LncRNA HEIH通过miR-98-5p调节肺癌细胞增殖和凋亡

目的探讨lncRNA HEIH对肺癌细胞增殖和凋亡的影响及其作用机制。方法培养人正常肺上皮细胞BEAS-2B和肺癌细胞系A549、A427、H1299和TKB-1,RT-qPCR检测细胞中HEIH表达水平;分别转染si-HEIH和miR-98-5p mimics至A549细胞,沉默A549细胞中HEIH表达或过表达miR-98-5p;MTT法检测细胞增殖;流式细胞仪检测细胞凋亡;Western blot检测CCND1、caspase-3、SHH、GLI-1、PTCH和SUFU蛋白表达。双荧光素酶报告基因实验验证HEIH与miR-98-5p之间的关系。结果与正常肺上皮细胞BEAS-2B相比,肺癌细胞系A549、A427、H1299和TKB-1中HEIH表达水平显著升高(P<0.05).其中A549细胞中的HEIH表达最高。因此,后续实验选择A549细胞为研究对象。沉默HEIH表达或过表达miR-98-5p均可降低A549细胞培养12、48和72 h后吸光度值(A值)(P<0.05)(MTT法);升高凋亡率(P<0.05);抑制CCND1蛋白表达(P<0.05),促进caspase-3蛋白表达(P<0.05)。并且过表达miR-98-5p还抑制了A549细胞中SHH和GLI-1的mRNA和蛋白表达(P<0.05),促进了PTCH和SUFU的mRNA和蛋白表达水平(P<0.05)。过表达HEIH逆转了过表达miR-98-5p对A549细胞增殖、凋亡以及SHH、GLI-1、PTCH和SUFU的mRNA和蛋白表达的影响。结论沉默HEIH表达可能通过靶向miR-98-5p经Hedgehog信号通路抑制肺癌细胞的增殖,并促进其凋亡。

LncRNA HEIH regulates proliferation and apoptosis of lung cancer cells through miR-98-5p

Objective To investigate the effect of lncRNA HEIH on proliferation and apoptosis of lung cancer cells and its mechanism. Methods Human lung epithelial cells BEAS-2 B and lung cancer cell lines A549, A427, H1299 and TKB-1 were cultured, and the expression level of HEIH was detected by RT-qPCR. Si-HEIH and miR-98-5 p mimics were transfected into A549 cells inorder to silence the expression of HEIH in A549 cells or over-expressing miR-98-5 p. Then cell proliferation was detected by MTT assay, apoptosis was examined by flow cytometry, and the levels of CCND1, caspase-3, SHH, GLI-1, PTCH and SUFU proteins were measured by Western blot. The dual luciferase reporter gene assay verified the relationship between HEIH and miR-98-5 p. Results Compared with the normal lung epithelial cells BEAS-2 B, the levels of HEIH in lung cancer cell lines,A549, A427, H1299 andTKB-1 were significantly increased(P<0.05). Among them,the level of HEIH in lung cancer celllines A549 was the highest. Therefore, A549 cells were selected as the research object in the subsequent experiments. Silencing HEIH expression or over-expression of miR-98-5 p reduced the A value of A549 cells after 12, 48 or 72 hours(P<0.05)(MTT assay), increased apoptosis rates(P<0.05) and inhibited CCND1 protein expression(P<0.05), and promoted caspase-3 protein expression(P<0.05). Over-expression of miR-98-5 p also inhibited the mRNA and protein expression of SHH and GLI-1 in A549 cells(P<0.05), and promoted the mRNA and protein expression of PTCH and SUFU(P<0.05). Over-expression of HEIH reversed the effect of over-expression of miR-98-5 p on proliferation and apoptosis of A549 cells and mRNA and protein expression of SHH, GLI-1, PTCH and SUFU. Conclusions Silencing HEIH expression may inhibit the proliferation of lung cancer cells and promote apoptosis by targeting miR-98-5 p through Hedgehog signaling pathway.