摘要
目的探究miR-452-5p、YTH结构域家族成员2(YTH domain family,member 2,YTHDF2)调控肝细胞癌细胞凋亡、铁死亡的作用及其机制。方法运用实时荧光定量聚合酶链式反应(real time fluorescence quantitative polymerase chain reaction,RT-qPCR)法检测L02、SMMC-7721、HepG2、Huh7、Hep3B细胞中miR-452-5p、YTHDF2的表达;脂质体法将anti-miR-con组(转染anti-miR-con)、anti-miR-452-5p组(转染anti-miR-452-5p)、pcDNA组(转染pcDNA)、pcDNA-YTHDF2组(转染pcDNA-YTHDF2)、anti-miR-452-5p+si-con组(转染anti-miR-452-5p+si-con)、anti-miR-452-5p+si-YTHDF2组(转染anti-miR-452-5p+si-YTHDF2)转染HepG2细胞;膜联蛋白V-异硫氰酸荧光素(Annexin V-FITC)和碘化丙锭(PI)双染法检测细胞凋亡率;细胞计数试剂盒(cell counting kit,CCK8)法检测细胞活性;蛋白免疫印迹(western blotting,WB)实验检测细胞中YTHDF2、溶质载体家族7成员11(SLC7A11)、谷胱甘肽过氧化物酶4(GPX4)和铁蛋白重链(FTH1)的蛋白表达;双荧光素酶报告基因检测实验检测细胞的荧光活性。结果与正常肝细胞比较,肝细胞癌细胞(SMMC-7721、HepG2、Huh7、Hep3B)中miR-452-5p显著升高,YTHDF2显著降低(P<0.05);抑制miR-452-5p明显抑制HepG2细胞活性,促进凋亡,促进ROS、SLC7A11、GPX4和FTH1表达(P<0.05)。过表达YTHDF2具有与抑制miR-452-5p相类似的HepG2细胞调控作用。miR-452-5p显著抑制野生型YTHDF2细胞的荧光活性,并负向调控YTHDF2表达。敲减YTHDF2部分逆转抑制miR-452-5p对HepG2细胞凋亡、铁死亡的调控作用。结论miR-452-5p在肝细胞癌细胞中高表达,抑制miR-452-5p促进癌细胞凋亡、铁死亡,其机制与靶向YTHDF2有关。
Objective To investigate the effects of miR-452-5p and YTH domain family, member 2(YTHDF2) in regulating apoptosis and ferroptosis of hepatocellular carcinoma cell and its action mechanism.Methods Real time fluorescence quantitative polymerase chain reaction(RT-qPCR) was used to detect the expression levels of miR-452-5p and YTHDF2 in L02,SMMC-7721,HepG2,Huh7 and Hep3B cells;anti-miR-con group(transfected anti-miR-con),anti-miR-452-5p group(transfected anti-miR-452-5p),pcDNA group(transfected pcDNA),pcDNA-YTHDF2 group(transfected pcDNA-YTHDF2),anti-miR-452-5p+si-con group(co-transfected anti-miR-452-5p and si-con),anti-miR-452-5p+si-YTHDF2 group(co-transfection anti-miR-452-5p and si-YTHDF2),which were transfected into HepG2 cells by Liposome.And annexin V-FITC and propidium iodide(PI) double staining were used to detect the apoptosis rate;cell counting Kit(CCK8) was used to detect the cell activity;Western Blot was used to detect the protein expression levels of YTHDF2,solute vector family 7 member 11(SLC7A11),glutathione peroxidase 4(GPX4) and ferritin heavy chain(FTH1).Moreover the fluorescence activity of cells was detected by dual luciferase reporter gene detection.Results Compared with those in normal hepatocytes, the expression levels of miR-452-5p were significantly increased and YTHDF2 were significantly decreased in hepatoma cells(SMMC-7721,HepG2,Huh7,Hep3B).To inhibit the expression of miR-452-5p significantly inhibited the activity of HepG2 cells, promoted cell apoptosis, and promoted the expressions of ROS,SLC7A11,GPX4 and FTH1(P<0.05).And the overexpression of YTHDF2 had regulatory effects which were similar to those by the inhibition of miR-452-5p in HepG2 cells.The miR-452-5p inhibited significantly the fluorescence activity of wild-type YTHDF2 cells and negatively regulated the expression of YTHDF2. Moreover the knockdown of YTHDF2 partially reversed the regulatory effects of inhibition of miR-452-5p on the cell apoptosis and ferroptosis in HepG2 cells.Conclusion The expression levels of miR-452-
作者
王雪梅
WANG Xuemei(Zhangye People’s Hospital Affiliated to Hexi College,Gansu,Zhangye 734000,China)
出处
《河北医药》
CAS
2022年第23期3544-3548,3553,共6页
Hebei Medical Journal
基金
甘肃省卫生厅医药科研计划(编号:1292GS2016B14)。
