LncRNA FTX Attenuates Hypoxia/Reoxygenation-induced H9c2 Cell Damage By Regulating MiR-410-3p/Fmr1 Molecular Axis

Background and purpose:Ischemic heart disease was a common clinical disease,which can promote the ischemic myocardial tissue to restore blood supply through surgery and other methods.However,the occurrence of myocardial ischemia-reperfusion injury can reduce the therapeutic effect.The mechanism played an important role in preventing myocardial ischemia-reperfusion injury.LncRNA could regulate the expression of target genes by acting as a sponge molecule of miRNA,thereby participating in the process of cardiovascular disease development.LncRNA FTX was under-expressed in myocardial cell injury and may be involved in the process of myocardial ischemia-reperfusion injury,but its mechanism of action had not been fully elucidated.Bioinformatics analysis showed that miR-410-3p may be a target gene of FTX,and miR-410-3p was highly expressed in cardiac muscle cells and may play an important regulatory role.Bioinformatics analysis showed that Fmr1 may be a target gene of miR-410-3p.Fmr1 was low-expressed in lipopolysaccharide-induced cardiomyocytes and may have protective effects on cardiomyocyte injury.But whether FTX could regulate the molecular axis of miR-410-3p/Fmrl and thus play a role in cardiomyocyte injury has not been elucidated.This study includes the following three parts:Part 1:The effects of LncRNA FTX on H/R-induced H9c2 cell viability,apoptosis,oxidative stress and inflammation.Part 2:LncRNA FTX targeted regulation of miR-410-3p regulates H/R-induced H9c2 cell injury.Part 3:miR-410-3p targets Fmr1 to regulate H/R induced H9c2 cell damage.Method:Twenty patients with myocardial ischemia-reperfusion injury were selected as I/R group,and 20 healthy volunteers were selected as Normal group.H9c2 cells of rat cardiomyocytes normal culture were used as Normoxia group.H9C2 cells were treated with hypoxia/reoxygenation and divided into H/R group.H9C2 cells were transfected with pcDNA,pcDNA-FTX,si-NC,and si-FTX and then treated with hypoxia/reoxygenation,and divided into H/R+ pcDNA group,H/R+FTX group,and H/R+si-NC Group,H/R+ si-FTX group.H9c2 cells were transfected with pcDNA-FTX and miR-NC,pcDNA-FTX and miR-410-3p mimics,and then treated with hypoxia/reoxygenation,and divided into H/R+FTX+miR-NC group,H/R+FTX+miR-410-3p group.H9c2 cells were transfected with miR-NC,miR-410-3p mimics,anti-miR-NC,anti-miR-410-3p,and then treated with hypoxia/reoxygenation.They were divided into H/R+miR-NC group,H/R+miR-410-3p group,H/R+anti-miR-NC group,H/R+anti-miR-410-3p group.H9C2 cells were transfected with anti-miR-410-3p and si-NC,anti-miR-410-3p and si-Fmr1,si-FTX and anti-miR-NC,si-FTX and anti-miR-410-3p.After hypoxia/reoxygenation treatment,they were divided into H/R+anti-miR-410-3p +si-NC group,H/R+anti-miR-410-3p+si-Fmr1 group,and H/R+si-FTX+anti-miR-NC group,H/R+si-FTX+anti-miR-410-3p group.The dual luciferase report experiment verified the targeted binding relationship between FTX and miR-410-3p,and the targeted binding relationship between miR-410-3p and Fmr1.Detect LDH1 MDA,SOD,GSH-PX according to the kit instructions.CCK-8 method was used to detect cell viability,and flow cytometry was used to detect the apoptosis rate.Transwell chamber experiments were used to detect cell migration and invasion.qRT-PCR was used to detect the expression of FTX,miR-410-3p,and Fmr1 mRNA.Western blot was used to detect Bcl-2,Bax,cleaved-casp-3 protein expression.The levels of inflammatory factors IL-1β,IL-6 and TNF-α were detected by ELISA.Result:Compared with the Normal group,the expression of FTX in serum in the I/R group was significantly reduced(P<0.05).After H/R treatment,the expression of FTX in myocardial