MicroRNA-21Protects From Cardiomyocytes Apoptosis In Vitro Via The Regulation Of PTEN/AKT/FOXO3a Pathway

Background and Objective: Cardiomyocytes apoptosis is one of the importantpathological responses to a number of cardiovascular diseases. MicroRNAs (miRNAs)are endogenous, non-coding, single-stranded RNAs of approximately twenty-twonucleotides which negatively regulate their target gene expression in a cell viadegradation or translational inhibition of their target mRNA and have a key role in thepathophysiological mechanism of various diseases. Among several microRNAs,microRNA-21is not only highly expressed in the cardiovascular system,but also beaberrantly expressed in many cardiovascular diseases, such as myocardial infract,heart failure and cardiac hypertrophy, plays an important role in thepathophysiological and molecular mechanism of various cardiovascular diseases. Sofar, four target genes of miR-21are found which mediated cardiovascular effects,such as programmed cell death4(PDCD4), phosphatase and tensin homologuedeleted on chromosome10(PTEN), Sprouty1(SPRY1) and Sprouty2(SPRY2). Itshould be noted that the major target genes of miR-21may be cell-specific andcondition-specific. PTEN/AKT/FOXO3a signal pathway is one of the importantpathways mediated cell apoptosis. In this study, an eukaryotic expression vector forlenti-miRNA-21is to construct and transfer into cadiomyocytes. We will detect theeffect of microRNA-21on TNF-α (10ng/ml)-induced cardiomyocytes apoptosis; Inaddition, we will further explore whether PTEN is one of the target genes responsiblefor miR-21-mediated TNF-α-induced cardiomyocytes apoptosis and whethermicroRNA-21could protect from TNF-α-induced cardiomyocytes apoptosis throughthe regulation of PTEN/AKT/FOXO3a pathway on the level of gene and protein. Inthe future, microRNA-21could be a new target for the diagnosis and therapy ofvarious cardiovascular diseases.Methods: MicroRNA-21genomic sequence was amplified by PCR and clonedinto pGC-FU-GFP lentivirus vector, an eukaryotic expression vector forlenti-microRNA-21was constructed. Neonatal cardiomyocytes were cultured in vitroand identified by cTnI. After transferred lenti-microRNA-21into the cardiomyocytes, we observed the transferred efficiency by fluorescent microscope and examined theexpression of microRNA-21by quantitative real time RT-PCR (qRT-PCR).48h afterprimary cardiomyocytes culture, we transferred lenti-microRNA-21into thecardiomyocytes, then constructed the apoptotic model of cardiomyocytes which weretreated with TNF-α (10ng/ml) after24h transfection and examined the apoptotic rateby Hoechst33342/PI and Annexin V-FITC detection kit. We examined thecardiomyocytes apoptosis which was determined by the cardiomyocytes apoptoticindex to evaluate the effect of microRNA-21on TNF-α (10ng/ml)-inducedcardiomyocytes apoptosis. MicroRNA-21and PTEN mRNA were examined byqRT-PCR. Intracellular signal molecules, such as the expression of PTEN,phosphorylated PTEN, AKT, phosphorylated Akt, FOXO3a, phosphorylatedFOXO3a and FasL were detected by Western blot. All datas were normalized by theircorresponding control and presented as the group means±S.E.M. The differencesamong experimental groups were compared by unpaired t-test or one-way ANOVAused for statistical evaluation of the datas, P values <0.05were consideredstatistically significant.Results: PCR identification and DNA sequencing showed that the eukaryoticexpression vector for lenti-microRNA-21was constructed successfully. Primarycardiomyocytes culture showed pleomorphism, such as triangle, spindle, polygon andso on. The cardiomyocytes pseudopoded out, touched each other, gradually formedcell clusters and radial concentric circles of the arrangement in shape, beatedsynchronicitily and shrank significantly and powerfully, so it called functional fitcells. The beating frequency of cardiomyocytes is approximately from70times/min to100times/min. Immunofluorescence showed that more than90%of thecardiomyocytes were positive for cTnI which is a significant marker of thecardiomyocytes. After transferred lenti-microRNA-21into the cardiomyocytes, weobserved that95%of the cardiomyocytes expressed green fluorescent protein (GFP)and the strongest fluorescence by fluorescent microscope and examined theexpression of microRNA-21was significantly upregulated compared with the controlgroup by quantitative real time RT-PCR (qRT-PCR).48h after primarycardiomyocytes culture, we transferred lenti-microRNA-21into the cardiomyocytes, ·then constructed the apoptotic model of cardiomyocytes were treated with TNF-α(10ng/ml) for24h after24h transfection and then examined the apoptotic rate byHoechst33342/PI and Annexin V-FITC detection kit. The results showed that thecardiomyocytes apoptotic rate decreased significantly (P<0.05). The results suggestedthat microRNA-21could protect from TNF-α-induced cardiomyocytes apoptosis. Theresults showed that TNF-α could downregulate the expression of microRNA-21andupregulate the expression of PTEN mRNA. The changing trends of the expression ofPTEN mRNA and miR-21changing trends were opposite. On the level of gene, theresults showed that PTEN might be one of the target genes of miR-21which mightprotect from TNF-α-induced cardiomyocytes apoptosis. Compared with the controlgroup, the protein level of PTEN decreased in the group of the cardiomyocytes whichlenti-microRNA-21transferred into (P<0.05) and pPTEN, pAKTser473, pFOXO3a andFasL increased in the group of the cardiomyocytes which lenti-microRNA-21transferred into (P<0.05), but the protein level of pAKTThr308was not changed(P>0.05). Compared with the control group, the protein level of PTEN elevated in thegroup of the cardiomyocytes which treated with TNF-α (10ng/ml)(P<0.05) andpPTEN, pAKTser473, pAKTThr308, pFOXO3a and FasL in the group of thecardiomyocytes which treated with TNF-α (10ng/ml) increased significantly (P<0.05).Compared with the group of the cardiomyocytes which treated with TNF-α (10ng/ml),the protein level of PTEN decreased in the group of the cardiomyocytes whichlenti-microRNA-21transferred into and then treated with TNF-α (10ng/ml)(P<0.05)and pPTEN, pAKTser473, pAKTThr308, pFOXO3a and FasL decreased in the group ofthe cardiomyocytes which lenti-microRNA-21transferred into and then treated withTNF-α (10ng/ml)(P<0.05). Total AKT and FOXO3a were not changed among allgroups (P>0.05). The changing trends of the expression of PTEN mRNA and PTENprotein were consistent. On the level of the protein, the results also showed thatPTEN might be one of the target genes of miR-21which might protect fromTNF-α-induced cardiomyocytes apoptosis. The change trends of the expression ofpPTEN, pAKTser473, pFOXO3a and FasL were consistent. The results suggestedthat microRNA-21might protect from TNF-α-induced cardiomyocytes apoptosisthrough the regulation of PTEN/AKT/FOXO3a pathway. Conclusion: The eukaryotic expression vector for microRNA-21is constructedsuccessfully. MicroRNA-21could protect from TNF-α-induced cardiomyocytesapoptosis through the regulation of PTEN/AKT/FOXO3a pathway and could be anew target for the diagnosis and therapy of various cardiovascular diseases in thefuture.