Role of miR-1 in regulating cardiomyocyte growth / 第二军医大学学报

ABSTRACT

Objective To construct and identify miR-1 adenovirus vector, and to analyze its effect on cardiac hypertrophy. Methods The primers of miR-1 precursor were designed for PCR amplification, and the PCR products were cloned into adenovirus shuttle plasmid pAdTrack and linearized by enzyme Pme I the resultant plasmid was co-transfected into E. coli BJ5183 cells with adenovirus backbone plasmid pAdEasy-1 for homologous recombination. Then the recombinant plasmid was identified, linearized and packaged into QBI-293A cells to amplify the recombinant adenovirus Ad-miR-1, which was then used to infect cardiomyocytes. Real-time quantitative PCR was used to observe the expression of miR-1 and two hypertrophic markers, the atrial natriuretic peptide ( Nppa ) and ß-myosin heavy chain ( myh7 ), in cultured primary cardiomyocytes. Cell surface area was analyzed using software AxioVision 4. 7. 1 (Carl Zeiss). Results Sequencing and enzyme digestion showed that the miR-1 recombinant plasmid was successfully constructed. Real-time quantitative PCR confirmed that adenovirus Ad-miR-1 significantly enhanced intracellular miR-1 expression in cardiomyocytes and reduced cell surface area and the expression of Nppa and myh7. Conclusion The adenovirus expressing miR-1 has been successfully constructed and it can be transfected into cardiomyocytes to increase the expression of miR-1, thus inhibiting cardiomyocyte growth.