摘要目的:初步探究微小RNA-203（miR-203）调控锌指蛋白281（ZNF281）对黑素瘤细胞增殖、迁移的影响。方法:常规培养人血管内皮细胞系ECV304、人黑素瘤细胞系A375、M14、SK-MEL-28、SK-MEL-2，实时荧光定量PCR检测各细胞系中miR-203表达情况，Western免疫印迹法检测各细胞系中ZNF281蛋白水平。A375、M14细胞分别分为5组：对照组（细胞正常培养）、miR-203模拟物对照组（加入miR-203模拟物阴性对照）、miR-203抑制剂对照组（加入miR-203抑制剂阴性对照）、miR-203模拟物组（加入miR-203模拟物）、miR-203抑制剂组（加入miR-203抑制剂）。实时荧光定量PCR检测各组A375、M14细胞中miR-203的表达，CCK8法检测各组A375、M14细胞增殖活性，Transwell实验检测各组A375、M14细胞迁移数量，Western免疫印迹法检测各组A375、M14细胞中ZNF281蛋白的表达。双荧光素酶验证miR-203与ZNF281的靶向关系。多组比较采用单因素方差分析，两两比较采用SNK- q法。 结果:与血管内皮细胞系ECV304相比，黑素瘤细胞系A375、M14、SK-MEL-28、SK-MEL-2中miR-203表达水平较低（均 P < 0.05），ZNF281蛋白水平较高（均 P < 0.05）。与对照组、miR-203模拟物对照组、miR-203抑制剂对照组相比，miR-203模拟物组A375、M14细胞中miR-203水平均显著升高（ F = 487.632、68.454，均 P < 0.05），细胞迁移数量均显著降低（均 P < 0.05），ZNF281蛋白表达均显著降低（均 P < 0.05）；miR-203抑制剂组A375、M14细胞中miR-203表达均显著降低（均 P < 0.05），细胞迁移数量均显著增加（均 P < 0.05），A375细胞中ZNF281蛋白表达显著升高（均 P < 0.05），36、48、60、72 h A375细胞增殖活性显著升高（均 P < 0.05），24、36、48、60、72 h M14细胞增殖活性均显著升高（均 P < 0.05）。miR-203与ZNF281之间存在靶位点。 结论:上调miR-203表达可抑制黑素瘤细胞增殖、迁移，下调miR-203表达可促进黑素瘤细胞增殖、迁移，可能通过调控ZNF281的表达实现。

abstractsObjective:To evaluate the effect of regulation of zinc finger protein 281 (ZNF281) expression by miR-203 on the proliferation and migration of melanoma cell lines.Methods:The human vascular endothelial cell line ECV304, as well as human melanoma cell lines A375, M14, SK-MEL-28 and SK-MEL-2 were subjected to conventional culture. Real-time fluorescence-based quantitative PCR was performed to measure the miR-203 expression, and Western blot analysis to determine the ZNF281 protein expression in the above cell lines. Both A375 and M14 cells were divided into 5 groups: control group (normally cultured) , miR-203 mimic control group transfected with the miR-203 mimic negative control, miR-203 inhibitor control group transfected with the miR-203 inhibitor negative control, miR-203 mimic group transfected with a miR-203 mimic, miR-203 inhibitor group transfected with a miR-203 inhibitor. Real-time fluorescence-based quantitative PCR was conducted to measure intracellular miR-203 expression, cell counting kit (CCK) -8 assay to assess cellular proliferation activity, Transwell assay to determine the number of migratory cells, and Western blot analysis to measure the protein expression of ZNF281 in A375 and M14 cells in the above groups. Dual-luciferase reporter assay was conducted to verify the targeting relationship between miR-203 and ZNF281. One-way analysis of variance was used for comparison of multiple groups, and the Student-Newman-Keuls- q (SNK- q) test for multiple comparisons. Results:Lower miR-203 expression and higher ZNF281 protein expression were observed in the melanoma cell lines A375, M14, SK-MEL-28, SK-MEL-2 compared with the vascular endothelial cell line ECV304 (all P < 0.05) . Compared with the control group, miR-203 mimic control group and miR-203 inhibitor control group, the miR-203 mimic group showed significantly increased miR-203 expression in A375 and M14 cells ( F = 487.632, 68.454, respectively, both P < 0.05) , significantly decreased number of migratory A375 and M14 cells (both P < 0.05) , and significantly decreased ZNF281 protein expression (both P < 0.05) , while the miR-203 inhibitor group showed significantly decreased miR-203 expression in A375 and M14 cells (both P < 0.05) , significantly increased number of migratory A375 and M14 cells (both P < 0.05) , significantly increased ZNF281 protein expression in A375 cells ( P < 0.05) , significantly increased proliferative activity of A375 cells at 36, 48, 60 and 72 hours (all P < 0.05) , and significantly increased proliferative activity of M14 cells at 24, 36, 48, 60 and 72 hours (all P < 0.05) . Dual-luciferase reporter assay showed interaction sites between miR-203 and ZNF281. Conclusion:Up-regulating the miR-203 expression can inhibit the proliferation and migration of melanoma cell lines, and vice versa, likely by regulating the expression of ZNF281.