DNMT3B通过Hippo信号通路促进肝癌细胞的增殖与侵袭

High expression of DNMT3B promotes proliferation and invasion of hepatocellular carcinoma cells via Hippo signaling pathway

目的探索DNMT3B对肝癌细胞的增殖与侵袭的影响。方法收集2008年5月~2013年5月在重庆医科大学附二院确诊的175例肝癌患者并制作成组织芯片,分析DNMT3B蛋白表达水平的差异与患者的预后情况及患者肿瘤无瘤生存率与肿瘤特异性生存率的关系。使用单因数与多因素Cox回归分析DNMT3B的表达对肝癌患者预后的影响。使用小干扰RNA(siRNA)与慢病毒过表达干扰DNMT3B的表达,利用CCK-8及EDU染色检测肝癌细胞增殖情况,对细胞进行Transwell实验检测细胞迁移侵袭能力分析。结果免疫组化显示DNMT3B蛋白在肝癌中的表达率(67.4%)高于配对癌旁组织的表达率(41.1%)。DNMT3B的高表达与肿瘤大小(P=0.001)、血管侵犯(P=0.004)、肝内转移(P=0.018)密切相关。DNMT3B高表达患者其肿瘤无瘤生存率与肿瘤特异性生存率低于DNMT3B低表达患者(P<0.005)。沉默DNMT3B显著抑制Huh-7细胞增殖,Transwell实验检测结果表明,与对照组相比沉默DNMT3B抑制Huh-7细胞的迁移与侵袭能力。Western blot检测显示,沉默DNMT3B的表达升高了LATS1的表达水平,降低了YAP1的表达,激活了Hippo信号通路。同时,甲基化特异性PCR显示LATS1的甲基化水平降低。结论肝癌中DNMT3B的表达高于癌旁组织,并且DNMT3B的高表达与患者的低生存率密切相关。沉默DNMT3B抑制细胞增殖、迁移和侵袭能力。DNMT3B主要通过甲基化LATS1并抑制其表达,促进癌基因YAP1的表达进而抑制Hippo信号通路的抑癌作用,从而促进肝癌恶性发展。

Objective To explore the role of DNMT3 B in regulating the proliferation and invasion of hepatocellular carcinoma(HCC) cells. Methods We collected the tumor tissues and adjacent tissues from a total of 175 patients with HCC diagnosed in the Second Affiliated Hospital of Chongqing Medical University between May, 2008 and May, 2013 to prepare the tissue microarrays. The association of the expression of DNMT3 B with the prognosis and the tumor-free survival and tumor-specific survival rates of the patients was analyzed. Univariate and multivariate Cox regression analyses were used to analyze the effect of DNMT3 B expression on the prognosis of HCC. We used RNA interference technique to knock down the expression of DNMT3 B in Huh-7 hepatoma cells and observed the changes in cell proliferation using CCK-8 assay and EDU staining and in cell migration and invasion ability using Transwell assay. Results The positive rates of DNMT3 B was significantly higher in HCC tissues than in paired adjacent tissues(67.4% vs 41.1%, P=0.015). A high DNMT3 B expression in HCC was significantly associated with the tumor size(P=0.001), vascular invasion(P=0.004), and intrahepatic metastasis(P=0.018). The patients with high DNMT3 B expressions had significantly lower tumor-free and tumor-specific survival rates than those with low DNMT3 B expressions(P<0.005). In Huh-7 cells, silencing DNMT3 B significantly inhibited the cell proliferation and inhibited cell migration and invasion. Western blotting showed that silencing DNMT3 B obviously increased LATS1 expression, decreased the expression of YAP1, and activated Hippo signaling pathway. Methylation-specific PCR showed that the methylation level of LATS1 was decreased in the cells with DNMT3 B silencing. Conclusion The expression level of DNMT3 B is significantly higher HCC tissues than in the adjacent tissues, and the high expression of DNMT3 B is closely related to the low survival rate of the patients. Silencing DNMT3 B inhibits the proliferation, migration and invasion of HCC cells. DNMT3 B promotes the progression of HCC primarily by enhancing the expression of YAP1 through methylation of LATS1 and inhibition of its expression, which inhibits the anti-cancer effect of Hippo signaling pathway.