[1]吴　琼,郑志存.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[J].现代检验医学杂志,2022,37(02):115-120.[doi:10.3969/j.issn.1671-7414.2022.02.024]
　WU Qiong,CHENG Zhi-cun.Effects of UBE2T on Proliferation, Clone Formation and Cell Cycle of Hepatocellular Carcinoma Cells and Its Molecular Mechanism[J].Journal of Modern Laboratory Medicine,2022,37(02):115-120.[doi:10.3969/j.issn.1671-7414.2022.02.024]
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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()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第37卷
期数:
2022年02期
页码:
115-120
栏目:
论　著
出版日期:
2022-03-15

文章信息/Info

Title:
Effects of UBE2T on Proliferation, Clone Formation and Cell Cycle of Hepatocellular Carcinoma Cells and Its Molecular Mechanism
文章编号:
1671-7414（2022）02-115-07
作者:
吴　琼; 郑志存
（江苏省海安市人民医院消化内科，江苏海安 226600）
Author(s):
WU Qiong1;  CHENG Zhi-cun2
（Department of Digestive Medicine, Haian People’s Hospital, Jiangsu Hai’an 226600, China）
关键词:
肝癌; 泛素结合酶E2T; PI3K-AKT信号通路
分类号:
R735.7；R730.43
DOI:
10.3969/j.issn.1671-7414.2022.02.024
文献标志码:
A
摘要:
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。
Abstract:
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.

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备注/Memo

备注/Memo:
基金项目：江苏省卫生计生委医学科研课题（H2019087）。
作者简介：吴琼（1984-），女，主治医师，研究方向：肝癌靶向治疗基础研究，E-mail:tcl59986205@126.com。
通讯作者：郑志存（1963-），男，主任医师，研究方向：肝癌靶向治疗。
更新日期/Last Update: 1900-01-01