miRNA211在人皮肤黑素瘤的表达及功能研究

Expression and function of miRNA211 in human cutaneous melanoma

目的 探讨miRNA211（miR-211）在恶性黑素瘤进展过程中的表达以及靶分子MMP-16与miR-211表达的相关关系。方法 实验分阴性对照组、空载体对照组、miR-211过表达组。TaqMan荧光定量PCR检测miR-211在黑素细胞、黑素瘤细胞的表达，以及在痣、黑素瘤组织中的表达。SRB试验和流式细胞仪分别检测细胞增殖和细胞周期分布。甲基纤维素克隆形成试验和Transwell迁移试验研究细胞克隆形成和细胞迁移。用集落大小衡量克隆形成力，而同一迁移距离下的相对细胞数用来衡量细胞迁移能力。TaqMan荧光定量PCR检测比较miR-211表达增加前后MMP-16 mRNA表达的变化。结果 miR-211在黑素瘤细胞G361、C32和A375表达量分别是0.09 ± 0.02，0.000 52 ± 0.000 20，0.000 03 ± 0.000 01（F = 10410，P 〈 0.01）。miR-211在黑素瘤标本的表达量（0.17 ± 0.03）显著低于痣的表达量（0.87 ± 0.08），t = 9.118，P 〈 0.01。在体外，miR-211表达上调的黑素瘤细胞miR-211过表达组与阴性对照组及空载体对照组比较，细胞增殖与细胞周期分布，差异无统计学意义。miR-211过表达组、空载体对照组的克隆形成力分别是0.49 ± 0.05、0.85 ± 0.09，两组比较，差异有统计学意义（t = 2.19，P 〈 0.05）。miR-211过表达组、空载体对照组的克隆形成力分别是0.49 ± 0.06、0.82 ± 0.09，两组比较，差异有统计学意义（t = 3.15，P 〈 0.05）。而空载体对照组与阴性对照组比较，差异无统计学意义。转染miR-211 mimics后24 h，在miR-211过表达组和空载体对照组的MMP-16 mRNA水平比较分别是0.33 ± 0.02和0.91 ± 0.03，两组比较，t = 11.30，P 〈 0.01；48 h分别是0.52 ± 0.01和0.96 ± 0.02，两组比较，t = 5.02，P 〈 0.05；72 h分别是0.71 ± 0.01和0.97 ± 0.03，两组比较，t = 3.85，P 〈 0.05。空载体对照组与阴性对照组在3个时间点的比较，差异均无统计学意义。结论 miR-211在恶性黑素瘤的细胞水平和组织水平低表达。miR-211抑制黑素瘤细胞的锚着非依赖性生长及细胞的迁移。miR-211表达上调后，MMP-16 mRNA表达下调，可能是miR-211下游的靶分子从而影响黑素瘤的侵袭转移。

Objective To determine the 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 groups： miR-211 over 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 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 in A375 cells before and after the over 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 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 of miR-211 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 over group, mock-vehicle group and negative control group. Compared with the mock-vehicle group, the miR-211 over 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 of MMP-16 significantly decreased in the miR-211 over group compared with the mock-vehicle group after transfection （24 hours： 0.33 ± 0.02 vs. 0.91 ± 0.03, t = 11.30, P 〈 0.01; 48 hours： 0.52 ± 0.01 vs. 0.96 ± 0.02, t = 5.02, P 〈 0.05; 72 hours： 0.71 ± 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 , the mRNA of MMP-16 decreased in A375 cells, suggesting that MMP-16 may be a downstream target of miR-211, and can influence melanoma metastasis.