miR-2135对P19细胞增殖及向心肌细胞分化的作用

Effect of miR-2135 on the P19 cell proliferation and differentiation into cardiomyocytes

背景:既往研究发现,向心肌分化P19细胞中的miR-2135表达水平显著高于未分化P19细胞,但miR-2135对P19细胞增殖、向心肌细胞分化和心脏发育的影响尚不明确。目的:探索mi R-2135对P19细胞增殖及向心肌细胞分化的影响。方法:分别以miR-2135沉默及空载质粒转染P19细胞,培养0-4 d,MTT法检测细胞增殖;培养0,24,48 h,流式细胞仪检测细胞周期。诱导两组转染的P19细胞向心肌细胞分化,诱导第0,10天,采用qR T-PCR检测P19细胞中miR-2135的表达;诱导第10天,采用蛋白免疫印迹检测心肌分化相关标志物心肌肌钙蛋白T、心房钠尿肽和心肌转录因子的表达。结果与结论:(1)转染结果:转染miR-2135沉默质粒的P19细胞低表达mi R-2135;(2)细胞增殖与周期:miR-2135沉默质粒转染P19细胞的增殖速度快于空载质粒转染P19细胞(P<0.05),培养24,48 h的S期细胞比例明显高于空载质粒转染P19细胞(P<0.05);(3)qR T-PCR检测结果:miR-2135沉默质粒转染P19细胞诱导10 d的miR-2135表达明显高于诱导0 d水平(P<0.01);(4)蛋白免疫印迹检测结果:诱导10 d,mi R-2135沉默质粒转染P19细胞心肌肌钙蛋白T、心房钠尿肽和心肌转录因子的表达明显低于空载质粒转染P19细胞;(5)结果表明:miR-2135抑制P19细胞的增殖,可促进其向心肌细胞分化。

BACKGROUND：Previous studies have found that the expression level of miR-2135 in differentiated P19 cel s is significantly higher than that in undifferentiated P19 cel s. However, the effects of miR-2135 on P19 cel proliferation and differentiation into cardiomyocytes as wel as cardiomyogenesis remain unclear. OBJECTIVE：To explore the effect of miR-2135 on P19 cel proliferation and differentiation into cardiomyocytes. METHODS：P19 cel s were transferred with miR-2135 knockdown plasmid or vector plasmid. After 4 days of culture, cel proliferation was detected by MTT assay. Flow cytometry was used to detect cel cycle at 0, 24, 48 hours of culture. P19 cel s in the two groups were induced to differentiate into cardiomyocytes. The expression of miR-2135 in differentiated and undifferentiated P19 cel s was detected by quantitative real-time PCR at 0 and 10 days of induction. Levels of cardiac troponin T, atrial natriuretic peptide and myocardial transcription factor were detected using western blot assay at 10 days of induction. RESULTS AND CONCLUSION：Expression level of miR-2135 in miR-2135-downexpressed P19 cel s was obviously reduced. Compared with control cel s, miR-2135 knockdown significantly upregulated P19 cel proliferation （P〈0.05） as wel as induced a cel cycle rest in S stage after 24 and 48 hours of culture （P〈0.05）. PCR findings showed that in the miR-2135 knockdown group, the expression level of miR-2135 at 10 days of induction was significantly higher than that at 0 day of induction （P〈0.01）. Western blot assay showed that miR-2135-downexpressed P19 cel s had obvious decreases in the protein levels of cardiac troponin T, atrial natriuretic peptide and myocardial transcription factor at 10 days of induction. Our findings confirm that miR-2135 inhibits P19 cel proliferation, but promotes the differentiation of P19 cel s into cardiomyocytes.