The Research And Identification Of The Expression And Function Of MicroRNAs In Normal And Hypertrophy Cardiac Tissue

In this study,the variation of microRNA expression in normal and hypertrophy myocardial cells was found by microarray analysis.Combining with bioinformatics prediction,the clues of microRNAs involving in cardiac hypertrophy was studied.It was found the microRNAs play a regulation role in cardiac hypertrophy by research methods for microRNA function.The theoretical basis for illustrating the function of microRNA in cardiac development,hypertrophy and arrhythmia was provided.It may be the new idea and strategy for the treatment of cardiovascular diseases.Research Purpose:(1) The model of cardiac hypertrophy in rats was performed by partial narrowing abdominal aortic surgery(2) Looking for specific microRNAs by microarray analysis.(3) The effects of specific microRNA on H₉c₂ cells.(4) To explore the mechanism of specific microRNA involved in cardiac hypertrophy.Methods:(1) Twenty male rats were randomly divided into normal,operation and sham-operation groups.Partial coarctation operation performed in the abdominal aorta.At the seven week after banding,the ratio of weight of heart to body was calculated and HE staining methods was used to display collagen content of heart in rats.(2) After seven weeks,the hypertrophy cardiac tissue was taken out,RNA was exacted by Trizol Method and the expression of microRNA was quantitative detected by microarray and miRNA assay kit.(3) After H₉c₂ cells being exposed to Angâ…¡(10^-6mol / L) for 48 hr,the diameter of myocardial cells and he expression of microRNA were measured.(4) To construct specific microRNA expression vector,using a mock vector as control,then to transfect into H₉c₂.Real-time PCR method was employed to detect the expression ofβ—myosin,α-actin,ANF,BNF genes.Immunocytochemical method was used to detect the variation of cytoskeleton proteinsα-actinin.AnnexinV-PE Kit was used to detect the apoptosis of H₉c₂.Bioinformatics method was used to predict the target gene of miR-350,Using western blot to detect the variation of target gene in protein level in H₉c₂.RT-PCR method was used to detect the variation of target gene in gene level.Immunocytochemical method was used to observe the transferring nuclear of NFATc regμlated by target genes.Results(1)After seven weeks,the significant change were found in IVSTd (2.40±0.03vs 1.87±0.21,mm),LVPWTd(2.52±0.24vs 1.77±0.17,mm) and LVDd(6.83±0.28vs4.50±0.19,mm)(P＜0.01) comparing with the normal group.However,to contrast to normal group,the sham-operation group have no significant difference in that three index(P＞0.05);HE showed that cadiocyte hypertrophy in heart tissue of the operation group.(2) The expression of micro350 in the hypotrophy model was significant higher than that in control by Microarray analysis and Real-time PCR.However,there is no significant change from expression of miR-350 in H₉c₂ induced by Angâ…¡than that not induced by Angâ…¡by real-time PCR.(3) After seventy-two hours,there are increase of over-expression of miR-350 in cardiac hypertrophy marker genes of theβ—myosin,α-actin,ANF and BNF by real-time quantitative PCR.the cytoskeletal proteinsα-actinin appeared obvious were increased by immunocytochemical detection.There are apoptosis of cardiac myocyte detected by Annexin V-PE kit(4) Using bioinformatics methods to predict the differences in the expression of miR-350 target genes of the JNK and MAPK14.(5)The level of JNK and MAPK14 protein was decreased by Western blot in H₉c₂ cells with microRNA-350 over-expression,while the mRNA level has no change.At the same time,JNK regulation of nuclear NFATc to the markedly was increased.Conclusions(1) The expression of microRNA has a significant difference change between the cardiac hypertrophy and control.(2) Overexpression of miR-350 can found in cardiac hypertrophy in H₉c₂. (3) The target gene(JNK and MAPK14) of miR-350 can be involved in cardiac hypertrophy regulated by miR-350.