摘要
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PNPLA3148I/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PNPLA3148M/M组比过表达PNPLA3148I/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PNPLA3148M/M组细胞增殖活性较过表达PNPLA3148I/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PNPLA3148M/M组较过表达PNPLA3148I/I组PGC1αmRNA表达水平,总PI3K、PThr-308蛋白激酶B(PThr-308 AKT)、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 PNPLA3148M/M细胞增殖作用较PNPLA3148I/I强.
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 PNPLA3148I/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 PNPLA3148M/M group was about 1 /3 times higher than that of overexpression of PNPLA3148I/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 PNPLA3148M/M group and negative control group [(98.02 ±1.29)% vs (100 ±2.61)%, P =0.181]. The proliferative activity of overexpression of PNPLA3148M/M group was about 1/3 times higher than that of overexpression of PNPLA3148I/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 PNPLA 3148M/M group and negative control group (46.46 ±1.83 vs 46.64 ±7.33, P =0.965) .The PGC1αmRNA expression, total PI3K, PThr-308 AKT, PSer2448-mammalian target of rapamycin (PSer2448-mTOR) and PGC1αprotein expression levels in the overexpression of PNPLA 3148M/M group were higher than those in the overexpression of PNPLA3148I/I group, but there were no significant differences in AA and LPA levels , as well as PTGS2 mRNA expression levels.Conclusion PNPLA3148M/M cell proliferation was stronger than PNPLA3148I/I.
作者
颜雪梅
袁淑华
许晓
艾鹤英
梁华
Yan Xuemei;Yuan Shuhua;Xu Xiao;Ai Heying;Liang Hua(Department of Endocrinology,the Third Affiliated Hospital,Sun Yat-sen University,Guaugdong Provincial Key Laboratory of Diabetology,Guangzhou 510630,China)
出处
《中华医学杂志》
CAS
CSCD
北大核心
2018年第34期2754-2759,共6页
National Medical Journal of China
基金
国家自然科学基金(81370909)