Expression and function of miRNA211 in human cutaneous melanoma / 中华皮肤科杂志

ABSTRACT

Objective To determine the expression of miRNA211 (miR-211) in the development of malignant melanoma,and to investigate the correlation between miR-211 and its target molecule,matrix metalloproteinase 16 (MMP-16).Methods Cultured A375 melanoma cells were divided into 3 groupsmiR-211 overexpression group and mock-vehicle group transfected with miR-211 mimics and empty vehicle respectively,and negative control group receiving no treatment.TaqMan fluorescence-based quantitative PCR was performed to determine the expression of miR-211 in HER1 primary melanocytes,A375,C32 and G361malignant melanoma cell lines,as well as in nevus tissues (n =18) and melanoma tissues (n =41),and to evaluate changes of MMP-16 mRNA expression in A375 cells before and after the overexpression of miR-211.Sulforhodamine B (SRB) assay and flow cytometry were conducted to evaluate cellular proliferative activity and determine cell cycle distribution respectively,and methylcellulose assay and Transwell assay to evaluate colony formation and cell migration abilities respectively.The size of selected colonies was used to represent colony formation ability,while the ratio of the number of migrating cells to that of non-migrating cells to represent cell migration ability.Results There were significant differences in the expression level of miR-211 among the G361,C32 and A375 cells (0.09 ± 0.02 vs.0.000 52 ± 0.000 20 vs.0.000 03 ± 0.000 01,F =10 410,P ＜ 0.01).The expression of miR-21 1 was significantly decreased in melanoma tissues compared with nevus tissues (0.17 ± 0.03 vs.0.87 ± 0.08,t =9.118,P ＜ 0.01).No significant differences were observed in cellular proliferative activity or cell cycle distribution among the miR-211 overexpression group,mock-vehicle group and negative control group.Compared with the mock-vehicle group,the miR-211 overexpression group showed significantly suppressed colony formation (0.49 ± 0.05 vs.0.85 ± 0.09,t =2.19,P ＜ 0.05) and cell migration (0.49 ± 0.06 vs.0.82 ± 0.09,t =3.15,P ＜ 0.05) abilities,while no significant difference was observed between the mock-vehicle group and negative control group.Additionally,the mRNA expression of MMP-16 significantly decreased in the miR-211 overexpression group compared with the mock-vehicle group after transfeetion (24 hours0.33 ± 0.02 vs.0.91 ± 0.03,t =11.30,P ＜ 0.01;48 hours0.52 ± 0.01 vs.0.96 ± 0.02,t =5.02,P ＜ 0.05;72 hours0.71 ± 0.01 vs.0.97 ± 0.03,t =3.85,P ＜ 0.05),with no significant difference between the mock-vehicle group and negative control group at the above time points.Conclusions miR-211 was lowly expressed in both malignant melanoma cells and tissues,and it could inhibit both anchorage-independent growth and migration of melanoma cells.After up-regulation of miR-211 expression,the mRNA expression of MMP-16 decreased in A375 cells,suggesting that MMP-16 may be a downstream target of miR-211,and can influence melanoma metastasis.