miR-149-3p在先天性心脏病胎鼠心脏组织中的表达及其对P19细胞分化的影响

The expression of miR-149-3p in heart tissue of newborn mice with congenital heart disease and its effect on differentiation of P19 cells

目的 探讨miR-149-3p在先天性心脏病(CHD)胎鼠心脏组织中的表达及其对小鼠畸胎瘤P19细胞分化的影响。方法 建立CHD室间隔缺损胎鼠模型,于妊娠第19天,HE染色观察心脏组织病理学变化,实时荧光定量PCR(qPCR)检测心脏组织中miR-149-3p和热休克蛋白蛋白家族B成员6(HSPB6)mRNA表达水平。二甲基亚砜(DMSO)诱导P19细胞向心肌细胞分化,并收集分化第0、5、10天的细胞,qPCR检测诱导分化过程中心肌分化标志物GATA结合蛋白4(GATA4)、心肌肌钙蛋白T(cTnT)、心房利钠尿多肽(ANP)的mRNA表达水平以及HSPB6 mRNA和miR-149-3p表达水平。miR-149-3p过表达慢病毒和空载慢病毒感染P19细胞,DMSO诱导分化第10天,免疫荧光染色检测细胞中cTnI蛋白表达情况,qPCR检测心肌分化相关标志物mRNA表达水平。双荧光素酶报告基因实验验证miR-149-3p与HSPB6之间的靶向关系。结果 与正常组比较,CHD组胎鼠出现心脏室间隔缺损,心脏组织中miR-149-3p高表达,而HSPB6低表达(P<0.01)。P19细胞在向心肌细胞分化的过程中,GATA4、cTnT、ANP和HSPB6mRNA水平均逐渐上升,而miR-149-3p表达水平逐渐下降(P<0.05)。诱导分化第10天,miR-149-3p过表达可明显降低心肌细胞分化率,并下调GATA4、cTnT、ANP mRNA表达水平(P<0.01)。双荧光素酶报告基因实验证实,miR-149-3p可以靶向调控HSPB6表达。结论 miR-149-3p可能通过靶向下调HSPB6抑制P19细胞向心肌细胞分化。

Objective To explore the expression of miR-149-3p in heart tissues of newborn mice with congenital heart disease(CHD) and its effect on differentiation of mouse embryonic teratoma P19 cells.Methods The newborn mouse model of ventricular septal defect CHD was established.On the 19th day of pregnancy,HE staining was used to observe pathological changes of fetal heart.Then qPCR was used to detect the expression levels of miR-149-3p and heat shock protein family B in fetal mouse heart tissue member 6(HSPB6) mRNA in heart tissue of fetal mice.Dimethyl sulfoxide(DMSO) was used to induce P19 cells to differentiate into cardiomyocytes,and cells on the 0 th,5 th and 10 th days of differentiation were collected.qPCR was used to detect markers of myocardial differentiation GATA4,cTnT,the mRNA expression level of atrial natriuretic peptide(ANP) and HSPB6 mRNA and miR-149-3p expression levels during the induction of differentiation.P19 cells were infected with miR-149-3p overexpression lentivirus and empty lentivirus.After induction for 10 days by DMSO,the expression of cTnI protein in cells was detected by immunofluorescence staining.mRNA expression levels of myocardial differentiation markers were detected by qPCR.The dual-luciferase reporter gene experiment was used to verify the targeting relationship between miR-149-3p and HSPB6.Results Compared with the normal group,the newborn mice in the CHD group developed cardiac ventricular septal defect,with high expression of miR-149-3p and low expression of HSPB6 in cardiac tissue(P<0.01).During the differentiation of P19 cells into cardiomyocytes,the expression levels of GATA4,cTnT,ANP and HSPB6 mRNA were gradually increased,while the expression level of miR-149-3p was gradually decreased(P <0.05).After 10 days of differentiation,the overexpression of miR-149-3p significantly reduced the differentiation rate of cardiomyocytes,and down-regulated the mRNA expression levels of GATA4,cTnT and ANP(P <0.01).The dual luciferase reporter gene experiment confirmed that miR-149-3p could target the expression of HSPB6.Conclusion miR-149-3p might inhibit the differentiation of P19 cells into cardiomyocytes through targeted down-regulation of HSPB6.