MicroRNA-27a-3p靶向调控SLC7A11抑制结直肠癌细胞铁死亡

MicroRNA-27a-3p inhibits colorectal cancer cell ferroptosis-induced by Erastin through targeting on SLC7A11

目的:探究微小RNA-27a-3p(microRNA-27a-3p,miR-27a-3p)对结直肠癌细胞系HT-29和SW480铁死亡的调控及作用机制。方法:分别用0、2.5和5μmol/L Erastin处理结直肠癌细胞系HT-29和SW480,采用实时荧光定量PCR(qRT-PCR)法检测各组细胞溶质载体家族7成员11(solute carrier family 7 member 11,SLC7A11)mRNA表达水平,筛选Erastin的合适浓度;将HT-29和SW480细胞各分为siControl组、siSLC7A11-1组和siSLC7A11-2组,分别转染对照siRNA、siSLC7A11-1和siSLC7A11-2,与5μmol/L Erastin共培养0、24、48、72 h,检测不同时间点细胞增殖活性,并检测细胞内丙二醛水平。另将HT-29和SW480细胞各分为miR-NC组、miR-27a-3p组和anti-miR-27a-3p组,分别转染miR-NC、miR-27a-3模拟物和miR-27a-3p抑制物,采用qRT-PCR法检测SLC7A11 mRNA表达水平,采用荧光素酶报告基因实验检测SLC7A113′UTR序列(SLC7A11-WT)及突变序列(SLC7A11-Mut)荧光素酶活力;与5μmol/L Erastin共培养0、24、48、72 h,检测不同时间点细胞增殖活性,并检测细胞内丙二醛含量。结果:随Erastin浓度升高,结直肠癌HT-29和SW480细胞SLC7A11 mRNA表达水平显著升高(P均<0.01);在结直肠癌HT-29和SW480细胞中,与siControl组相比,siSLC7A11-1组和siSLC7A11-2组48 h和72 h细胞增殖活性显著增强(P<0.05),丙二醛含量显著下降(P<0.05);与miR-NC组相比,miR-27a-3p组细胞增殖活性显著增强(P<0.05),丙二醛水平和SLC7A11-WT荧光素酶活性显著下降(P<0.05或P<0.01),SLC7A11-Mut荧光素酶活性没有显著变化(P>0.05),anti-miR-27a-3p组细胞活力显著下降(P<0.01),丙二醛含量和SLC7A11-WT荧光素酶活性显著升高(P均<0.01),SLC7A11-Mut荧光素酶活性没有显著变化(P>0.05)。结论:miR-27a-3p可能通过靶向SLC7A11 mRNA 3′UTR,调控Erastin诱导的结直肠癌细胞铁死亡。

Objective:To explore the regulation and mechanism of microRNA-27a-3p(miR-27a-3p)on ferroptosis of colorectal cancer cell line HT-29 and SW480.Methods:Colorectal cancer cell lines HT-29 and SW480 were treated with 0,2.5 and 5μmol/L Erastin,respectively.Real-time fluorescence quantitative PCR(qRT-PCR)was used to detect the mRNA expression of solute carrier family 7 member 11(SLC7A11)in each group and the optimal concentration of Erastin was screened.HT-29 and SW480 cells were divided into siControl group,siSLC7A11-1 group and siSLC7A11-2 group,and transfected with control siRNA,siSLC7A11-1 and siSLC7A11-2,respectively.The cells were co-cultured with 5μmol/L Erastin for 0,24,48 and 72 h to detect the cell proliferation activity and the level of malondialdehyde in the cells at different time points.HT-29 and SW480 cells were divided into miR-NC group,miR-27a-3p group and anti-miR-27a-3p group,respectively,and transfected with miR-NC,miR-27a-3 mimics and miR-27a-3p inhibitors,respectively.The mRNA expression level of SLC7A11 was detected by qRT-PCR,and the luciferase reporter gene assay was used to detect the luciferase activity of SLC7A113′UTR sequence(SLC7A11-WT)and mutant sequence(SLC7A11-Mut).The cells were co-cultured with 5μmol/L Erastin for 0,24,48 and 72 h,and the cell proliferation activity was detected at different time points,and the intracellular malondialdehyde level was detected.Results:The mRNA expression of SLC7A11 in colorectal cancer HT-29 and SW480 cells was significantly increased with the increase of Erastin concentration(P<0.01).In colorectal cancer HT-29 and SW480 cells,compared with the siControl group,the cell proliferation activity in siSLC7A11-1 and siSLC7A11-2 groups was significantly increased at 48 h and 72 h(P<0.05),with a significant decrease in malondialdehyde levels(P<0.05).Compared with the miR-NC group,cell proliferation activity in miR-27a-3p group was significantly enhanced(P<0.05),while malondialdehyde levels and luciferase activity of SLC7A11-WT group were significantly decreased(P<0.05 or P<0.01),and luciferase activity of SLC7A11-Mut group showed no significant changes(P>0.05);and the cell viability of anti-miR-27a-3p group was significantly decreased(P<0.01),the level of malondialdehyde and the luciferase activity of SLC7A11-WT were significantly increased(P<0.01),and the luciferase activity of SLC7A11-Mut was not significantly changed(P>0.05).Conclusion:miR-27a-3p may regulate Erastin-induced ferroptosis in colorectal cancer cells by targeting the 3′UTR of SLC7A11 mRNA.