大鼠心肌细胞缺氧后miR-133a-3p的表达及其下游靶基因预测

Preliminary study on miRNA-133a-3p and hypoxia in rat cardiomyocytes

目的检测大鼠心肌细胞缺氧后微小RNA(miRNA,miR)-133a-3p的表达,并预测其潜在靶基因和进行初步验证。方法分离培养SD大鼠乳鼠心肌细胞,给予缺氧(37℃,5%CO_2,1%O_2)0 h、4 h、8 h、12 h、16 h、20 h和24 h,采用qRT-PCR检测其在上述不同时间点的miRNA-133a-3p的表达水平,运用miRanda、pic Tar和terget Scan等靶基因预测分析软件及其他生物信息学方法,筛选miR-133a-3p潜在调控的靶基因,并对关键候选基因采用qRT-PCR和Western blot进行初步验证。结果大鼠心肌细胞中miRNA-133a-3p的表达在缺氧4 h出现高表达,与其他缺氧时间比较差异有统计学意义(P=0.000);4 h后,其表达逐渐下降,12 h后趋于稳定;生物信息学预测发现miRNA-133a-3p的靶基因可能为TGF-β1;荧光定量检测发现在4 h时TGF-β1的表达较0 h、24 h的差异都有统计学意义(P<0.05);且4 h时心肌细胞TGF-β1蛋白的表达较0 h组和24 h组都明显升高,差异具有统计学意义(P<0.05)。结论 miRNA-133a-3p可能参与大鼠心肌细胞的缺氧坏死过程,并且可能通过调控TGF-β1而发挥作用。

Objective To study the relationship of miRNA-133a-3p and hypoxia in rat cardiomyocytes,and to detect the expression of the probable target genes. Methods SD rats cardiomyocytes were cultivated,and treated in hypoxia condition（ 37 ℃,5% CO2,1% O2） for0 hour,4 hours,8 hours,12 hours,16 hours,20 hours and 24 hours. The miRNA-133a-3p level was detected by qRT-PCR,and its biological information and target genes were predicted by target Scan pic Tar and miRanda,etc. Then we carried out qRT-PCR and Western blot to detect the expression of the probable target genes. Results miRNA-133a-3p expression was much higher in 4 hours hypoxia（ P = 0. 000）,then its expression gradually declined,and stabilized after 12 hours hypoxia. Bioinformatics prediction found that TGF-β1 may be its target. The mRNA expression of TGF-β1 was obviously higher in 4 hours hypoxia than those in 0 hour and 24 hours（ P〈0. 05）. And the protein expression of TGF-β1 was significantly higher in 4 hours hypoxia（ P〈0. 05）. Conclusion miRNA-133a-3p may participate in cardiomyocytes necrosis in rats through regulating TGF-β1.