人参皂苷Rb1通过KEAP1/PGAM5/AIFM1通路促进肝细胞癌HepG2细胞发生氧死亡

Ginsenoside Rb1 promotes oxeiptosis in hepatocellular carcinoma HepG2 cells through KEAP1/PGAM5/AIFM1 pathway

目的:探讨人参皂苷Rb1(Gn-Rb1)对肝细胞癌(HCC)HepG2细胞氧死亡的影响及其可能的分子机制。方法:采用生物信息学方法分析氧死亡的关键基因PGAM5表达对HCC患者生存期的影响。选取辽宁省肿瘤医院收治的8例HCC患者的HCC组织与癌旁组织,通过WB法及qPCR检测氧死亡相关基因蛋白与mRNA的表达情况。将HepG2细胞随机分为对照组与Gn-Rb1组(予以200μmol/L Gn-Rb1干预),采用细胞克隆形成实验、划痕愈合实验分别检测Gn-Rb1对HepG2细胞的集落形成能力、迁移能力的影响,ELISA检测对细胞ROS生成水平的影响,微板法检测对细胞LDH释放水平的影响;WB法、qPCR法检测Gn-Rb1对HepG2氧死亡关键基因蛋白质与mRNA水平表达的影响。结果:生物信息学分析发现,PGAM5高表达肝癌患者总生存时间较低表达患者更长(P<0.05)。在临床HCC组织与癌旁组织样本中发现,相较于癌旁组织,在蛋白质与mRNA水平上,肿瘤组织KEAP1与PGAM5表达显著降低,NRF2表达显著升高(均P<0.01),p-AIFM1蛋白水平显著升高(P<0.05)。对HepG2细胞予以200μmol/L Gn-Rb1干预后,相较于对照组,Gn-Rb1组HepG2细胞的迁移能力与集落形成能力显著降低(均P<0.01),而LDH水平显著升高(P<0.05);相比于对照组,在mRNA和蛋白质水平上,Gn-Rb1组细胞中KEAP1、PGAM5表达均显著升高而NRF2表达均显著降低(均P<0.05),p-AIFM1蛋白表达显著降低(P<0.01)。结论:HCC组织中氧死亡被抑制,而Gn-Rb1能够通过调控KEAP1/PGAM5/AIFM1通路促进HepG2细胞氧死亡的发生,抑制细胞增殖和迁移能力。

Objective:To investigate the effect of ginsenoside Rb1(Gn-Rb1)on oxeiptosis in hepatocellular carcinoma(HCC)HepG2 cells and its possible molecular mechanism.Methods:Bioinformatic was used to analyse the effect of the expression of PGAM5,a key gene of oxeiptosis,on the survival time of HCC patients.HCC tissues and paracancerous tissues of 8 HCC patients admitted into Liaoning Provincial Tumor Hospital were selected,and the expressions of oxeiptosis-related gene proteins and mRNAs were detected by WB assay and qPCR.HepG2 cells were randomly divided into the control group and the Gn-Rb1 group(intervened with 200μmol/L Gn-Rb1).The effects of Gn-Rb1 on the colony formation ability and the migration ability of HepG2 cells were detected by the cell clone formation assay and the cell scratch assay,respectively.ELISA was used to detect the effect on the level of ROS.Microplate was used to detect the effect on the level of LDH;the effects of Gn-Rb1 on the expressions of key gene proteins and mRNAs were detected by WB assay and qPCR.Results:Bioinformatic analysis revealed that,compared with low-expression patients,patients with PGAM5 high-expression hepatocellular carcinoma had longer overall survival time(P<0.05).In clinical hepatocellular carcinoma and paracancerous tissue samples,it was found that compared with paracancerous tissues,at protein and mRNA levels,the expressions of KEAP1 and PGAM5 in tumor tissues were significantly lower;the expression of NRF2 was significantly higher(all P<0.01);and the expression of p-AIFM1 was significantly higher(P<0.05).After HepG2 cells were intervened with 200μmol/L Gn-Rb1,compared with those in the control group,the migratory and colony-forming abilities of HepG2 cells in the Gn-Rb1 group reduced significantly(all P<0.01),and the level of LDH increased significantly(P<0.05).Compared with the control group,at mRNA and protein levels,the expressions of KEAP1 and PGAM5 in the Gn-Rb1 group were significantly higher(all P<0.05);the expression of NRF2 was significantly lower(all P<0.05);and the expression of p-AIFM1 protein was significantly lower(P<0.01).Conclusion:Oxeiptosis is inhibited in hepatocellular carcinoma tissues,and Gn-Rb1 promotes oxeiptosis in HCC HepG2 cells by regulating the KEAP1/PGAM5/AIFM1 pathway,thus suppressing the proliferation and migration abilities of HepG2 cells.