| miR-122-5p通过靶向NOP14抑制黑素瘤的细胞增殖 |
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| 引用本文： | 李璟蓉,赵瑞,方锐华,王建琴. miR-122-5p通过靶向NOP14抑制黑素瘤的细胞增殖[J]. 南方医科大学学报, 2018, 38(11): 1360. DOI: 10.12122/j.issn.1673-4254.2018.11.14 |
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| 作者姓名： | 李璟蓉 赵瑞 方锐华 王建琴 |
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| 摘 要： | 目的探讨在黑素瘤组织中miR-122-5p的表达情况，同时研究miR-122-5p对人黑素瘤细胞株SK-MEL-110和A375细胞增殖、细胞周期及凋亡的调控效应。方法荧光定量PCR检测miR-122-5p在黑素瘤和色素痣组织中的表达；培养SK-MEL-110和A375细胞，瞬时转染miR-122-5p inhibitor及阴性对照inhibitor后，荧光定量PCR检测miR-122-5p的表达，MTT及流式细胞仪方法检测对细胞增殖、周期和凋亡的影响，荧光定量PCR和Western Blot法检测NOP14的mRNA和蛋白水平；应用双荧光素酶基因报告法进一步验证NOP14是否为miR-122-5p的靶基因。结果miR-122-5p在人色素痣和黑素瘤组织中的相对表达量分别为1.23±0.270和7.65±1.37。转染miR-122-5p inhibitor后，miR-122-5p在SK-MEL-110和A375细胞中的相对表达量分别为0.21±0.08和0.17±0.05。miR-122-5p inhibitor可以显著抑制黑素瘤细胞株SK-MEL-110和A-375细胞的增殖能力，明显增加G1期细胞的比例，但对SK-MEL-110和A-375细胞的凋亡没有明显影响。转染miR-122-5p inhibitor对NOP14 mRNA水平没有明显的影响，但可以显著提高NOP14的蛋白水平表达。荧光素酶报告基因检测显示，共转染miR-122-5p mimics和野生型psi-CHECK2-3′UTR质粒组相对荧光素酶活性为0.21±0.14，较NC和野生型psi-CHECK2-3′UTR质粒转染组（0.56±0.1）显著下降（P<0.01）。结论miR-122-5p在黑素瘤组织中高表达，提示miR-122-5p可能参与黑素瘤的发生发展过程。miR-122-5p 可能通过NOP14影响SK-MEL-110和A-375细胞的周期，进而抑制细胞的增殖。
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miR-122-5p inhibits the proliferation of melanoma cells by targeting NOP14 |
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| Abstract: | Objective To investigate the expression profile of miR-122-5p in melanoma tissues and the effect of miR-122-5p onthe proliferation, cell cycle and apoptosis of human melanoma cell lines SK-MEL-110 and A375. Methods The expressionprofiles of miR-122-5p in melanoma and pigmented nevus tissues were detected using real-time fluorescence quantitative PCR(qRT-PCR). SK-MEL-110 and A375 cells transfected with miR-122-5p inhibitor or negative control inhibitor (NC) I wereexamined for miR-122- 5p expression using qRT-PCR and changes in cell proliferation, cell cycle and apoptosis using MTTassay or flow cytometry. NOP14 mRNA and protein expressions in the cells were detected using qRT- PCR and Westernblotting, respectively. Luciferase reporter assay was used to confirm the identity of NOP14 as the direct target of miR-122-5p.Results The relative expression of miR-122-5p in human pigmented nevus tissues and melanoma tissues was 1.23±0.270 and7.65 ± 1.37, respectively. The relative expression of miR-122-5p in SK-MEL-110 and A375 cells transfected with miR-122-5pinhibitor was 0.21 ± 0.08 and 0.17 ± 0.05, respectively. miR-122-5p inhibitor obviously inhibited the cell proliferation andincreased the percentage of cells in G1 stage in both SK-MEL-110 and A-375 cells, but did not cause obvious changes in theapoptosis of the two cells. miR-122-5p inhibitor did not significantly affect the expression level of NOP14 mRNA, butobviously increased the expression level of NOP14 protein. Luciferase reporter assay revealed a significantly lower luciferaseactivity in cells co-transfected with miR-122-5p mimics and wild-type psi-CHECK2-3’UTR plasmid than in the cells cotransfectedwith NC and wild-type psi-CHECK2-3’UTR plasmid (0.21 ± 0.14 vs 0.56 ± 0.1, P<0.01). Conclusion miR-122-5pexpression is upregulated in melanoma tissues, indicating its involvement in the development of melanoma. miR-122-5pinhibits the proliferation of SK-MEL-110 and A-375 cells possibly by affecting the cycle through NOP14. |
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