miRNA-19对葡萄膜黑色素瘤细胞增殖、凋亡、迁移和侵袭的影响及其机制研究

Effects of miRNA-19 on proliferation,apoptosis,migration and invasion of uveal melanoma cells and its mechanisms

目的探讨miR-19在葡萄膜黑色素瘤(UM)细胞中的表达及其对UM细胞增殖、凋亡、迁移和侵袭的影响及其作用机制。方法qRT-PCR检测正常视网膜上皮细胞系ARPE-19和UM细胞系SP6.5、M23中miR-19的表达。将M23细胞分为miR-19 inhibitor组(转染miR\|19\|inhibitor)及miR-19 NC组(转染scramble),MTT法检测各组细胞增殖,流式细胞术检测细胞凋亡,细胞划痕实验检测细胞迁移能力,Transwell实验检测细胞侵袭能力,Western blot检测表皮生长因子受体(EGFR)/丝氨酸\|苏氨酸激酶(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路蛋白表达。结果正常视网膜上皮细胞系ARPE-19中miR-19相对表达量为1.35±0.13,均显著低于UM细胞系SP6.5与M23中miR-19相对表达量8.35±0.73和9.35±0.43(均为P<0.01)。M23细胞转染后,miR-19 inhibitor组中miR-19表达量低于miR-19 NC组(1.05±0.33 vs 8.05±0.64,P<0.01)。MTT法检测结果显示,转染24 h,miR-19 inhibitor组与miR-19 NC组光密度值比较,差异无统计学意义(P>0.05);转染48 h、72 h、96 h,miR-19 inhibitor组光密度值均低于miR-19 NC组(均为P<0.05)。miR-19 inhibitor组细胞凋亡率高于miR-19 NC组[(15.34±2.35)%vs(8.23±0.72)%,P<0.05]。miR-19 inhibitor组细胞划痕愈合率低于miR-19 NC组[(23.7±2.1)%vs(68.9±5.1)%,P<0.05]。miR-19 inhibitor组侵袭细胞数少于miR-19 NC组[(45.1±3.9)个vs(115.3±8.9)个,P<0.05]。miR-19 inhibitor组UM细胞系M23 EGFR蛋白表达量低于miR-19 NC组(0.43±0.03 vs 1.02±0.02,P<0.05)。miR-19 inhibitor组AKT蛋白表达量低于miR-19 NC组(0.52±0.04 vs 1.12±0.05,P<0.05)。miR-19 inhibitor组mTOR蛋白表达量低于miR-19 NC组(0.63±0.05 vs 1.41±0.06,P<0.05)。结论敲低miR-19表达可抑制UM细胞增殖、迁移和侵袭能力并促进凋亡,其机制可能与EGFR/AKT/mTOR信号通路被抑制有关。

Objective To investigate the expression of miR-19 in uveal melanoma(UM)and its effects on cell proliferation,apoptosis,migration and invasion and its mechanisms.Methods Real-time quantitative polymerase chain reaction(qRT-PCR)was used to detect the expression of miR-19 in normal retinal pigment epithelial cell lines ARPE-19 and UM cell lines SP6.5 and M23.The M23 cell line was divided into miR-19 inhibitor group,in which the cells were transfected with miR-19 inhibitor,and miR-19 NC group,in which the cells were transfected with scramble.MTT assay was used to measure cell proliferation.Flow cytometry was used to measure apoptosis.Cell scratch test was used to measure cell migration ability.Transwell assay was used to measure cell invasion.Western blot was used to measure the expression of epithelial growth factor receptor(EGFR)/serine/threonineproteinkinase(AKT)/mammalian target of rapamycin(mTOR)signaling pathway protein.Results The expression level of miR-19 in normal uveal epithelial cell line ARPE-19 was 1.35±0.13,and 8.35±0.73 and 9.35±0.43 in uveal melanoma cell lines SP6.5 and M23,respectively.The expression level of miR-19 in SP6.5 and M23 was significantly higher than that in ARPE-19(both P<0.01).After transfection,the expression level of miR-19 in M23 cells of the miR-19 inhibitor group was lower than that in miR-19 NC group(1.05±0.33 vs 8.05±0.64,P<0.01).The results of MTT showed that after 24 hours of transfection,the optical density value was not statistically difference between miR-19 inhibitor group and miR-19 NC group(P>0.05).After transfection for 48 hours,72 hours,96 hours,the optical density value of miR-19 inhibitor group was significantly lower than miR-19 NC group(all P<0.05).The apoptosis rate of miR-19 inhibitor group was higher than that of miR-19 NC group[(15.34±2.35)%vs(8.23±0.72)%,P<0.05].The scratch healing rate in the miR-19 inhibitor group was lower than that in the miR-19 NC group[(23.7±2.1)%vs(68.9±5.1)%,P<0.05].The number of invasive cells in the miR-19 inhibitor group was less than that in the miR-19 NC group(45.1±3.9 vs 115.3±8.9,P<0.05).The expression levels of EGFR protein,AKT protein,and mTOR protein in the miR-19 inhibitor group were lower than those in the miR-19 NC group(0.43±0.03 vs 1.02±0.02,0.52±0.04 vs 1.12±0.05,and 0.63±0.05 vs 1.41±0.06,respectively,all P<0.05).Conclusion Knockdown of miR-19 expression can inhibit the proliferation,migration and invasion and promote apoptosis in UM cells.The mechanism may be related to the inhibition of EGFR/AKT/mTOR signaling pathway.