MiR-194对恶性黑色素瘤细胞增殖和凋亡的影响及机制

Effect of miR-194 on cell proliferation and apoptosis in melanoma cells and potential mechanism

摘要:

目的研究miR-194对人恶性黑色素瘤细胞的增殖和凋亡的影响及机制。方法应用实时荧光定量聚合酶链反应（qRT-PCR）分别检测miR-194在原代正常人皮肤黑色素细胞系PIG1 和5种恶性黑色素瘤细胞系（A375、SK-MEL-1、SK-MEL-2、SK-MEL-5及SK-MEL-28）中的相对表达量。利用Lipofectamine TM 2000分别将A375 细胞系转染miR-194 mimics 和阴性对照质粒，从而将A375 细胞系分为miR-194 模拟物组和阴性对照组；应用MTT法和流式细胞术分别测定miR-194 模拟物组和阴性对照组细胞的增殖能力及凋亡率，蛋白印迹（Western blot）分别检测miR-194 模拟物组和阴性对照组的细胞周期蛋白D1（Cyclin D1）、裂解型半胱氨酸天冬氨酸蛋白酶3（Cleaved Caspase-3）蛋白表达情况。结果miR-194 在5 种黑色素瘤细胞系A375、SKMEL-1、SK-MEL-2、SK-MEL-5及SK-MEL-28中的表达水平低于正常人皮肤黑色素细胞系PIG1，依次分别是正常细胞系的（0.18±0.03）、（0.25±0.05）、（0.37±0.03）、（0.39±0.04）及（0.45±0.03）倍（均p<0.05）。转染后第0、1、2、3、4 及5 天，miR-194 模拟物组vs阴性对照组的OD 值分别为：（0.18±0.02）vs（0.19±0.03）（p >0.05）、（0.29±0.02）vs（0.27±0.03）（p >0.05）、（0.42±0.08）vs（0.45±0.07）（p >0.05）、（0.63±0.09）vs（1.17±0.12）（p <0.05）、（1.05±0.15）vs（2.15±0.21）（p <0.05）及（1.87±0.23）vs（3.18±0.27）（p <0.05）。miR-194 模拟物组和阴性对照组的细胞凋亡率分别为24.2%和9.3%（p <0.05）。Cyclin D1 和Cleaved Caspase-3 蛋白在miR-194模拟物组中的表达量分别是阴性对照组的（0.36±0.04）和（3.2±0.28）倍（均p<0.05）。结论miR-194 在黑色素瘤细胞中低表达，miR-194表达上调可促进黑色素瘤细胞凋亡并抑制黑色素瘤细胞的增殖，其机制可能为下调Cyclin D1 蛋白及上调Cleaved Caspase-3 蛋白表达。

Abstract:

Objective To investigate the effect of microRNA-194 (miR-194) on cell proliferation and apoptosis in melanoma cells and potential underlying mechanism. Methods Real-time fluorescence quantitative PCR (qRT-PCR) was utilized to measure miR-194 in normal primary human skin melanocytes (PIG1) and five kinds of malignant melanoma cells (A375, SK-MEL-1, SK-MEL-2, SK-MEL-5 and SK-MEL-28). A375 melanoma cells were transfected with miR-194 mimics or negative control plasmid via lipofectamine 2000 system. The proliferation capability was measured by MTT assay, and apoptosis rate was determined by flow cytometry. Expressions of Cyclin D1 and cleaved Caspase-3 were detected by Western blot. Results The miR-194 level among A375, SK-MEL-1, SK-MEL-2, SK-MEL-5 and SK-MEL-28 was significantly lower than that in PIG1, and fold of PIG1 was (0.18 ±0.03), (0.25 ±0.05), (0.37 ±0.03), (0.39 ±0.04), and (0.45 ±0.03) (p < 0.05), respectively. OD values at 490 nm on day 0, 1, 2, 3, 4, and 5 post transfection in the miR-194 mimics group vs the negative control group were (0.18 ±0.02) vs (0.19 ±0.03) (p > 0.05), (0.27 ±0.02) vs (0.29 ±0.03) (p > 0.05), (0.42 ±0.08) vs (0.45 ±0.07) ( > 0.05), (0.63 ±0.09) vs (1.17 ±0.12) (p <0.05), (1.05 ±0.15) vs (2.15 ±0.21) (p < 0.05) and (1.87 ±0.23) vs (3.18 ±0.27) (p < 0.05), respectively. The apoptosis rate in the miR-194 mimics group vs the negative control group was 24.2% vs 9.3% (p < 0.05).Compared with the negative control group, the miR-194 mimics group had (0.36 ±0.04)-fold change and (3.2±0.28) -fold change in Cyclin D1 and cleaved Caspase-3, respectively (p < 0.05). Conclusions There is low expression of miR-194 in melanoma cells. Up-regulation of miR-194 can promote apoptosis rate and inhibit proliferation of melanoma cells by down -regulation of Cyclin D1 protein and up -regulation of cleaved Caspase-3 protein.