过表达miR-32削弱多氯联苯抑制P19细胞向心肌细胞的分化

摘要

BACKGROUND:Polychlorinated biphenyls inhibit the differentiation of P19 cels into cardiomyocytes. In the meanwhile, microRNAs play an important role in regulating cel differentiation. OBJECTIVE:To explore the effect of microRNA-32(miR-32) on the polychlorinated biphenyls-inhibited differentiation of P19 cels into cardiomyocytes. METHODS:P19 cels were co-cultured with 2.5 μmol/L polychlorinated biphenyls and 1% dimethyl sulfoxide. Afterwards, α-actinin, desmin, cTnI and GATA4 were identified by western blot assay.Wddwxrof polychlorinated biphenyls on the expression of miR-32 was evaluated by real-time PCR. Mouse eukaryotic vector expressing miR-32 was constructed by gene recombination technology,andtransfected into P19 celsfolowed byco-culturedwith2.5 μmol/Lpolychlorinated biphenylsand1% dimethyl sulfoxide. Then, expressions of differentiation-related proteins were detected by western blot assay. RESULTS AND CONCLUSION:Polychlorinated biphenylsinhibited the differentiation of P19 cels into cardio myocytes anddecreased the miR-32 expression. Cel lines overexpressing miR-32 was successfuly established in mice.Furthermore, we found thatoverexpression of miR-32 weakens the inhibition of polychlorinated biphenyls to the differentiation of P19 cels into cardiomyocytes.%背景：多氯联苯对P19细胞分化成心肌细胞有抑制作用。miRNAs在多氯联苯抑制P19细胞分化成心肌细胞过程中发挥了重要的作用。　　目的：探索miR-32在调节多氯联苯对P19细胞分化成心肌细胞中的作用。　　方法：将P19细胞与1%二甲基亚砜和2.5μmol/L多氯联苯共孵育，通过Western blot鉴定分化标志物α-actinin、desmin、cTnI以及早期心脏发育的重要转录因子GATA4的变化。qRT-PCR鉴定多氯联苯对miR-32表达的影响。应用基因重组技术成功构建了小鼠miR-32真核表达载体，将其转染至P19细胞，与2.5μmol/L多氯联苯和1%二甲基亚砜共孵育，通过Western blot实验观察分化相关蛋白表达。　　结果与结论：①多氯联苯降低了 miR-32的表达水平，抑制 P19细胞向心肌细胞的分化；②成功构建了小鼠miR-32真核表达载体，建立了稳定过表达miR-32的P19细胞系；③过表达miR-32发挥了削弱多氯联苯对P19细胞向心肌细胞分化的抑制作用。