微 RNA-130a靶向人类 RUNT相关转录因子 3对肺癌 A549细胞增殖和转移的影响

目的 探讨微RNA(miR)-130a与人类RUNT相关转录因子3(RUNX3)的靶向关系及其对非小细胞肺癌A549细胞增殖、侵袭、迁移和上皮间质转化的影响.方法 将体外培养的A549细胞分为对照组(未处理)、anti-miR-NC组(转染miR-130a inhibi?tor阴性对照)、anti-miR-130a组(转染miR-130a inhibitor)、anti-miR-130a+siNC组(转染miR-130a inhibitor和NC-siRNA)和anti-miR-130a+siRUNX3组(转染miR-130a inhibitor和RUNX3-siRNA),采用实时定量PCR检测各组细胞中miR-130a和RUNX3mRNA的表达水平,蛋白质印迹法(Western Blot)检测各组细胞中RUNX3蛋白和上皮型钙黏蛋白(E-cadherin)、神经型钙黏蛋白(N-cadherin)和波形蛋白(Vimentin)的表达水平,四甲基偶氮唑盐微量酶反应比色法(MTT法)检测各组细胞增殖能力,克隆形成实验检测各组细胞的克隆形成能力,Transwell小室实验检测各组细胞侵袭与迁移能力,荧光素酶实验检测miR-130a和RUNX3的靶向关系.结果 荧光素酶实验证实,RUNX3是miR-130a的潜在靶基因.与anti-miR-NC组相比,下调miR-130a可抑制A549细胞增殖(94.72±6.05)％比(51.28±3.24)％]、克隆(36.15±2.06)％比(15.46±1.25)％]、侵袭(90.25±5.48)个比(42.18±3.26)个]、迁移(118.55±9.86)个比(73.48±6.75)个](P<0.05),促进E-cadherin蛋白表达(0.26±0.03)比(0.48±0.04)](P<0.05),而抑制N-cadherin、Vimentin蛋白表达(0.52±0.03)比(0.25±0.02);(0.68±0.05)比(0.35±0.03)](P<0.05);与anti-miR-130a+siNC组相比,共转染anti-miR-130a与siRUNX3可成功增强A549细胞增殖(52.16±3.12)％比(72.23±6.13)％]、克隆(16.08±1.05)％比(22.12±1.34)％]、侵袭(41.59±3.02)个比(68.32±4.26)个]、迁移能力(72.26±5.18)个比(98.25±6.02)个](P<0.05),抑制E-cad?herin蛋白表达(0.52±0.04)比(0.33±0.03)](P<0.05),而促进N-cadherin、Vimentin蛋白表达(0.27±0.03)比(0.46±0.03);(0.33±0.02)比(0.53±0.04)](P<0.05).结论 miR-130a可通过靶向RUNX3抑制A549细胞增殖、侵袭、迁移和上皮间质转移.

Effect of miR-130a targeting RUNX3 on proliferation and metastasis of lung cancer A549 cells

Objective To investigate the targeting relationship between miR-130a and RUNX3 and its effects on proliferation, invasion, migration and epithelial-mesenchymal transition of lung cancer A549 cells.Methods A549 cells cultured in vitro were assigned into control group (untreated), anti-miR-NC group (transfected negative control of transfected miR-130a inhibitor), anti-miR-130a (transfected miR-130a inhibitor), anti-miR-130a+siNC (transfected miR-130a inhibitor and NC-siNC) and anti-miR-130a + siRUNX3 groups (transfected miR-130a inhibitor and RUNX3-siNC). The expression of miR-130a and RUNX3 mRNA in each group were detected by real-time quantitative PCR. Western Blot was used to detect the expression levels of RUNX3 protein and epithelial cadherin (Ecadherin), neural cadherin (N-cadherin) and vimentin (Vimentin) proteins in each group of cells. Cell proliferation abilities of eachgroup were checked by MTT method, the clone formation abilities of each group of cells were detected by clone formation, invasion andmigration abilities of each group were checked by Transwell lab assay. The targeting relationship between miR-130a and RUNX3 was detected by luciferase assay.Results Luciferase assay confirmed that RUNX3 was a potential target gene of miR-130a. Compared with the anti-miR-NC group, downregulation of miR-130a could inhibit the proliferation of A549 cells (94.72±6.05)% vs. (51.28± 3.24)%], clone (36.15±2.06)% vs. (15.46±1.25)% ], invasion (90.25±5.48) vs. (42.18±3.26)], migration (118.55±9.86) vs. (73.48± 6.75)] (P<0.05), promote the protein expression of E-cadherin (0.26±0.03) vs. (0.48±0.04)] (P<0.05), while inhibit the expression of Ncadherin and Vimentin (0.52±0.03) vs. (0.25±0.02); (0.68±0.05) vs. (0.35±0.03)] (P<0.05). Compared with the anti-miR-130a+siNC group, co-transfection of anti-miR-130a and siRUNX3 could successfully enhance the proliferation of A549 cells (52.16±3.12)% vs. (72.23±6.13)%], clone (16.08±1.05) )% vs. (22.12±1.34)%], invasion (41.59±3.02) vs. (68.32±4.26)], migration ability (72.26±5.18) vs. (98.25±6.02)] (P<0.05), inhibit the protein expression of E-cadherin (0.52±0.04) vs. (0.33±0.03)] (P<0.05), and promote the protein expression of N-cadherin, Vimentin (0.27±0.03) vs. (0.46±0.03); (0.33±0.02) vs. (0.53±0.04)] (P<0.05).Conclusion miR-130a can inhibit proliferation, invasion, migration and epithelial-mesenchymal metastasis of A549 cells by targeting RUNX3.