LncRNA HOTTIP调节miR-615/IGF2分子轴促进肺癌细胞增殖和侵袭的实验研究

LncRNA HOTTIP promotes proliferation and metastasis of lung cancer cell via regulating miR-615/IGF2 axis

目的探讨长链非编码RNA同源异型基因簇A远端转录本(lncRNA HOTTIP)对肺癌细胞增殖、侵袭和迁移的影响及可能机制。方法体外培养A549细胞,构建HOTTIP敲降载体、miR-615 inhibitor转染A549细胞,分为空白对照组、阴性对照组、HOTTIP shRNA组、HOTTIP shRNA+无序序列组、HOTTIP shRNA+miR-615 inhibit表达ors组。采用MTT、Transwell、免疫荧光法检测下调HOTTIP表达对A549细胞增殖、侵袭和迁移的影响。采用双荧光素酶报告实验检测lncRNA HOTTIP、miR-615及IGF2的靶向调控关系。采用实时荧光定量PCR(qPCR)和Western blotting检测HOTTIP、miR-615和IGF的表达量。结果A549细胞中HOTTIP相对表达量为1.74±0.08,高于BEAS-2B细胞的1.00±0.05(P<0.001)。HOTTIP shRNA组,HOTTIP相对表达量为0.43±0.04,低于空白对照组和阴性对照组(P<0.05)。下调HOTTIP表达后,A549细胞增殖、侵袭和迁移活性低于空白对照组(P<0.05),细胞凋亡率相应增加(P<0.05)。双荧光素酶报告实验证实HOTTIP靶向作用miR-615,而miR-615可负调控IGF2的表达。HOTTIP shRNA组细胞miR-615表达量高于空白对照组,IGF2 mRNA表达量低于阴性对照组,而HOTTIP shRNA+miR-615 inhibitors组IGF2 mRNA和蛋白表达量较HOTTIP shRNA组升高(P<0.05)。结论敲除A549细胞中lncRNA HOTTIP表达后可通过上调miR-615并下调IGF2进而抑制肺癌细胞的增殖、迁移和侵袭活性。

Objective To discuss the effect of long non-coding RNA homeobox gene atranscript at the distal tip(lncRNA HOTTIP)on proliferation,invasion and migration of lung cancer cell and it s possible mechanism.Methods The lung cancer cell line A549 was cultured in vitro for the study,and HOTTIP-knockdown vectors and miR-615 inhibitors vectors were constructed and used to transfect A549 cells,respectively,which were divided into CTL group,negative control(NC)group,HOTTIP shRNA group,HOTTIP shRNA+vector group and HOTTIP shRNA+miR-615 inhibitors group.MTT,transwell assay,immunofluorescence assays and flow cytometry were used to detect the proliferation,invasion,migration and apoptosis.The interaction between HOTTIP,miR-615 and IGF2 were verified by dual luciferase reporter gene assay.Quantitative real time polymerase chain reaction(qPCR)and western blot were used to detect miR-615 and IGF2 expressions.Results LncRNA HOTTIP expression level in A549 cells was 1.74±0.08,that was higher than BEAS-2B cells(P<0.001).The expression of HOTTIP in HOTTIP shRNA group was 0.43±0.04,which was lower than CTL group and NC group(P<0.05).After silencing lncRNA HOTTIP,the proliferation,invasion and migration activities of A549 cells in HOTTIP shRNA group declined while the apoptosis rate increased,compared with CTL group,there was significant difference(P<0.05).Moreover,dual-luciferase reporter gene assay showed that HOTTIP directly interacted with miR-615 and down-regulated its expression,while miR-615 negatively regulated IGF2 mRNA expression.qPCR and Western blotting results showed that miR-615 expression level in HOTTIP shRNA group was higher than that in CTL group,while IGF2 expression level was up-regulated(P<0.05).Besides,IGF2 in HOTTIP shRNA+miR-615 inhibitor group was higher than that in HOTTIP shRNA group(P<0.05).Conclusion LncRNA HOTTIP gene silencing can inhibit the proliferation and migration activities and enhance the apoptosis activity of A549 cells by regulating miR-615/IGF2 axis.