Objective: To explore the influence of patatin-like phospholipase domain containing-3 (PNPLA3) wild type 148I/I and mutant type 148M/M on HepG2 cell proliferation and the relative mechanisms. Methods: HepG2 cell line stably overexpressing PNPLA3 148I/I, 148M/M and negative control (NC) were set up. Cell counting kit-8 (CCK8) assay was used to measure cell viability. Edu assay was used to determine the ability of cell proliferation. Western blot was used to detect the protein levels in the phosphatidylinositol 3-kinases (PI3K) pathway. Enzyme-linked immunosorbent assay (ELISA) was used to detect proliferation-related PNPLA3 metabolites[arachidonic acid (AA) and lysophosphatidic acid (LPA)]. Quantitative real-time PCR was used to detect the expression level of prostaglandin G/H synthase 2 (PTGS2) and proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) associated with PNPLA3. Results: The cell viability of overexpression of PNPLA3 148M/M group was about 1/3 times higher than that of overexpression of PNPLA3 148I/I group, and the difference was statistically significant[(98.02±1.29)% vs (71.51±2.89)%, P<0.001]. There was no significant difference between overexpression of PNPLA3 148M/M group and negative control group[(98.02±1.29)% vs (100±2.61)%, P=0.181]. The proliferative activity of overexpression of PNPLA3 148M/M group was about 1/3 times higher than that of overexpression of PNPLA3 148I/I group, and the difference was statistically significant(46.46±1.83 vs 35.96±2.65, P=0.001). There was no significant difference between overexpression of PNPLA3 148M/M group and negative control group(46.46±1.83 vs 46.64±7.33, P=0.965). The PGC1α mRNA expression, total PI3K, PThr-308AKT, PSer2448-mammalian target of rapamycin (PSer2448-mTOR) and PGC1α protein expression levels in the overexpression of PNPLA3 148M/M group were higher than those in the overexpression of PNPLA3 148I/I group, but there were no significant differences in AA and LPA levels, as well as PTGS2 mRNA expression levels. Conclusion: PNPLA3 148M/M cell proliferation was stronger than PNPLA3 148I/I.
目的： 探讨含patatin样磷脂酶结构域蛋白3(PNPLA3)野生型148I/I及突变型148M/M对人肝癌细胞株HepG2增殖的影响及相关机制。 方法： 构建PNPLA3 148I/I及148M/M慢病毒稳定过表达及阴性对照细胞株即过表达PNPLA3野生型148I/I HepG2细胞、过表达PNPLA3突变型148M/M HepG2细胞和阴性病毒对照(NC)HepG2细胞。细胞增殖/毒性检测试剂盒(CCK8)检测不同细胞株的活力，5－乙炔基－2′脱氧尿苷(EdU)法检测细胞增殖情况，Western印迹检测磷脂酰肌醇－3激酶(PI3K)信号通路的蛋白水平，采用酶联免疫吸附法(ELISA)检测与增殖相关的PNPLA3代谢产物花生四烯酸(AA)、溶血磷脂酸(LPA)，实时荧光定量PCR检测PNPLA3相关增殖指标前列腺素过氧化物合酶2(PTGS2)和过氧化物酶体增殖物激活受体γ辅助活化因子1α(PGC1α)mRNA表达水平。 结果： 过表达PNPLA3 148M/M组比过表达PNPLA3 148I/I组细胞活力高，差异有统计学意义[(98.02±1.29)%比(71.51±2.89)%，P<0.001]，与NC相比差异无统计学意义[(98.02±1.29)%比(100±2.61)%，P＝0.181]。过表达PNPLA3 148M/M组细胞增殖活性较过表达PNPLA3 148I/I组高，差异有统计学意义(46.46±1.83比35.96±2.65，P＝0.001)，与NC组相比差异无统计学意义(46.46±1.83比46.64±7.33，P＝0.965)。过表达PNPLA3 148M/M组较过表达PNPLA3 148I/I组PGC1α mRNA表达水平，总PI3K、PThr－308蛋白激酶B(PThr－308AKT)、PSer2448－哺乳动物雷帕霉素靶蛋白(PSer2448－mTOR)、PGC1α蛋白表达水平增高，但AA和LPA水平以及PTGS2 mRNA表达水平两组差异无统计学意义。 结论： PNPLA3 148M/M细胞增殖作用较PNPLA3 148I/I强。.
Keywords: Cell proliferation; Fatty liver, non-alcoholic; Genotype; Hep G2 cells; PNPLA3.