Research Paper Volume 12, Issue 10 pp 9014—9030

Figure 4. The expression of each microRNA in primary PASMCs of PH rats induced by MCT was verified by RT-qPCR. (A) RT-qPCR was used to detect the expression of six microRNAs that may act on HK-II in PASMCs of PH: rno-miR-411-5p, rno-miR-210-3p, rno-miR-532-3p, rno-miR-125a-5p, rno-miR-322-3p, rno-miR-708-5p. (B) Dual luciferase assay confirmed that miR-210-3p and miR-125a-5p can inhibit luciferase activity. The 3'-UTRs containing normal HK-II mRNA were cloned into the MCS (Multiple cloning site) in the pmirGLO vector to construct the HK2-125/210-pmirGLO vector. This vector was then co-transfected into 293T cells with these two microRNA mimics to determine whether the microRNA acts on the target gene of plasmid by detecting luciferase activity. (C) The bioinformatics analysis targeting algorithms (TargetScan and http://microRNA.org) showed that miR-125a-5p contained candidate binding sites in the 3’-UTR of HK-II mRNA. (D) The results of luciferase reporter gene assay showed that miR-125a-5p mimic significantly inhibited firefly luciferase activity of wild-type plasmid vector, while firefly luciferase activity of mutant plasmid vector hardly changed. (E) Transfection with miR-125a-5p mimic significantly increased the mRNA expression of miR-125a-5p. (F) The expression of HK-II protein in PASMCs was significantly increased in PH group, and the expression of HK-II in PH+miR-125a-5p mimic group was significantly decreased. The results demonstrated that miR-125a-5p negatively regulates the expression of HK-II in PASMCs. (*P<0.05, compared with control group, #P<0.05, compared with WT-miR-125a-5p mimics group, ¥P<0.05, compared with PH group).