A Preliminary Study Of The Effect Of MicroRNA On LPPR4 In The Pathogenesis Of Coronary Artery Disease

BackgroundCoronary artery disease,abbreviated CAD,is a main source of medical and economic burden world-wide.Early onset coronary artery disease,EOCAD,is defined as CAD onset before 55 years old for male or 65 years old for female patients,respectively.Hereditary factor plays a significant role in EOCAD pathogenesis.We collected a pedigree of Han EOCAD patients showing euchrosomal dominant heredity features.Previous works elucidated that single nucleotide deletion in the 3' untranslated region(3'UTR)of LPPR4 may be the driver gene of this pedigree,and such mutation caused decreased expression of LPPR4 protein.In order to explore the mechanism of EOCAD in this pedigree,we first determined the microRNA regulation of LPPR4 gene expression.The search result provided by target prediction tool microRNA.org-Targets and Expression validated that the candidate mutation was located in the cognitive sequence of miR-450-5p.Additionally,previous research report that miR-103a and miR-155-5p are closely related with CAD.Analysis of miRNA-DNA target-effect relationship implied their potential interactions with LPPR4 3'UTR region.ObjectiveDetermine the relationship between the three target miRNA and LPPR4 mRNA and protein expression and phenotype.Validate the interaction between target miRNA and LPPR4 3'UTR fragment and downstream expression regulating mechanism.MethodsDue to the high-level correlation between the expression level and its corresponding downstream effects of miRNA,the primary expression levels of the three target miRNAs were first measured by quantitative real-time PCR.Interventions were then carried out by transfecting HASMC cells and HEK-293 cells with miRNA mimics.Western blot and transwell migration assay were then subsequently conducted to validate the correlation between miRNA and LPPR4 expression level.In the next section we tried to ascribe the effect miRNAs have on LPPR4 expression to the interaction between miRNA and LPPR43'UTR region,hence the dual-luciferase assay was conducted and corresponding plasmids were built.In the last section we investigated whether mRNA stability was affected during the interaction of miRNA and 3'UTR region by carrying out the mRNA decaying assay.ResultsqPCR showed that miR-103a has the highest expression level among the three target miRNAs.In the follow-up western blot and transwell assay in the cell lines transfected with miR-103a and miR-450-5p mimics,results indicate that miR-450-5p may impose an down-regulating effect on the expression of LPPR4 while miR-103a seem to be having an upregulating effect.In the dual-luciferase assay,results indicate that both miR-103 a and miR-450-5p showed a tendency of binding with the target 3'UTR region of LPPR4.In the mRNA decay assay aimed at testing mRNA stability,the decaying rate of LPPR4 mRNA seem to be lower than that of the control group,but no significant difference was observed between the miR-450-5p group and control group.ConclusionmiR-103a may upregulate the protein expression level of LPPR4 and hinder the migration of vascular smooth muscle cells,while miR-450-5p seem to be having an opposing effect.Both miR-103a and miR-450-5p seem to be interacting with LPPR4 gene through the specific binding sites on the 3'UTR region.miR-103a is correlated with stabilizing LPPR4 mRNA.