ESET通过lncRNA GMDS-AS1调控肝癌细胞增殖、凋亡、糖酵解的研究

Histone methyltransferase ESET regulates the proliferation,apoptosis and glycolysis of hepatoma cells by inhibiting lncRNA GMDS-AS1

目的探究组蛋白甲基转移酶集合域分叉1(ESET)、长链非编码RNA甘露糖4,6-脱水酶反义RNA1(lncRNA GMDSAS1)对肝癌细胞增殖、凋亡、糖酵解的调控及二者之间的潜在关系。方法收集右江民族医学院附属医院和百色市人民医院2019年1月至2020年1月期间手术切除的肝癌组织及癌旁组织49例。脂质体法将空白组(不做任何处理)、空载体组(转染pcDNA或si-con)、敲减ESET组(转染si-ESET)、过表达lncRNA GMDS-AS1组(转染pcDNA-GMDS-AS1)转染至HepG2细胞;实时荧光定量逆转录聚合酶链反应(qRT-PCR)实验、蛋白质印迹法实验检测ESET、lncRNA GMDS-AS1、细胞增殖抗原标志物Ki-67、葡萄糖转运蛋白1(GLUT-1)、乳酸脱氢酶A(LDHA)、活化胱天蛋白酶-3(C-caspase-3)的表达;细胞计数试剂盒(CCK8)法、流式细胞术检测细胞增殖、凋亡;RNA结合蛋白免疫沉淀(RIP)实验检测ESET与lncRNA GMDS-AS1的关系。结果肝癌组织中ESETmRNA和蛋白表达量为5.18±0.94、0.69±0.14,显著高于癌旁组织的0.99±0.33、0.23±0.07,肝癌组织lncRNA GMDS-AS1表达量为0.63±0.20,显著低于癌旁组织的1.02±0.31(P<0.05),二者呈负相关性;与空载体组相比,敲减ESET组细胞ESET的表达明显降低,细胞增殖抗原标志物Ki-67蛋白表达显著降低,细胞活性显著降低,GLUT-1、LDHA的蛋白表达明显降低,C-caspase-3蛋白表达显著升高,凋亡率显著升高(P<0.05);过表达lncRNA GMDS-AS1组细胞具有与敲减ESET组相似的上述检测指标的变化。RIP实验显示,ESET与lncRNA GMDS-AS1具有结合能力。结论甲基转移酶ESET参与肝癌细胞的增殖、凋亡和糖酵解过程,其潜在的机制可能与负向调节lncRNA GMDS-AS1有关。

Objective To explore the regulation of histone methyltransferase SET domain bifurcated 1(ESET)and long noncoding RNA GDP-mannose 4,6-dehydratase antisense RNA1(lncRNA GMDS-AS1)on hepatoma cell proliferation,apoptosis and glycolysis and the potential relationship between them.Methods A total of 49 cases of hepatocellular carcinoma tissues and adjacent tissues surgically resected from the Affiliated Hospital of Youjiang Medical University and Baise People's Hospital for Nationalities between January 2019 and January 2020 were collected.The blank group(without any treatment),empty group(transfected with pcDNA or si-con),ESET knockdown group(transfected with si-ESET)and lncRNA GMDS-AS1 overexpression group(transfected with pcDNA-GMDSAS1)were transfected into HepG2 cells by the liposome method.The expression levels of ESET,lncRNA GMDS-AS1,cell proliferation antigen marker Ki-67,glucose transporter 1(GLUT-1),lactate dehydrogenase A(LDHA)and activated cysteine protease(C-caspase-3)were detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction(qRT‒PCR)and Western blotting.Cell proliferation and apoptosis were detected by the cell counting kit(CCK-8)method and flow cytometry.An RNA immunocoprecipitation(RIP)assay was used to detect the relationship between ESET and lncRNA GMDS-AS1.Results The expression levels of ESET mRNA and protein in hepatocellular carcinoma tissues were 5.18±0.94 and 0.69±0.14,respectively,which were significantly higher than those in adjacent tissues[(0.99±0.33)and(0.23±0.07)],and the expression level of lncRNA GMDS-AS1 in hepatocellular carcinoma tissues was 0.63±0.20,which was significantly lower than that in adjacent tissue(1.02±0.31)(P<0.05),and both were negatively correlated.Compared with the empty vector group,the expression levels of ESET,Ki-67,GLUT-1 and LDHA protein as well as cell activity in the knockdown ESET group were significantly decreased,and C-caspase-3 protein expression and apoptosis rate were significantly increased(P<0.05).The lncRNA GMDS-AS1 overexpression group had similar index changes as the ESET knockdown group.RIP experiments showed that ESET had the ability to interact lncRNA GMDS-AS1.Conclusion The histone methyltransferase ESET was involved in the proliferation,apoptosis,and glycolysis processes of hepatoma cells,and the underlying mechanism may be related to the negative regulation of lncRNA GMDS-AS1.