Effect of regulation of zinc finger protein 281 expression by miR-203 on the proliferation and migration of melanoma cell lines
10.35541/cjd.20200170
- VernacularTitle:miR-203调控锌指蛋白281表达对黑素瘤细胞增殖、迁移的影响
- Author:
Xiaobo CHEN
1
;
Qianying DOU
;
Lixin HUANG
Author Information
1. 南阳市中心医院整形外科，河南 473000
- From:
Chinese Journal of Dermatology
2020;53(9):720-724
- CountryChina
- Language:Chinese
-
Abstract:
Objective: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.
