Effect Of Circulating MiRNA And MiR-31 On Essential Hypertensive Left Ventricular Hypertrophy

Objective:To investgate the characteristic expression of circulating mi RNA in patients of primary hypertension with left ventricular hypertrophy and search for a new kind of biological marker for diagnosis and prediction of hypertensive left ventricular hypertrophy.Methods:Screening 5 patients of primary hypertension with left ventricular hypertrophy and 5 patiets of primary hypertension without left ventricular hypertrophy as study objects.mi RNA was extracted from plasma samples, which were collected from the two groups of patients.Then,expression of circulating mi RNA was detected by high-throughput next-generation sequencing technology. Sequencing data were analyzed to filter out the differentially expressed mi RNAs, whose function was preliminary investgated by bioinformatics analysis and functional enrichment analysis to their target genes.Results:A total of 64 mi RNAs expressed differentially in patients of essential hypertension with left ventricular hypertrophy were screened out, including 35 upregulated and 29 downregulated.Bioinformatics analysis revealed function of the differentially expressed mi RNAs was mainly related to adrenergic signaling in cardiomyocytes,neuroactive ligand-receptor interaction,complement and coagulation cascades,fatty acid degradation,which are closely related to left ventricular hypertrophy. Of the differentially expressed mi RNAs, hsa-mir-100, hsa-mir-202, hsa-mir-181a-2, hsa-mir-450 b were proven to have more functions by functional enrichment analysis.Conclusion:mi RNA expression profile is present in peripheral circulation of patients of essential hypertension with left ventricular hypertrophy,which provide candidate biomarkers for diagnosis and prediction of left ventricular hypertrophy in patients with essential hypertension.Objective:To observe expression of mi R-31 and LATS2 in the hypertrophy process of cardiomyocytes to provide information for subsequent experiments.Methods:Primary rat cardiomyocytes were isolated and cultured in vitro, and hypertrophic model of cardiomyocyte was constructed by administering 10-6 mol/L Ang II. Hypertrophy genes ANP、β-MHC were detected by q RT-PCR and cell morphology was observed by fluorescence staining to F-actin to confirm the hypertrophic model of cardiomyocyte. Expression of mi R-31 and LATS2 was detected by q RT-PCR in hypertrophic cardiomyocytes,and LATS2 protein was further detected by western blot.Results: Hypertrophic model of cardiomyocyte was successfully constructed by administering 10-6 mol/L Ang II, with hypertrophy genes ANP, β-MHC up-regulated on the third day and cell surface area increased significantly on the fifth day after administration of Ang II. Accompanied with hypertrophic change of cardiomyocytes, mi R-31 was significantly up-regulated, while LATS2 in gene and protein levels were reduced obviously.Conclusion: Hypertrophic model of cardiomyocyte could be successfully constructed by administering Ang II; Accompanied with hypertrophic change of cardiomyocytes, mi R-31 was significantly up-regulated, while LATS2 in gene and protein levels were reduced obviously.Objective:To investgate regulation of mi R-31 on LATS2 and cardiomyocyte hypertrophy by down-regulating its expression in hypertrophic cardiomyocytesMethods : Lentiviral vector expressing inhibitor of mi R-31(LV-mi R-31-inhibitor) was constructed, and then transfected into the hypertrophic model of cardiomyocyte in vitro. Hypertrophy genes ANP 、 β-MHC were detected by q RT-PCR and cell morphology was observed by fluorescence staining to F-actin to analyze the changes of hypertrophic cardiomyocytes. Expression of mi R-31 and LATS2 was detected by q RT-PCR in the changing process of hypertrophic cardiomyocytes,and LATS2 protein was further detected by western blot.Results: Mi R-31 expression of the hypertrophic model of cardiomyocyte was down-regulated sharply after transfection with LV-mi R-31- inhibitor,which caused hypertrophy genes ANP, β-MHC down-regulated and cell surface area significantly reduced. Accompanied with the changes of hypertrophic cardiomyocytes, LATS2 increased slightly at gene level with no significant difference, but increased significantly in protein level.Conclusion: Hypertrophic cardiomyocytes could be reversed to some extent by down-regulating expression level of mi R-31;Mi R-31 may regulate the genesis of cardiac hypertrophy by acting on LATS2.Objective:To confirm directly target regulation of mi R-31 on LATS2Methods:Dual luciferase gene reporter vectors LATS2-3 ’UTR and LATS2-3’ UTR-MU were constructed by respectively inserting wild-type and mutant-type gene fragments of LATS2-3 ’UTR to double luciferase tool vector. Luciferase reporter plasmids or dual luciferase empty vector(LATS2-3 ’UTR-NC) were transfected into 293 T cells with the plasmid overexpressing mi R-3l or empty vector without mi R-3l, then luciferase activity was examed to identify target regulation of mi R-31 on LATS2.Results: The luciferase activity from 293 T cells co-transfected with the plasmid overexpressing mi R-3l and LATS2-3 ’UTR dual luciferase reporter plasmid was significantly reduced compared with 293 T cells co-transfected with the plasmid overexpressing mi R-3l and LATS2-3 ’UTR-MU dual luciferase reporter plasmid.Conclusion: Mi R-3l achieve its target regulation on LATS2 through derectly binding to 3 ’UTR of LATS2.