miR-194在葡萄膜黑色素瘤细胞系中的表达及对细胞增殖、凋亡、迁移和侵袭的影响

Expression of miR-194 and its effects on tumor cell proliferation,apoptosis,migration and invasion of uveal melanoma cells

目的探讨miR-194在葡萄膜黑色素瘤细胞系中的表达及对细胞增殖、凋亡、迁移和侵袭的影响。方法RT-PCR检测miR-194在正常视网膜上皮细胞系ARPE-19及葡萄膜黑色素瘤细胞系SP6. 5、M23及OM431中的表达。取SP6. 5、M23及OM431细胞分成两组,miR-194过表达组(miR-194组)及阴性对照组(NC组),经Lipofectamine^(TM) 2000分别转染miR-194 mimic及阴性对照序列scramble。CCK-8实验及流式细胞仪分别测定细胞增殖和凋亡能力,细胞划痕及Transwell实验分别测定细胞迁移和侵袭能力。Western blot测定叉头盒蛋白A1 (forkhead box,FOXOA1)、多聚ADP-核糖聚合酶(poly ADP-ribose polymerase,PARP)蛋白表达。结果正常视网膜上皮细胞系ARPE49中miR-194的相对表达量为1.03±0.04,葡萄膜黑色素瘤细胞系SP6.5、M23及OM431相对表达量分别为0.25±0.02、0.37±0.02及0.47±0.03,葡萄膜黑色素瘤细胞系SP6.5、M23及OM431中miR-194相对表达量低于正常视网膜上皮细胞系ARPE-19(均为P<0.001)。转染0 h、24 h、48 h、72 h、96 h后,miR-194组和NC组A值分别为:0.23±0.02、0.21±0.03(t=0.960,P=0.391),0.38±0.02、0.37±0.03(t=0.480,P=0.656),0.75±0.04、0.78±0.06(t=-0.720,P=0. 511),0.87±0. 09、1.59±0. 12 (t=-8. 313,P=0.001),1.53±0. 14、3. 05±0. 22 (t=-10. 096,P=0.000);流式细胞仪检测结果显示:miR494组细胞凋亡率为23. 30%±2. 10%,NC组为2. 47%±0.30%,miR-194组细胞凋亡率高于NC组(t=17.007,P=0.000)。细胞划痕实验结果显示:miR-194组细胞划痕愈合率为25.3%±3.5%,NC组为72.9%±6.4%,miR-194组细胞划痕愈合率低于NC组(t=-11.302,P=0.000);Transwell实验结果显示:200倍视野下,miR-194组侵袭细胞数为(188.6±20.7)个,NC组为(352.9±32.8)个,miR-194组侵袭细胞数少于NC组(t=-7.337,P=0.001)。miR-194组FOX A1蛋白相对表达量为0.29±0.03,NC组为1.03±0.06,miR-194组FOX A1蛋白相对表达量低于NC组(t=-19.106,P=0.000);miR-194组裂解型PARP蛋白相对表达量为2.87±0.24,NC组为1.03±0.06,miR-194组裂解型PARP蛋白相对表达量高于NC组(t=12.882,P=0.000)。结论miR-194在葡萄膜黑色素瘤细胞系中低表达,上调miR-194表达可抑制葡萄膜黑色素瘤细胞增殖,促进凋亡,抑制迁移和侵袭,其机制可能与下调FOX A1及上调裂解型PARP蛋白表达有关。

Objective To explore the expression of miR-194 and its effect on cell proliferation,apoptosis,migration and invasion of uveal melanoma cells.Methods Real time quantitative PCR(qRT-PCR)was carried out to examine miR-194 expression in normal cell line,ARPE-19,and human uveal melanoma cell lines,SP6.5,M23,and OM431.SP6.5 cell line was divided into two groups,miR-194 over-expression group(miR-194 group)and negative control group(NC group),which was transfect with miR-194 mimics and scramble by LipofectamineTM 2000,respectively.CCK-8 and flow cytometry assay were used to detect the proliferation and apoptosis ability,respectively.Scratch test and transwell assay were performed to detect the migration and invasion ability,respectively.Western blot was used to measure the expression of forkhead box A1(FOXA1)and cleavage of poly(ADP-ribose)polymerase(PARP)protein.Results The expression level of miR-194 in ARPE-19 cell line was 1.03±0.04,while the uveal melanoma cell lines,SP6.5,M23 and OM431,was 0.25±0.02,0.37±0.02 and 0.47±0.03,respectively.The expression level of miR-194 in SP6.5,M23 and OM431 was significantly lower than that in ARPE-19(all P<0.05).After transfect for 0 h,24 h,48 h,72 h and 96 h,the A value of miR-194 group and NC group was 0.23±0.02 vs.0.21±0.03(t=0.960,P=0.391),0.38±0.02 vs.0.37±0.03(t=0.480,P=0.656),0.75±0.04 vs.0.78±0.06(t=-0.720,P=0.511),0.87±0.09 vs.1.59±0.12(t=-8.313,P=0.001),1.53±0.14 vs.3.05±0.22(t=-10.096,P=0.000),respectively.Flow cytometry assay showed that the apoptosis rate of miR-194 group was 23.3%±2.1%,while 2.47%±0.30%in the NC group.The apoptosis rate of miR-194 group was significantly higher than that in NC group(t=17.007,P=0.000).Scratch test showed that the wound healing rate of miR-194 group was 25.3%±3.5%,while 72.9%±6.4%in the NC group.The wound healing rate of miR-194 group was significantly less than that in the NC group(t=-11.302,P=0.000).Transwell assay showed that the invasive cell number of miR-194 group was 188.6±20.7,while 352.9±32.8 in the NC group under the field of vision with 200 times.The invasive cell number of miR-194 group was significantly less than that in the NC group(t=-7.337,P=0.001).The expression level of FOX A1 protein in miR-194 group was 0.29±0.03,while 1.03±0.06 in the NC group.The expression level of FOX A1 protein in miR-194 group was significantly lower than that in the NC group(t=-19.106,P=0.000).The expression level of PARP protein in miR-194 group was 2.87±0.24,while 1.03±0.06 in the NC group.The expression level of PARP protein in miR-194 group was significantly higher than that in the NC group(t=12.882,P=0.000).Conclusion MiR-194 is down-regulated in uveal melanoma cell line.Over-expression of miR-194 can inhibit the proliferation,migration and invasion,as well as promote apoptosis of uveal melanoma cell line,of which the mechanisms may be associated with up-regulation of FOX A1 and down-regulation of PARP.