microRNA-9-5p靶向MEF2C对腺泡状横纹肌肉瘤细胞生物学行为的影响

doi:10.13481/j.1671-587x.20200309

摘要/Abstract

摘要： 目的：探讨microRNA-9-5p （miR-9-5p）靶向肌细胞增强因子2C （MEF2C）对腺泡状横纹肌肉瘤（ARMS）细胞生物学行为的影响，为ARMS的分子诊断和靶向治疗提供依据。方法：qRT-PCR法检测ARMS组织和细胞中miR-9-5p和MEF2CmRNA表达水平，CCK-8法检测细胞增殖率，流式细胞术检测细胞凋亡率，Transwell小室法检测细胞侵袭和迁移能力，双荧光素酶报告基因检测293T细胞中荧光素酶活性，Western blotting法检测细胞中MEF2C蛋白表达水平。结果：ARMS组织和细胞中miR-9-5p表达水平高于正常骨骼肌组织和HSKMC细胞（P<0.05）。与miR-NC组比较，miR-9-5p抑制剂组RH30细胞中miR-9-5p表达水平降低（P<0.01），细胞增殖率降低（P<0.05），细胞凋亡率升高（P<0.05），细胞侵袭和迁移数降低（P<0.05）。加入miR-9-5p后，共转染MEF2C-3'-UTR-WT的293T细胞中荧光素酶活性降低（P<0.01）；与miR-NC组比较，miR-9-5p抑制剂组RH30细胞中MEF2C mRNA和蛋白表达水平升高（P<0.05）。ARMS组织中MEF2C mRNA表达水平低于正常骨骼肌组织（P<0.01），且与ARMS组织中miR-9-5p表达呈负相关关系（r=-0.542，P<0.05）；RH30细胞和PLA802细胞中MEF2CmRNA表达水平低于HSKMC细胞（P<0.01）。与转染对照质粒（EV）比较，转染MEF2C过表达质粒后RH30细胞中MEF2C mRNA表达水平升高（P<0.01），细胞增殖率降低（P<0.01），细胞凋亡率升高（P<0.05），细胞侵袭数降低（P<0.05），细胞迁移数降低（P<0.01）。与转染si-NC比较，转染MEF2CsiRNA后RH30细胞中MEF2C mRNA表达水平降低（P<0.05），细胞增殖率升高（P<0.05），细胞凋亡率降低（P<0.05），细胞侵袭数升高（P<0.01），细胞迁移数升高（P<0.01）。结论：miR-9-5p通过直接靶向MEF2C mRNA的3'-UTR诱导mRNA降解而抑制MEF2C表达，从而促进RH30细胞的增殖、侵袭、迁移以及抗凋亡能力。

关键词: 腺泡状横纹肌肉瘤, miR-9-5p, 肌细胞增强因子2C, 细胞增殖, 细胞凋亡

Abstract: Objective: To investigate the effect of microRNA-9-5p (miR-9-5p) targeting the myocyte enhancer factor 2C (MEF2C) on the biologicals behaviors of the alveolar rhabdomyosarcoma (ARMS) cells, and to provide the basis for the molecular diagnosis and targeted therapy of ARMS. Methods: The expression levels of miR-9-5p and MEF2C mRNA in ARMS tissue and cells were detected by qRT-PCR method, the proliferation rate of cells was detected by CCK-8 method, the apoptotic rate was detected by flow cytometry, the numbers of invasion and migration cells were detected by Transwell chamber assay, the luciferase activity in 293T cells was detected by double luciferase reporter gene, and the expression level of MEF2C protein in the cells was detected by Western blotting method. Results: The expression levels of miR-9-5p in ARMS tissue and cells were higher than those in normal skeletal muscle tissue and HSKMC cells (P<0.05). Compared with miR-NC group, the expression level of miR-9-5p in RH30 cells in miR-9-5p inhibitor group was decreased (P<0.01),the proliferation rate of RH30 cells in miR-9-5p inhibitor group was decreased (P<0.05), the apoptotic rate was increased (P<0.05), and the numbers of invasion and migration cells were decreased (P<0.05). The luciferase activity in the 293T cels after co-transfection with MEF2C-3'-UTR-WT and added with miR-9-5p was decreased (P<0.01);compared with miR-NC group, the expression levels of MEF2C mRNA and protein in the RH30 cells in miR-9-5p inhibitor group were increased (P<0.05).The expression level of MEF2C mRNA in the ARMS tissue was lower than that in normal skeletal muscle tissue (P<0.01), which was negatively correlated with the expression of miR-9-5p in ARMS tissue (r=-0.5420, P<0.05);the expression level of MEF2C mRNA in the RH30 cells and PLA802 cells was lower than that in the HSKMC cells (P<0.01).Compared with transfection with control plasmid(EV), the expression level of MEF2C mRNA in the RH30 cells after transfection with MEF2C over-expression plasmid was increased (P<0.01), the proliferation rate was decreased (P<0.01), the apoptotic rate was increased (P<0.05), the number of invasion cells was decreased (P<0.05), and the number of migration cells was decreased (P<0.01).Compared with transfection with si-NC, the expression level of MEF2C mRNA in the RH30 cells after transfection with MEF2C siRNA was decreased (P<0.01), the proliferation rate was increased (P<0.05), the apoptotic rate was decreased (P<0.01), the number of invasion cells was increased (P<0.01),and the number of migration cells was increased (P<0.01). Conclusion: MiR-9-5p can directly target the 3'-UTR of MEF2C mRNA to induce mRNA degradation and inhibit the expression of MEF2C, and promote the proliferation, invasion, migration and anti-apoptosis ability of the RH30 cells.

Key words: alveolar rhabdomyosarcoma, miR-9-5p, myocyte enhancer 2C, cell proliferation, apoptosis

中图分类号:

R738.6

商浩, 李春森, 李锋, 刘春霞. microRNA-9-5p靶向MEF2C对腺泡状横纹肌肉瘤细胞生物学行为的影响[J]. 吉林大学学报(医学版), 2020, 46(03): 482-491.

SHANG Hao, LI Chunsen, LI Feng, LIU Chunxia. Effect of microRNA-9-5p targeting MEF2C on biological behaviors of alveolar rhabdomyosarcoma cells[J]. Journal of Jilin University(Medicine Edition), 2020, 46(03): 482-491.

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