cells was significantly reduced(P<0.05),the expression level of miR-410-3p was significantly increased(P<0.05),and the expression of Fmr1 mRNA and protein was significantly reduced(P<0.05).After H/R treatment,the myocardial cell viability was significantly reduced(P<0.05),the myocardial cell apoptosis rate was significantly increased(P<0.05),the number of migrating cells and invasive cells was significantly reduced(P<0.05),and the expression of Bcl-2,Bax and cleaved-casp-3 protein were significantly increased(P<0.05),the activity of LDH was significantly increased(P<0.05),and the content of MDA was significantly increased(P<0.05),the activities of SOD and GSH-PX were significantly reduced(P<0.05),and the levels of IL-1β,IL-6 and TNF-α were significantly increased(P<0.05).After FTX overexpression,the myocardial cell vitality increased significantly(P<0.05),the myocardial cell apoptosis rate was decreased significantly(P<0.05),the number of migrating cells and invasive cells were increased significantly(P<0.05),and the expression of Bcl-2 protein was significantly increased(P<0.05),the expression of Bax,cleaved-casp-3 protein were significantly reduced(P<0.05),the activity of LDH significantly reduced(P<0.05),the content of MDA was significantly reduced(P<0.05),the activitives of SOD,GSH-PX were significantly increased(P<0.05),and the levels of IL-1β IL-6 and TNF-α were decreased(P<0.05).The double luciferase reporting experiment confirmed that FTX can target miR-410-3p and negatively regulate the expression and activity of miR-410-3p.FTX was negatively correlated with miR-410-3p(r=-0.63 8,p=0.0025).After co-transfection with miR-410-3 pmimics and FTX overexpression,the cell viability was significantly reduced(P<0.05),the apoptosis rate was significantly increased(P<0.05),and the number of migrating cells and invading cells was significantly reduced(P<0.05),the expression of Bcl-2 protein was significantly reduced(P<0.05),the expression of Bax and cleaved-casp-3 protein were significantly increased(P<0.05),the activity of LDH was significantly increased(P<0.05),and the content of MDA was significantly increased(P<0.05),the activitives of SOD,GSH-PX were significantly reduced(P<0.05),and the levels of IL-1β,IL-6,and TNF-α were significantly increased(P<0.05).The double luciferase reporting experiment confirmed that miR-410-3p can target Fmrl and can negatively regulate Fmr1 expression and activity.Fmr1 was negatively correlated with miR-410-3p(r=-0.6753,p=0.0011).After inhibiting the expression of miR-410-3p,the cell viability was significantly increased(P<0.05),the apoptosis rate was significantly reduced(P<0.05),the expression of Bcl-2 protein was significantly increased(P<0.05),the expression of Bax,cleaved-casp-3 protein were significantly reduced(P<0.05),the number of migrating cells and invasive cells were significantly increased(P<0.05),the activity of LDH was significantly reduced(P<0.05),and the content of MDA was significantly reduced(P<0.05),the activities of SOD and GSH-PX were significantly increased(P<0.05),and the levels of IL-1β,IL-6,and TNF-α were significantly reduced(P<0.05).anti-miR-410-3p and si-Fmr1 co-existed after transfection,the effects of inhibiting miR-4 10-3p expression on myocardial cell viability,apoptosis,migration,oxidative stress and inflammatory response were significantly attenuated(P<0.05).Conclusion:1.The over expression of LncRNA FTX can alleviate the injury of cardiomyocytes induced by hypoxia/reoxygenation.2.LncRNA FTX can reduce the injury of cardiomyocytes induced by hypoxia/reoxygenation by inhibiting the expression of mir-410-3p.3.LncRNA FTX can alleviate the injury of cardiomyocytes induced by hypoxia/reoxygenation by regulating mir-410-3p/Fmr1 molecular axis.