The Effect Of MiR-22 In Rat Cardiomyocyte Hypertrophy

Objectives: To study the effect of miRNAs in rat cardiomyocyte hypertrophy.Methods: To understand the roles of miRNAs in hypertrophy, the model of rat cardiomyocytes hypertrophy was applied by angiotensin II (Ang II) or phenylephrine (PE). The quantitative real time-polymerase chain reaction(qRT-PCR) was used to detect several hypertrophic markers, including those encoding the atrial natriuretic peptide (ANP, Nppa),α-myosin heavy chain (αMHC, myh6) andβ-myosin heavy chain (βMHC, myh7). The cell surface size was detected with cardiomyocyte immunochemistry and cell surface area analysis software of Leica laser scanning confocal microscope. The expression change of miRNAs that significantly changed in rat cardiac hypertrophy model was detected by qRT-PCR in rat hypertrophic cardiomyocytes. The most significant changed miRNAs was choosed as target miRNAs to study. To explore the potential involvement of miRNAs in the regulation of cardiomyocyte hypertrophy, we specifically over-expressed or knockdown the target miRNAs in neonatal rat cardiomyocytes and detected the change of hypertrophic marker and cell surface size. The luciferase reporter gene and Western blot were used to define the target of miRNAs and clarify the mechanism of cardiac hypertrophy.Results: (1) Two cell models for cardiac myocyte hypertrophy were established successfully. The first model is cardiomyocyte hypertrophy stimulated with angiotensin II (AngII) (5 uM), and the second model is hypertrophy stimulated with phenylephrine (PE) (10 uM) in the Dulbecco's Modified Eagle Media(DMEM). The stimulation lasted 48 hours. (2)The expression of indicated miRNAs was analyzed by qRT-PCR. Compared with control, the expression of miR-1, miR-16, miR-26b, miR-30e and miR-133a was decreased in both models and miR-181b was decreased only in Ang II-induced hypertrophy model. However, miR-22, miR-23a and miR-214 were significantly up-regulated in both models, while miR-21 and miR-145a were increased only in Ang II-induced hypertrophic cardiomyocytes. We also found that several miRNAs, such as miR-24, miR-125b and miR-199a, were not altered in both hypertrophic models. Among these aberrantly expressed miRNAs, miR-22 was the most significantly up-regulated miRNA in both PE- and Ang II-induced hypertrophic cardiomyocytes.(3) We showed that over-expression of miR-22 in cardiomycytes markedly increased Nppa and decreased myh6 expression level and down-regulation of miR-22 attenuated either PE- or Ang II-induced increase of Nppa and decrease of myh6 in cardiomyocytes. (4)Bioinformatic analysis indicated that SIRT1, NAT5, PTEN, HDAC4 and SRF might be potential targets of miR-22. We showed that up-regulation of miR-22 in cardiomyocytes significantly down-regulated PTEN expression level, while inhibition of endogenous miR-22 level markedly increased PTEN protein level.Conclusions: Two rat cardiomyocytes hypertrophy models stimulated with PE/AngII were established successfully. The expression of some miRNAs was changed and miR-22 was the most significantly changed miRNAs in two models. Overexpression of miR-22 induced rat cardiomyocytes hypertrophy and inhibition of miR-22 attenuated either PE- or Ang II-induced rat cardiomyocytes hypertrophy.m We also found that miR-22 induced cardiomyocytes hypertrophy by directly repressing PTEN expression levels.