shRNA沉默环指蛋白146基因对非小细胞肺癌细胞迁移、侵袭及其相关蛋白表达的影响

Interfering RNF146 by a short hairpin RNA inhibits the migration and invasiveness and related regulatory proteins expression of non- small cell lung cancer

目的:shRNA沉默环指蛋白146(RNF146)基因,观察非小细胞肺癌细胞中RNF146沉默对肺癌细胞迁移和侵袭及其相关蛋白表达的影响。方法:逆转录聚合酶链反应(RT-PCR)和Western blot方法用于筛选RNF146高表达肺癌细胞系。构建shRNA-RNF146真核表达载体,瞬时转染A549和H1299肺癌细胞,Western blot检测转染效率。划痕实验评价肺癌细胞体外迁移能力;Buyden小室实验检测肺癌细胞体外侵袭能力。Western blot检测肺癌细胞中迁移和侵袭相关蛋白的表达。结果:RT-PCR和Western blot方法筛选RNF146高表达肺癌细胞系,显示A549和H1299细胞中RNF146蛋白均高于其它细胞系。shRNA-RNF146转染A549和H1299细胞系后,划痕实验显示转染组细胞24小时迁移率明显低于空载体组和未转染组(P<0.01)。Buyden小室实验结果表明转染组比空载体组和未转染组穿膜细胞数目明显减少(P<0.05)。Western blot检测与细胞迁移和侵袭相关的调控蛋白(MMP2、MMP7、MMP9、RhoA、RhoB、RhoC、Rock1、TIMP-1等)的表达情况,结果显示干扰RNF146后A549细胞中MMP2和MMP7表达明显减少,而MMP9、RhoA、RhoB、RhoC、Rock1、TIMP-1等蛋白的表达变化不明显。结论:RNF146可能通过调控MMP2和MMP7的蛋白水平促进肺癌细胞迁移和侵袭。

Objective：To investigate the role of RNF146 in migration and invasion and related regulatory proteins expression after silencing by a short hairpin RNA targeting-RNF146 in non-small cell lung cancer（ NSCLC）cell line. Methods：RT-PCR and Western blot were used to screen NSCLC cell line with high RNF146 expression at mRNA and protein levels. shRNA-RNF146 eukaryotic expression vector was constructed and transient transfected in-to A549 and H1299 cells. The transfection effeciency of RNF146 was evaluated by western blot. The cell migratory and invasive ability were detected by Wound-healing assay and matrigel invasion assay using Buyden chamber. All exper-iments contained shRNA - RNF146 - transfected clones,vector transfected clones and non - transfected parental cells. Migration and invasion related proteins expression was measured by Western blot. Results：Effective downregu-lating of RNF146 expression in A549 and H1299 cells decreased the ability of these cells to migrate as measured by the wound-healing assay（P〈0. 01）. The number of cell passing through the matrigel and multipore membrane was also decreased in the RNF146-transfected cell compared with those of the vector-transfected clones and parental cells（P〈0. 05）. Western blot results revealed correlations between the levels of RNF146 in lung cancer cells and the expression of migration and invasion related regulatory proteins including MMP2 and MMP7. Conclusions：The regula-tion of migration and invasion of RNF146 may correlate with MMP2 and MMP7 proteins in lung cancer cell line.