miRNA-31-5p在过量维甲酸抑制C2C12细胞增殖中的调控机制研究

Regulatory mechanisms of miRNA-31-5p in excess retinoic acid-induced abnormal C2C12 cell proliferation

关键词:  miRNA-31-5p在过量维miRNA-31-5p  维甲酸  C2C12细胞  细胞增殖甲酸抑制C2C12 细胞增殖中的调控机制研究[

Key words:  miRNA-31-5p  retinoic acid  C2C12  cell proliferation

摘要:

目的　探讨miRNA-31-5p在过量维甲酸（Retinoic acid，RA）抑制小鼠肌原细胞系C2C12增殖中的作用机制。　方法　在10 μmol/L RA及无RA条件下，qPCR检测C2C12细胞增殖过程中miRNA-31-5p及抗肌营养不良蛋白（dystrophine，Dmd)表达的水平；分别转染miRNA-31-5p mimics（miRNA-31-5p mimics组）、miRNA-31-5p inhibitor（miRNA-31-5p inhibitor组）、Duplex N.C（Duplex N.C组）及N.C inhibitor（N.C inhibitor组）。用细胞增殖/毒性检测实验（CCK-8）检测0 h，24 h,36 h及48 h的OD值， 5-溴脱氧尿嘧啶核苷掺入实验（BrdU）检测48 h细胞中BrdU阳性细胞比率。在10 μmol/L RA条件下，qPCR检测C2C12细胞在下调miRNA-31-5p后，Dmd表达水平变化。 　结果　在10 μmol/L RA条件下，细胞中miRNA-31-5p在36 h及48 h的表达水平均明显高于相同时间点正常条件下的表达水平，P<0.01；Dmd在36 h及48 h的表达水平均明显低于相同时间点正常条件下的表达水平，P<0.01。CCK-8结果显示在0 μmol/L RA条件下,miRNA-31-5p过表达抑制C2C12细胞增殖， 10 μmol/L RA条件下，细胞的增殖活性进一步降低；0 μmol/L RA条件下，miRNA-31-5p低表达促进C2C12细胞增殖，10 μmol/L RA作用下， miRNA-31-5p inhibitor组细胞增殖活性在第48 h显著高于N.C inhibitor组，P<0.01。10 μmol/L RA的作用下，miRNA-31-5p mimics组细胞BrdU阳性率与Duplex N.C组比降低27.6%,P<0.05；miRNA-31-5p inhibitor组细胞BrdU阳性率与N.C inhibitor组相比升高24.1%,P<0.05。转染48 h后，miRNA-31-5p inhibitor组Dmd的相对表达量较N.C inhibitor组明显上调,P<0.01；在10 μmol/L RA作用下，miRNA-31-5p inhibitor组细胞Dmd的表达升高，与N.C inhibitor组相比差异具有显著性意义。　结论　 miRNA-31-5p可参与10 μmol/L RA导致的C2C12细胞增殖抑制，而其可能的分子机制是通过对Dmd靶向作用实现的。

Abstract:

Objective　To investigate miRNA-31-5p regulation mechanism in decreased proliferation of C2C12 myoblast cell line which was caused by excess retinoic acid (RA). 　Methods Establish proliferation assay model of C2C12 cell lines in vitro. The expression level of miRNA-31-5p and dystrophine (Dmd) in C2C12 cells without RA or added with 10 μmol/L RA were detected using qPCR. Liposome of the miRNA-31-5p analogues (miRNA-31-5p mimics) or inhibitors (miRNA-31-5p inhibitor) were transfected into C2C12 cells, with the corresponding load (Duplex N.C or N.C inhibitor) measured as the control group. The absorbance value (OD index) of cells was monitored by cell proliferation / cytotoxicity detection reagent (Cell Counting Kit-8, CCK-8) in 0 h, 24 h, 36 h and 48 h. Using 5-bromodeoxyuridine incorporation assay (BrdU assay), BrdU-positive cells rate in 48h was detected. After miRNA-31-5p was down-regulated, the expression level of Dmd was analyzed by qPCR under the condition of 10 μmol/L RA. 　Results In the state of 10 μmol/L RA, expression levels of miRNA-31-5p had an increase tendency in 36 h and 48 h (P<0.01), and 10 μmol/L RA inhibited the expression of Dmd, with the significant differences in 36 h and 48 h (P<0.01). CCK-8 showed that miRNA-31-5p inhibitor was shown to reduce C2C12 cells proliferation activity, which was also suppressed by 10 μmol/L RA to a lower degree. In contrast, down-regulation of miRNA-31-5p significantly promoted cell proliferation (P<0.01). miRNA-31-5p inhibitor was shown to promote proliferation of C2C12 cells at 48 h comparing to N.C inhibitor under the stimulation of 10 μmol/L RA. Under the effect of 10 μmol/L RA, BrdU-positive cells rate of miRNA-31-5p mimics group was 27.6% lower than Duplex N.C group (P<0.05), and BrdU-positive cells rate of miRNA-31-5p inhibitor group was elevated by 24.1% compared to N.C inhibitor group (P<0.05). After 48 h of miRNA-31-5p inhibitor transfection, the relative expression of Dmd was significantly increased compared to N.C inhibitor group, P<0.01. Under the stimulation of 10 μmol/L RA, Dmd was measured the same increased tendency by the transfection of miRNA-31-5p inhibitor. 　Conclusion miRNA-31-5p participated in C2C12 cell proliferation decrescence induced by 10 μmol/L RA, which possible related molecular mechanism was achieved through targeting Dmd.