期,CCK-8检测各组细胞增殖抑制情况。结果 786-O较HEK-293 LATS1mRNA表达水平显著降低(P0.05),YAP mRNA表达水平显著升高(P0.05),BSP显示LATS1在人肾癌细胞系786-O中高度甲基化;DAC处理786-O后,LATS1 mRNA和蛋白表达水平显著升高(P0.05),YAP mRNA和蛋白表达水平显著降低(P0.05),周期停滞在G0/G1期(P0.05)、细胞增殖受到明显抑制(P0.05)、细胞凋亡显著增加(P0.05),而HEK-293组均无明显变化(P0.05)。结论 LATS1基因去甲基化后下调YAP基因表达,抑制786-O细胞增殖并诱导其凋亡。LATS1基因甲基化在肾癌发生中起重要作用。
[Abstract]:Objective to investigate the effect of LATS1 gene demethylation on the biological function and Hippo-YAP signaling pathway of human renal carcinoma cells. Methods RT-PCR was used to detect the expression of mRNA and yes-associated protein (Yap) mRNA in Hippo-YAP signaling pathway 1 (large tumor suppressor gene 1 and LATS1 in human renal clear cell carcinoma cell line 786-O and human embryonic kidney cell line HEK-293. Bisulfite sequence-PCRP-BSP was used to methylate LATS1 low-expression cells 786-O to analyze the mRNA and protein expression levels of LATS1 and YAP in each group treated with 5-aza-2na-deoxycytidine dinoside (5-aza-2ox-deoxycytidine DAC). The mRNA and protein expression levels of LATS1 and YAP in each group were detected by RT-PCRG Western blot. Apoptosis was detected by flow cytometry and inhibition of cell proliferation was detected by CCK-8. Results the expression level of 786-O was significantly lower than that of HEK-293 LATS1mRNA (P0.05). (P0.05) the expression of LATS1 in human renal cell carcinoma cell line 786-O was significantly higher than that of HEK-293 LATS1mRNA (P0.05). (P0.05) the expression of LATS1 mRNA and protein in human renal cell carcinoma cell line 786-O treated with hypermethylated LATS1 was significantly increased (P0.05). Significantly decreased (P0.05), cycle arrest in the G0/G1 phase (P0.05), cell proliferation was significantly inhibited (P0.05), apoptosis significantly increased (P0.05), but no significant change in HEK-293 group (P0.05). Conclusion LATS1 gene demethylation down-regulates the expression of YAP gene, inhibits the proliferation of 786-O cells and induces apoptosis. The methylation of LATS1 gene plays an important role in the pathogenesis of renal cell carcinoma.
【作者单位】：重庆医科大学附属第一医院泌尿外科;重庆市綦江区人民医院泌尿外科;重庆医科大学附属大学城医院消化神经中心;
【基金】：国家自然科学基金面上项目(30972999) 重庆市自然科学基金(cqpc2012jja1698) 重庆市卫生局基金(2013-2-082)~~
【分类号】：R737.11

【参考文献】