Down-regulation Of Apo(a) By Both MiR-23b-3p And MiR-125b-5p Targeting Ets1in HepG2Cells

Objective: High plasma concentrations of lipoprotein(a)[Lp(a)], which is encoded bythe APOA gene increase an individual’s risk of developing diseases, such as coronaryartery diseases, restenosis, and stroke. apo(a) is dispensable component of Lp(a) andcontributes the plasma concentration of Lp(a). Ets1as important positive trans-actingelement of apo(a) gene serves as a target gene of several miRNAs, as follows: miR-193b,miR-125b-5p, miRNA200b, microRNA-1and microRNA-499. To date, there is noeffective way to lower plasma Lp(a). The aim of our study is to screen one or few miRNAsnotably regulating apo(a) synthesis through modulating target gene Ets1. Certainly thesemiRNAs should be expressed in hepatic cells.Methods: Transfection of miRNA mimic, miRNA inhibitors, siRNA and Ets-1expressing plasmid was performed using Lipofectamine2000(Invitrogen). RT-PCR wasused to examine the level of apo(a) mRNA. Western Blot Analysis was for detection ofprotein level including apo(a) and Ets1. Immunocytochemistry was further for apo(a)analysis. apo(a) concentration of the medium was analysed by ELISA. Dual LuciferaseReporter Assays further was used to verify the miRNA’s target. miRNA microassayassayed the expression spectrum of miRNA. miRNA real time RT-PCR quantified themiRNA in cells.Results: In this study, we identified apo(a) and Ets1were differential expressed betweenSMMC7721and Hep G2cells, which were inverse correlated to the differential expressedmiRNAs predicted targeting the Ets1. These differential expressed miRNAs were asfollows, miR-125b-5p, miR-23b-3p, miR-26a-5p and miR-423-5p. On the basis, weconfirmed that miR-125b-5p and miR-23b-3p can lower apo(a) level by binding to target gene Ets1through the miRNA mimics delivery HepG2. We also performed a dualluciferase assay to verify the Ets1serving as a shared target gene of miR-125b-5p andmiR-23b-3p.Conclusion: As crucial transcriptional factor, Ets1regulated the expression of apo(a)gene. miR-125b-5p and miR-23b-3p lowered apo(a) through post-transcription suppress bymean of targeting Ets-13’UTR.