MiR-143-3p Targeting Hexokinase 2 Inhibits Hypoxia-Induced Proliferation Of Pulmonary Arterial Smooth Muscle Cells

Objectives:、To investigate the effects and mechanism of microRNA and its target gene hexokinase 2(HK-Ⅱ)on hypoxia-induced proliferation of pulmonary artery smooth muscle cells.Methods:The primary PASMCs was detached from the normal male Sprague-Dawley rats,α-SMA antibody immunochemical fluorescence staining was performed to identify PASMCs.The microRNAs interacting with HK-Ⅱwere predicted by TargetScan,and identified by luciferase assay.After hypoxia treatment,PASMCs were intervened by the identified microRNA’s mimic.The protein level of HK-Ⅱin PASMCs was determined by WB(Western blot)analysis.The level of microRNA was detected by RT-qPCR(Real-time quantitative polymerase).The proliferative level of PASMCs was detect by CCK-8(Cell Counting Kit-8).Determinations of glucose desorption and glucose production by anthrone-sulfuric acid colorimetry and lactic acid detection kits to reflect glycolysis levels.Results:(1)The proliferation level of PASMCs was significantly increased after 48 hours hypoxia treatment;(2)The HK-Ⅱprotein level of PASMCs in hypoxic environment was increasing with time.The HK-Ⅱlevel was significantly increased at 6 hours,(P<0.05),and reach to peak at 12 hours;(3)TargetScan predicted 7 different microRNAs,the result of RT-qPCR shows 6 of them were down regulated during hypoxia in PASMCs.Combined with published articles,there are 3 microRNAs including miR-139-5p,miR-143-3p and miR-125a-5p were selected that targeted on HK-Ⅱ.Dual luciferase assay confirmed all these 3 microRNAs inhibit the Luciferase activity,directly target on HK-Ⅱ,and the miR-143-3p has the highest inhibition rate;(4)In PASMCs,hypoxia caused a down-regulation of miR-143-3p expression,an increase in HK-Ⅱ expression level,glucose consumption and lactate production.Transfection of miR-143-3p mimic,hypoxia-induced HK-Ⅱ expression was inhibited(p<0.05),glucose consumption and lactate production were significantly decreased(p<0.05);CCK8 detection suggested that up-regulation of miR-143-3p expression inhibits the hypoxia-induced proliferation of PASMCs(p<0.05).Conclusion:In hypoxia-induced PASMCs,the expression of miR-143-3p was decreased and HK-Ⅱ was increased;miR-143-3p can inhibit the glycolysis and proliferation by regulating the expression of HK-Ⅱ.