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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 in to cardiomyocytes through targeted down- regulation of HSPB6 [ABSTRACT FROM AUTHOR]