SAMHD1通过调控p27表达抑制肝细胞癌细胞增殖的研究

SAMHD1 inhibits the proliferation of hepatocellular carcinoma by regulating the expression of p27

背景与目的:不育α基序结构域和组氨酸/天冬氨酸残基双联体结构域包涵蛋白1(sterile alpha motif and histidine/aspartic acid domain-containing protein 1,SAMHD1)具有抑制多种肿瘤细胞生长的作用,但其调节肝细胞癌(hepatocellular carcinoma,HCC)细胞增殖的作用及机制尚未见报道。探究SAMHD1调控p27的表达对HCC细胞Huh7的增殖、凋亡和细胞周期的影响。方法:首先通过蛋白质印迹法(Western blot)检测正常肝细胞和不同类型HCC细胞中SAMHD1的表达情况。利用基因修饰技术构建过表达SAMHD1、dNTP酶活性位点突变体(SAMHD1-D207N)和磷酸化位点突变体(SAMHD1-T592E)的重组质粒,然后利用四甲基偶氮唑蓝(methyl thiazolyl tetrazolium,MTT)法检测过表达SAMHD1及其突变体或siRNA干扰沉默SAMHD1对HCC细胞增殖的影响,采用流式细胞术检测细胞周期与细胞凋亡情况。在癌症基因组图谱(The Cancer Genome Atlas,TCGA)数据库中分析SAMHD1和p27表达的相关性。结果:HCC细胞中SAMHD1表达上调,过表达SAMHD1、SAMHD1-D207N和SAMHD1-T592E可以抑制Huh7细胞增殖,细胞周期停滞在G1/G0期;相反,干扰SAMHD1后细胞增殖加快,细胞周期停滞在G2/M期。机制研究表明,SAMHD1上调细胞周期蛋白激酶抑制因子p27的表达。在HCC组织中,p27的表达与SAMHD1表达呈正相关。结论:过表达SAMHD1可以上调p27的表达,导致细胞周期停滞在G1/G0期,从而抑制HCC细胞增殖,这种抑制作用不依赖于其dNTP酶活性和磷酸化修饰。

Background and purpose: Sterile alpha motif and histidine/aspartic acid domain-containing protein 1(SAMHD1) is found to inhibit cell growth in a variety of tumor cells, however, whether it influences the growth of hepatocellular carcinoma(HCC) cells is unclear. This study aimed to investigate the effect of SAMHD1 on the proliferation, apoptosis and cell cycle of Huh7 cells by regulating the expression of p27. Methods: The expressions of SAMHD1 in normal hepatocytes and different types of HCC cells were detected by Western blot. Recombinant plasmids of overexpression SAMHD1, dNTPase mutants(SAMHD1-D207 N) and phosphorylation mutant(SAMHD1-T592 E) were constructed by gene recombination technology. The effects of overexpression of SAMHD1, D207 N and T592 E mutants or silencing the expression of SAMHD1 by siRNA on the cells proliferation were employed by methyl thiazolyl tetrazolium(MTT) assay, and the effects on the cell cycle and apoptosis of cells were measured by flow cytometry. Results: The expression of SAMHD1 was significantly increased in HCC cells. Overexpression of SAMHD1, SAMHD1-D207 N and SAMHD1-T592 E inhibited the proliferation of Huh7 cells, and induced cell cycle arresting in G1/G0 phase. Converse results were obtained by silencing the overexpression of SAMHD1. Mechanism studies indicated that SAMHD1 could up-regulate the expression of cyclin-dependent kinase inhibitor p27. Moreover, the mRNA expression of p27 was positively correlated with the expression of SAMHD1 in HCC samples derived from The Cancer Genome Atlas(TCGA) database. Conclusion: Overexpression of SAMHD1 can lead to an increase in the expression of p27, resulting in the cell cycle arrest in G1/G0 phase, and inhibits the proliferation of HCC cells, which is independent of dNTPase activity and phosphorylation.