Hsa-miR-626Regulates The Expression Of Lipoprotein(a) In Hep G2Cells

Objective: Lipoprotein(a)[Lp(a)], consisting of apolipoprotein B-100(apo B-100)and the unique constituent apolipoprotein(a)[apo(a)], is mainly synthesized in liver.Several clinical studies have shown that elevated levels of Lp(a) in plasma are anindependent risk factor for atherosclerosis-related events like prematurecardiovascular disease, stroke. Lp(a) plasma levels are relatively resistant to manypharmacologic and nonpharmacologic agents, plasmapheresis is the only able methodto reduce Lp(a) levels over50%.The only mechanism in vivo we know which caneffect Lp(a) plasma levels is some single nucleotide polymorphisms(SNPs) of apo(a)gene LPA. Seveval microRNAs(miRNAs) which relate to Atherosclerosis(As) andlipid metabolism have been found.The aim of this research is to screen miRNAswhich probably can regulate Lp(a) synthesis by bioinformatics analysis and verify ifthese miRNAs can down-regulate the expression level of apo(a) in hepatocyte.Methods:Analyze the secondary structure of the sequence of3’-untranslatedregion(3’-UTR) of LPA and predict miRNAs which probably can effect LPA bybioinformatics analysis.Select a cell line which highly express protein apo(a) bywestern blot, and check the transfection efficiency of miRNA mimics by fluorescencedetection.Detect LPA mRNA expression of Hep G2cells6h,12h and24h aftertransfection of negative control miRNA mimic and the other miRNA mimicsrespectively by RT-PCR; Detect apo(a) expression of Hep G2cells24h,36h and48hafter transfection of negative control miRNA mimic and the other miRNA mimicsrespectively by western blot.Detect apo(a) expression of Hep G2cells48h aftertransfection of negative control miRNA mimic, miR-626mimic,miR-626mimic+miR-626inhibitor and miR-626inhibitor respectively by western blot. Conduct the target gene verification experiment of miR-626by luciferase reportergene expression analysis.The experimental data are measured by mean±SD,statistical analysis results and graphs are generated by Graphpad Prism5.0.1,confidential interval is95%，a significant difference is measured by P<0.05.Results:Bioinformatics analysis prediction indicate the miRNAs which may effectthe expression level of LPA are as follows: miR-655, miR-590-3p, miR-519a,miR-519b-3p, miR-338-3p, miR-519c-3p, miR-590-5p, miR-425and miR-626.HepG2cell line highly express apo(a), so it can be used in this research.MiR-655mimicand miR-590-5p mimic can slightly down-regulate apo(a) expression of Hep G2;however, miR-626mimic can significantly down-regulate apo(a) expression of HepG2cells; No significant difference of LPA mRNA express level is detected inmiR-655, miR-590-3p, miR-519a, miR-519b-3p, miR-338-3p, miR-519c-3p,miR-590-5p, miR-425and miR-626mimics transfection groups when compared tonegative control.The apo(a) espression level of miR-626mimic+miR-626inhibitorcotransfection group is significantly down-regulated when compared to miR-626mimic transfection group, and the apo(a) espression level of miR-626inhibitortransfection group is significantly up-regulated when compared to negative controlgroup.After cell lysis, the fluorescent intensity of miR626transfection group issignificantly lower than that of control group.Conclusions:MiR-626can significantly down-regulate the apo(a) expression in HepG2cells.The mechanism of miR-626down-regulate the apo(a) expression in Hep G2cells is miR-626directly bonds to3’-UTR of LPA mRNA and inhibits the translationof LPA mRNA.