MEG3 Inhibits Apolipoprotein(a) Expression In HepG2 Cells Through MiR-125a-5p/TET2 Pathway

OBJECTIVES Previous studies found that miR-125a-5p inhibits the expression of demethylase TET2,and TET2 can inhibit the expression of apo(a)in HepG2 cells.Bioinformatics analysis shows that MEG3 can interact with miR-125a-5p.Due to the complementarity,there is a possibility that MEG3 inhibits mi R-125a-5p.It is speculated that MEG3 can inhibit the expression of apo(a)in HepG2 cells by inhibiting miR-125a-5p and up-regulating TET2.To detect the expression of MEG3 on HepG2 cells with high expression of apo(a),and to analyze the expression of apo(a)after transfection of MEG3,and to study the regulation of apo(from MEG3/mi R-125a-5p/TET2 pathway).a)The molecular mechanism.METHODS In this study,miRs targeted to TET2 mRNA 3'-UTR were firstly analyzed using the bioinformatics online software Targetscan.The binding of the miRs was determined by the degree of conservation of the binding and the free energy value,and the candidate miRs were obtained and then reported by luciferase.The gene system was analyzed and verified for its targeted targeting;the fluorescein reporter enzyme system was then used to analyze the targeted binding of MEG3 to miR-125a-5p.The cell viability was measured by MTT assay.The expression of MEG3 in Hep G2 cells with high expression of apo(a)and SMMC7721 cells with low expression of apo(a)was detected byreal-time quantitative PCR(qRT-PCR);MEG3 was transfected into Hep G2 cells,western blot The expression of apo(a)and TET2 was detected by qRT-PCR,and the expression of TET2 was silenced by small interfering RNA technology.The statistical analysis of all data was expressed as mean±standard deviation(±SD).Graphpad Prism5.0.1 was used to analyze and plot the data,and the 95% confidence interval was selected.P<0.05 was considered significant.RESULTS There were three strongly conserved hsa-miR-125a-5p binding sites in the 3'-UTR of human TET2 mRNA.The on-line software BiBiserv2 RNAhybrid showed the binding of TET2 mRNA 3'-UTR to hsa-miR-125a-5p.The free energies below the threshold(-10 kcal/mol)were-25.5 kcal/mol,indicating that the TET2 mRNA 3'-UTR has a stable binding to hsa-miR-125a-5p.The results of luciferase reporter gene system analysis showed that the transfection of hsa-miR-125a-5p significantly decreased the expression level of the wild-type psiCHECKTM-2-TET2-WT 3?UTR plasmid,while the mutant psiCHECKTM-2-TET2-Mut 3' UTR plasmid expression has no effect,suggesting that TET2 is the target gene for hsa-miR-125a-5p,hsa-miR-125a-5p inhibits wild-type recombination through targeted binding to TET2 mRNA 3'-UT Expression of the plasmid.Bioinformatics analysis showed that MEG3 and hsa-miR-125a-5 could complement each other.Systematic analysis of luciferase reporter gene confirmed the presence of MEG3 binding to hsa-miR-125a-5p.miR microarray results showed that the expression of hsa-miR-125a-5p was increased in Hep G2 cells,which was 1.5 times higher than that in the control group.qRT-PCR analysis showed that apo(a)highly expressed HepG2 cells and low expression of apo MEG3 is expressed in SMMC7721 cells of(a),but the expression level of MEG3 is significantly lower than that of the latter.MTT analysis results showed that MEG3 transfected 72 h before thetoxicity of HepG2 cells,MEG3 transfection inhibited apo(a)expression.mechanism study found that MEG3 transfection significantly down-regulated the expression of miR-125a-5p,significantly increased TET2 expression and activity;miR-125a-5p added mimics can reverse the down-regulation of MEG3 on apo(a),and inhibit the expression of TET2 And activity,but can be reversed by inhibitors of miR-125a-5p;TET2 silencing can reverse the down-regulation of apo(a)by MEG3.CONCLUSIONS MEG3 dose-and time-dependently down-regulated the expression of apo(a)in Hep G2 cells;MEG3down-regulated the expression of apo(a)in HepG2 cells through the miR-125a-5p/TET2 pathway.