miR-132调节非小细胞肺癌hedgehog信号通路受体基因PTCH1及细胞增殖、迁移和侵袭

 目的  研究miR-132在非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞系H1299中的作用及相应的靶基因。方法  real-time PCR检测7种细胞系中miR-132的表达量。用CCK-8检测稳定株细胞H1299-pEGP-miR-132与H1299-pEGP-miR-null 的生长速率的变化。使用稳定株细胞H1299检测miR 132对于细胞克隆形成、细胞迁移和细胞侵袭的影响。用Transwell结合matrigel的方法检测H1299-pEGP-miR-132细胞与H1299-pEGP-miR-null细胞在72 h时细胞迁移和侵袭的变化,同时在H1299-pEGP-miR-132细胞中使用anti-miR-132进行了相关的实验验证。运用生物信息学方法预测miR-132的靶基因,并运用双荧光实验、real-time PCR和Western blot验证。在H1299-pEGP-miR-132细胞中共同转染anti-miR-132和PTCH1 siRNA检测其对细胞的增殖、迁移和侵袭的影响。结果   miR-132在肺癌细胞系中呈现高表达,在NSCLC细胞H1299中miR-132通过靶向调节PTCH1基因的表达发挥作用。H1299-pEGP-miR-132细胞的增殖(P<0.01)、迁移(P<0.05)、侵袭(P<0.05)能力明显高于对照组。在H1299-pEGP-miR-132细胞中转入PTCH1 siRNA后细胞增殖能力升高(P<0.05),迁移和侵袭能力降低(P<0.001)。H1299-pEGP-miR-132细胞15天形成的克隆数目是对照组细胞的1.41倍(P<0.05)。Western blot法检测显示miR-132使H1299中PTCH1蛋白表达量显著降低(0.68倍),anti-miR-132使PTCH1表达量显著升高(1.97倍)。结论  miR-132可能参与NSCLC的细胞增殖、迁移和侵袭。它的致癌性部分归因于对Hh信号通路关键的负调控蛋白PTCH1的调节。

miR-132 regulates the hedgehog signaling pathway receptor PTCH1 and cell proliferation,migration and invasion in non-small cell lung cancer cells

Objective  To study the role of miR-132 in non-small cell lung cancer (NSCLC) cells H1299 and its target gene.Methods  We detected the expression of miR-132 in 7 cell lines by real-time PCR.We explored the cell growth rate of H1299 cells stably expressing miR-132 (H1299-pEGP-miR-132) and H1299 cells stably expressing miR-null (H1299-pEGP-miR-null) using Cell Counting Kit-8.We carried out experiments to assess the effect of miR-132 on colony formation in H1299-pEGP-miR-132 cells compared with H1299-pEGP-miR-null cells. We studied the influence of miR-132 on cell
 migration and cell invasion of H1299-pEGP-miR-132 compared with H1299-pEGP-miR-null using Transwell and Matrigel after 72 h since transfection.Besides, we also confirmed the results in H1299-pEGP-miR-132 cells transfected with anti-miR-132 or anti-miR-NC.We predicted the target gene of miR-132 in H1299 cells using biology information method and confirmed it by Dual luciferase assay, real time PCR and Western blot.We investigated cell proliferation, cell migration and cell invasion of H1299 pEGP-miR-132 cells co-transfected with anti-miR-132 and PTCH1 siRNA.Results     Relative expression level of miR-132 in NSCLC cell lines was significantly increased.miR-132 plays a role in NSCLC cell H1299 by targeting PTCH1.Overexpressing miR-132 promoted cell proliferation (P<0.01), cell migration (P<0.05) and cell invasion (P<0.05) in H1299 cells.Blocking PTCH1 expression by siRNA enhanced cell proliferation (P<0.05), but suppressed cell migration (P<0.001) and invasion (P<0.001) in H1299 cells.Overexpressing miR-132 improved anchorage-independent growth up to 1.41-fold (P<0.05) on day 15 in H1299 cells.Western blot revealed that the expression of PTCH1 protein was down-regulated to 0.68-fold by miR-132 and was up-regulated to 1.97-fold by anti-miR-132 in H1299 cells.Conclusions  miR-132 may involve in NSCLC cell proliferation, migration and invasion, and its oncogenic activity was partially due to the regulation of the key negative regulatory protein PTCH1 of Hh signaling pathway.