鸦胆亭上调miR-29a-3p抑制非小细胞肺癌H1299细胞增殖、迁移与侵袭

Bruceantin inhibits proliferation,migration and invasion of nonsmall cell lung cancer H1299 cells by up-regulating miR-29a-3p

本研究主要探讨鸦胆亭(bruceantin,BCT)对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞的增殖、侵袭与迁移的抑制作用及其机制。采用MTT法检测BCT对NSCLC细胞株的细胞毒作用;采用集落形成、划痕和Transwell实验分别检测细胞的增殖、迁移与侵袭能力;Western blot及RT-qPCR实验分别检测与细胞增殖、迁移与侵袭等的相关蛋白及miRNA、mRNA的表达情况;利用基因预测网站预测miRNA的下游靶基因。结果显示,BCT对NSCLC细胞株有强大的细胞毒作用,对H1299、PC-9和A549细胞的半数抑制浓度(IC50)分别为0.12±0.02、0.31±0.20和2.07±0.70μmol·L^(-1)。当0、0.03、0.15和0.75μmol·L^(-1)BCT分别作用H1299细胞24 h后,其增殖、迁移与侵袭能力呈现浓度依赖性地抑制。值得关注的是,多种与细胞迁移与侵袭相关的miRNA,如miR-29a-3p、miR-21-3p、miR-183-5p与miR-34b-5p的表达水平随着BCT给药浓度的增加而增加,尤其对miR-29a-3p的作用最为明显;通过基因预测网站预测整合素β1(integrinβ1,ITGB1)可能为miR-29a-3p的靶基因;Western blot结果进一步显示,多种与细胞增殖、迁移与侵袭相关的蛋白,如整合素家族多种蛋白以及下游β-catenin、p-Src和血管内皮生长因子(vascular endothelial growth factor,VEGF)蛋白的表达均呈浓度依赖性减低,其中ITGB1蛋白降低最明显;而RT-qPCR结果显示ITGB1的mRNA表达与给药浓度无关。由此推测,BCT有可能是通过上调miR-29a-3p抑制ITGB1蛋白的表达,且与其mRNA水平无关,深入的机制仍需进一步探讨。本研究初步提示BCT在NSCLC的治疗中具有继续开发的研究潜力。

We investigated the inhibitory effect and mechanism of action of bruceantin(BCT)on the proliferation,invasion and migration of non-small cell lung cancer(NSCLC)cells.The cytotoxic activity of BCT was measured by MTT assay;a colony forming assay,wound healing assay,and a Transwell assay were used to investigate the anti-proliferative,anti-migration,and anti-invasion effects,respectively;immunoblotting and RTqPCR were used to detect the expression of related proteins,miRNA,and mRNA,respectively,that were involved in cell proliferation,migration,and invasion.Two gene prediction websites were used to predict the downstream target gene of miRNA.Our results show that BCT has a potent cytotoxic effect on NSCLC cell lines,with a half maximal inhibitory concentration(IC50)of BCT against H1299,PC-9,and A549 of 0.12±0.02,0.31±0.20,and2.07±0.70μmol·L^(-1),respectively.When H1299 cells were treated with 0.03,0.15,and 0.75μmol·L^(-1) BCT for24 h,the proliferation,migration,and invasive ability were inhibited in a concentration-dependent manner.It is worth noting that the expression level of miRNAs related to cell migration and invasion,such as miR-29 a-3 p,miR-21-3 p,miR-183-5 p,and miR-34 b-5 p increased with the concentration of BCT,especially for miR-29 a-3 p.Using the two gene prediction websites,we predict that integrinβ1(ITGB1)may be the target gene of miR-29 a-3 p;immunoblot results further show that a variety of proteins related to cell proliferation,migration,and invasion,such as various proteins of the integrin family,β-catenin,p-Src,and vascular endothelial growth factor,all decreased in a concentration-dependent manner,among which the reduction of ITGB1 protein was the most obvious.RTqPCR results showed that there was no change in ITGB1 mRNA expression.We speculate that BCT might inhibit the expression of ITGB1 protein by up-regulating miR-29 a-3 p independent of its mRNA level.The in-depth mechanism needs to be further explored.This study suggests that BCT has the potential for further development in the treatment of NSCLC.