UBE2T对肝癌细胞增殖、克隆形成及细胞周期的影响及机制研究

Effects of UBE2T on Proliferation, Clone Formation and Cell Cycle of Hepatocellular Carcinoma Cells and Its Molecular Mechanism

目的 通过检测肝癌组织中泛素结合酶E 2T(ubiquitin binding enzyme E2T, UBE2T)表达,探究其对肝癌细胞增殖、克隆形成及细胞周期的影响及其潜在分子机制。方法 采用实时荧光定量PCR实验(qRT-PCR)检测50例临床肝癌组织及其癌旁正常组织中UBE2T相对表达量;通过转染靶向UBE2T基因的shRNA干扰慢病毒载体GV248-shUBE2T-1,GV248-shUBE2T-2及阴性对照GV248-NC至肝癌HepG2细胞,构建UBE2T基因稳定干扰细胞系,并验证其转染敲降效率;采用细胞增殖实验、克隆形成实验及细胞周期实验分别检测敲低UBE2T表达对肝癌细胞增殖、克隆形成及细胞周期的影响;蛋白免疫印迹实验检测PI3K/AKT信号通路关键靶基因PI3K和AKT磷酸化水平。结果 临床50例肝癌组织中UBE2T相对表达量为2.012±0.489,显著高于癌旁正常组织的1.102±0.533,差异有统计学意义(t=8.896,P<0.01)。与阴性对照组(1.000±0.001)相比,转染干扰慢病毒载体的shUBE2T-1组(0.201±0.003)和shUBE2T-2组(0.246±0.005)中UBE2T表达明显降低,差异有统计学意义(F=518.800,P <0.001)。shUBE2T-1组(0.392±0.028)和shUBE2T-2组(0.368±0.026)细胞增殖速率明显低于对照组(0.519±0.031),差异有统计学意义(F=24.477,P=0.001);shUBE2T-1组(5.018±0.521)和shUBE2T-2组(5.906±0.485)细胞克隆形成速率明显低于对照组(9.312±0.479),差异有统计学意义(F=62.819,P<0.001),并将细胞周期显著阻滞在G1期。shUBE2T-1组和shUBE2T-2组细胞中PI3K磷酸化水平为0.285±0.030和0.267±0.029,AKT磷酸化水平为0.454±0.037和0.411±0.039,均分别低于对照组的1.005±0.026和1.004±0.031,差异有统计学意义(F=659.930, 255.517,均P<0.001),但其本底表达保持不变。结论 肝癌中UBE2T显著高表达,其可能通过诱导PI3K,AKT的磷酸化进而参与调控PI3K-AKT信号通路来促进肝癌细胞增殖。

Objective To investigate the effects of UBE2T on the proliferation, clonogenesis and cell cycle of hepatoma cells and its potential molecular mechanism by detecting the expression of UBE2T hepatoma tissues. Methods Real-time quantitative PCR(qRT-PCR) was used to detect the relative expression level of UBE2T in 50 cases of clinical liver cancer and adjacent normal tissues. Lentiviral vector GV248-shUBE2T-1, GV248-shUBE2T-2 and negative control GV248-NC were transfected into HepG2 cells to construct UBE2T gene stable interference cell lines, and the knockdown efficiency of the transfection was verified. Cell proliferation assay, clone formation assay and cell cycle assay were used to detect the effect of knockdown UBE2T expression on proliferation, clone formation and cell cycle of hepatoma cells. Phosphorylation levels of PI3K and Akt, key target genes of the PI3K/Akt signaling pathway, were detected by Western blot assay. Results The relative expression level of UBE2T in 50 cases of liver cancer was 2.012±0.489, significantly higher than that in adjacent normal tissues(1.102±0.533),the difference was statistically significant(t=8.896, P<0.01). Compared with the negative control group(1.000±0.001), the expression of UBE2T in shUBE2T-1 group(0.201±0.003) and shUBE2T-2 group(0.246±0.005) was significantly decreased,the difference was statistically significant(F=518.800, P<0.001) after transfection with interfered lentiviral vector. The cell proliferation rate in shUBE2T-1 group(0.392±0.028) and shUBE2T-2 group(0.368±0.026) was significantly lower than that in control group(0.519±0.031),the difference was statistically significant(F=24.477, P=0.001).The cell clone formation rate in shUBE2T-1 group(5.018±0.521) and shUBE2T-2 group(5.906±0.485) was significantly lower than that in control group(9.312±0.479),the difference was statistically significant(F=62.819, P<0.001), and the cell cycle was significantly arrested in G1 phase. The phosphorylation level of PI3K in shUBE2T-1 group and shUBE2T-2 group was 0.285±0.030 and 0.267±0.029, and the phosphorylation level of Akt was 0.454±0.037 and 0.411±0.039. All of them were lower than those in the control group(1.005±0.026 and 1.004±0.031),the difference was statistically significant(F=659.930,255.517, P<0.001), respectively, but their background expression remained unchanged. Conclusion UBE2T was highly expressed in liver cancer, which may promote the proliferation of liver cancer cells by inducing the phosphorylation of PI3K and Akt and then participating in the regulation of PI3K-Akt signaling pathway.