摘要
目的探讨miRNA-26b(miR-26b)在肝癌细胞自噬调控中的作用及可能分子机制。方法采用荧光定量PCR(QPCR)检测人正常肝细胞系HL-7702和人肝癌细胞系Hep G2、Hep-3B、Bel-7402及SSMC-7721中miRNA-26b的表达情况。双荧光素酶基因报告实验评价miR-26b对IL-6的靶向调控作用。向Hep G2细胞转染miR-26b模拟物mimics(转染组)和阴性对照NC(阴性对照组),Western blotting检测两组IL-6/STAT3信号通路以及自噬相关蛋白的表达。向Hep G2细胞转染IL-6 siRNA1和IL-6 siRNA2,Western blotting检测干扰IL-6后STAT3激活及自噬相关蛋白表达。结果 Hep G2、Hep-3B、Bel-7402细胞系中miR-26b表达水平分别为0.34±0.063、0.69±0.092、0.57±0.086,显著低于正常肝细胞系HL-7702的1.15±0.189,差异具有统计学意义(P<0.05)。miR-26b可抑制野生型IL-6 3’UTR报告基因载体的荧光素酶活性,而对突变型IL-63’UTR-MUT的荧光素酶活性无影响。转染miR-26b mimics后IL-6、p-STAT3相对表达量显著降低(P<0.05),诱导自噬相关蛋白Beclin1相对表达量和LC3Ⅱ/LC3Ⅰ比值显著升高(P<0.05)。沉默IL-6导致p-STAT3相对表达量显著降低(P<0.05),诱导自噬相关蛋白Beclin1相对表达量和LC3Ⅱ/LC3Ⅰ比值显著升高(P<0.05)。结论 miRNA-26b靶向抑制IL-6/STAT3信号通路的激活,进而诱导肝癌细胞自噬发生。
Objective To investigate the role and possible molecular mechanism of miRNA-26b(miR-26b)in the regulation of autophagy in hepatocellular carcinoma cells.Methods The expression of miRNA-26b in human normal liver cell line HL-7702 and human hepatoma cell lines HepG2,Hep-3B,Bel-7402 and SSMC-7721 was detected by fluorescence quantitative PCR(QPCR).Dual luciferase reporter assay was used to evaluate the regulatory effect of miR-26b on IL-6.MiR-26b mimics(transfected group)and negative control NC(negative control group)were transfected into HepG2 cells,and Western blotting was used to detect the expression of IL-6/STAT3 signaling pathway and autophagy related protein in two groups.IL-6 siRNA1 and IL-6 siRNA2 were transfected into HepG2 cells and Western blotting was used to detect STAT3 activation and autophagy related protein expression after interfering IL-6.Results The expression levels of miR-26b in HepG2,Hep-3B and Bel-7402 cell lines were 0.34±0.063,0.69±0.092,0.57±0.086,which was significantly lower than that of normal liver cell line HL-7702(1.15±0.189),and the difference was statistically significant(P<0.05).MiR-26b inhibited the luciferase activity of wild-type IL-6 3’UTR reporter gene vector,but had no effect on the luciferase activity of mutant IL-6 3’UTR-MUT.After transfection of miR-26b mimics,the relative expression of IL-6 and p-STAT3 decreased significantly(P<0.05),and the relative expression of autophagy related protein Beclin1 and the ratio of LC3 II/LC3 I increased significantly(P<0.05).After the silence IL-6 expression,the relative expression of p-STAT3 decreased significantly(P<0.05),and the relative expression of autophagy related protein Beclin1 and the ratio of LC3 II/LC3 I increased significantly(P<0.05).Conclusion MiRNA-26b can inhibit the activation of IL-6/STAT3 signaling pathway and induce autophagy in hepatoma cells.
作者
夏婷
胡衡
朱群安
熊勰
XIA Ting;HU Heng;ZHU Qunan;XIONG Xie(Department of Oncology,No.94 Hospital of PLA,Nanchang 331100,China)
出处
《临床肿瘤学杂志》
CAS
北大核心
2018年第7期593-597,共5页
Chinese Clinical Oncology
