NS5ATP3通过调节胆固醇代谢促进HepG2细胞生长  

NS5ATP3 promotes HepG2 cell growth by regulating cholesterol metabolism

作　　者：刘雨薇 韩凯 韩铭[3] 袁晓雪 梁璞[3] 成军[3] Liu Yuwei;Han Kai;Han Ming;Yuan Xiaoxue;Liang Pu;Cheng Jun(Department of Emergency,Beijing Ditan Hospital,Capital Medical University,Beijing 100015,China;Biomedical Inovation Center,Beijing Shijitan Hospital,Capital Medical University,Beijing 100038,China;Institute of Infectious Diseases,Beijing Ditan Hospital,Capital Medical University,Beijing 100015,China)

机构地区：[1]首都医科大学附属北京地坛医院急诊科,北京100015 [2]首都医科大学附属北京世纪坛医院生物医学创新中心,北京100038 [3]首都医科大学附属北京地坛医院传染病研究所,北京100015

出　　处：《中国肝脏病杂志（电子版）》2022年第2期1-10,共10页Chinese Journal of Liver Diseases：Electronic Version

基　　金：国家自然科学基金项目(81670547、81700508)。

摘　　要：目的探讨NS5ATP3在非酒精性脂肪性肝病(nonalcoholic fatty liver disease,NAFLD)相关肝细胞癌(hepatic cell carcinoma,HCC)发生发展中的作用机制。方法转染pNS5ATP3或si-NS5ATP3后检测HepG2细胞内总胆固醇(total cholesterol,TC)水平,分别采用CCK8法、划痕实验、Caspase-3活性检测法检测细胞增殖、凋亡及迁移情况。通过聚合酶链式反应(quantitative real-time polymerase chain reaction,qRT-PCR)和Western blot检测胆固醇合成、细胞增殖、凋亡及细胞周期相关基因和蛋白(SREBP2、HMGCR、FoxM1)的表达。比较共转染pNS5ATP3和si-HMGCR与单纯转染pNS5ATP3时下游HCC关键基因FoxM1的表达水平及细胞增殖能力。结果NS5ATP3过表达增加了细胞内TC水平[(0.044±0.008)pmol/mg vs(0.034±0.004)pmol/mg;t=3.231,P=0.023)],而NS5ATP3沉默后降低了TC水平[(0.025±0.002)pmol/mg vs(0.033±0.003)pmol/mg;t=3.846,P=0.009)]。Western blot表明,与对照组相比,过表达NS5ATP3可引起SREBP2和HMGCR蛋白水平增加,p-AMPKα水平显著降低;沉默NS5ATP3后,SREBP2和HMGCR蛋白水平下降,p-AMPKα水平增高。qRT-PCR结果也显示,与对照组相比,NS5ATP3过表达上调SREBP2 mRNA(2.45±0.75 vs 1±0.33;t=5.159,P=0.037)和HMGCR mRNA(2.30±0.30 vs 1±0.10;t=4.432,P=0.047)的表达,而沉默NS5ATP3后,SREBP2 mRNA(0.24±0.21 vs 1±0.26;t=4.769,P=0.041)和HMGCR mRNA(0.47±0.13 vs 1±0.21;t=4.522,P=0.046)表达水平显著下降。在48 h和72 h时,过表达NS5ATP3组细胞相对活力显著高于对照组(1.85±0.06 vs 1.56±0.12,t=4.583,P=0.010;3.08±0.19 vs 2.61±0.21,t=4.790,P=0.009),24 h时差异无统计学意义(1.10±0.06 vs 1±0.08,t=1.873,P=0.088)。在24 h、48 h和72 h时,沉默NS5ATP3组细胞相对活力均显著低于对照组(0.90±0.07 vs 0.98±0.09,t=3.378,P=0.020;1.57±0.05 vs 1.63±0.11,t=2.717,P=0.035;1.82±0.23 vs 2.61±0.21,t=5.010,P=0.004)。与对照组相比,过表达NS5ATP3后,细胞中caspase-3/7水平降低(0.69±0.09 vs 1±0.15;t=3.128,P=0.026),而沉默siNS5ATP3后,caspase-3/7活性增�Objective To investigate the mechanism of NS5ATP3 in the occurrence and development of nonalcoholic fatty liver disease(NAFLD)-related hepatocellular carcinoma(HCC).Methods The HepG2 cell line was transfected with pNS5ATP3 or si-NS5ATP3,and the concentration of total cholesterol(TC),cell proliferation,apoptosis and migration were detected by TC test kit,CCK8 method,scratch test and caspase-3 activity detection,respectively.Meanwhile,key genes and proteins(SREBP2,HMGCR,FoxM1)involved in cholesterol synthesis,cell proliferation,apoptosis and cell cycle were dectected by quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot assay.Expression of FoxM1,which had important roles in the tumorigenesis of HCC and cell proliferation were compared between HepG2 cell line transfected with pNS5ATP3 or co-transfected with pNS5ATP3 and si-HMGCR.Results NS5ATP3 overexpression increased the intracellular TC level[(0.044±0.008)pmol/mg vs(0.034±0.004)pmol/mg;t=3.231,P=0.023)],while NS5ATP3 silencing decreased the TC level[(0.025±0.002)pmol/mg vs(0.033±0.003)pmol/mg;t=3.846,P=0.009)].Western blot showed that overexpression of NS5ATP3 could increase the protein levels of SREBP2 and HMGCR and decrease the protein level of p-AMPKα;after silencing NS5ATP3,the protein levels of SREBP2 and HMGCR decreased and p-AMPKαincreased.qRT-PCR results also showed that compared with the control group,NS5ATP3 overexpression could up-regulate the mRNA expression of SREBP2(2.45±0.75 vs 1±0.33;t=5.159,P=0.037)and HMGCR(2.30±0.30 vs 1±0.10;t=4.432,P=0.047).After NS5ATP3 was silenced,the mRNA level of SREBP2(0.24±0.21 vs 1±0.26;t=4.769,P=0.041)and HMGCR(0.47±0.13 vs 1±0.21;t=4.522,P=0.046)decreased.At 48 h and 72 h,the relative viability of cells in NS5ATP3 overexpression group was significantly higher than that in control group(1.85±0.06 vs 1.56±0.12,t=4.583,P=0.010;3.08±0.19 vs 2.61±0.21,t=4.790,P=0.009)and there was no significant difference at 24 h(1.10±0.06 vs 1±0.08,t=1.873,P=0.088).At 24 h,48 h and 72 h,the re

关 键 词：肝细胞癌 胆固醇代谢 NS5ATP3 FOXM1 HMGCR 

分 类 号：R735.7[医药卫生—肿瘤]