The Role Of MiR-301b-3p Targeting PPARG/NF-κB Signal Pathway In Myocardial Ischemia/Reperfusion Injury

Acute myocardial infarction(AMI)is one of the major diseases that cause death in patients in the worldwide,usually due to sudden occlusion of the epicardial coronary artery.The main treatment for acute myocardial infarction is myocardial reperfusion.However,myocardial reperfusion itself induces myocardial damage,a phenomenon known as myocardial ischemia/reperfusion injury(MI/RI).MI/RI may cause myocardial stunning,ventricular arrhythmia,hemodynamic abnormalities,long-term heart failure,etc.The inflammatory response plays an important role in MI/RI.Peroxisome proliferator-activated receptor gamma(PPARG)is a subtype of peroxisome proliferator-activated receptors(PPARs)and belongs to the nuclear transcription factor superfamily.NF-κB is a class of transcription factors involved in the regulation of inflammatory responses.PPARG can alleviate inflammatory response by inhibiting NF-κB signaling pathway.MicroRNA(miRNA)are small,non-coding RNAs of about 22 nucleotides in length,and are widely found in animals,and can regulate gene expression by degradation of m RNA or translational repression.MiRNAs are involved in the regulation of inflammation in rat MI/RI.We predicted that PPARG may be a target gene for miR-301b-3p by using TargetScan,DIANA and mirDB bioinformatics websites.In addition,our previous study found that expression ofmiR-301b-3p was increased in rat MI/RI.Therefore,we proposed the following hypothesis: low expression of miR-301b-3p may alleviate inflammatory response induced by myocardial ischemia/reperfusion in rats,and improve cardiac function through inhibition of NF-κB signaling pathway via targeting PPARG.To validate this hypothesis,the study was divided into three parts.In the first part,to detect the expression of miR-301b-3p and PPARG in rat MI/RI.In the second part,to demonstrate whether PPARG was a target gene for miR-301b-3p.In the third part,we investigated the role of low expression of miR-301b-3p targeting PPARG/NF-κB signaling pathway in rat MI/RI.Part I Detection of PPARG and miR-301b-3p expression in rat myocardial ischemia/reperfusion injuryObjective: To detect expression of miR-301b-3p and PPARG in rat MI/RI.Methods: First,mirDB,Target Scan,and DIANA websites were used to predict miRNA that might bind to PPARG.Second,16 SD rats were randomized into two groups(8 in each group),sham group and ischemia/reperfusion(I/R)group.The SD rat MI/RI model was constructed by ligation and loosening of the left anterior descending of the rat coronary artery.The sham group threaded but did not ligature the left anterior descending coronary artery,while the I/R group ligated the left anterior descending coronary artery for 1 hour and then released for 6 hours.Finally,after the establishment of the model of MI/RI,HE staining was used to observe the myocardial tissue damage.The serum content of cTnT were detected by ELISA,and the expression of miR-301b-3p and PPARG were detected by qRT-PCR.Results: PPARG might be a potential target gene for miR-301b-3p in targetscan,DIANA,and mirDB websites.The results of HE staining showed that neat and orderly myocardial fibers,no myocardial necrosis,no interstitial edema,and no neutrophil infiltration were observed in the sham group,while myocardial fiber disorder,myocardial cell necrosis,interstitial edema,and neutrophil infiltration observed in the I/R group.The results of ELISA showed that compared with the sham group,the serum content of cTnT were significantly increased in the I/R group(P < 0.05).The results of qRT-PCR showed that compared with the sham group,the expression of PPARG was significantly decreased in the I/R group(P< 0.05),while the expression of miR-301b-3p was significantly increased(P <0.05).Conclusion: 1.Myocardial tissue damage was observed and serum content of cTnT increased after myocardial ischemia/reperfusion in rats.2.The expression of PPARG was decreased in SD rat MI/RI,while the expression of miR-301b-3p was increased.Part Ⅱ Verify whether PPARG was the target gene of miR-301b-3pObjective: To verify whether PPARG was the target gene of miR-301b-3p.Methods: First,TargetScan bioinformatics website was used to predict the binding site of miR-301b-3p to PPARG.Secondly,the dual-luciferase reporter gene assay was used to verified whether PPARG is the target gene of miR-301b-3p.Finally,H9C2 cardiomyocytes were transfected with miR-301b-3p negative control and miR-301b-3p inhibitor,respectively.Theexpression of miR-301b-3p and PPARG were detected by qRT-PCR,and PPARG protein expression was further detected by western blot.Results: PPARG-3’UTR region has a binding site to miR-301b-3p in targetscan website.The results of the dual-luciferase reporter gene assay showed that compared with the miR-301b-3p mimics+mutant-type PPARG group,the luciferase activity of the miR-301b-3p mimics+wild-type PPARG group was significantly reduced(P < 0.05).The results of qRT-PCR showed that compared with the miR-301b-3p negative control group,the expression of miR-301b-3p in the miR-301b-3p inhibitor group was significantly decreased(P < 0.05),while the m RNA expression of PPARG were significantly increased(P < 0.05).The results of western blot showed that compared with miR-301b-3p negative control group,the protein expression of PPARG in miR-301b-3p inhibitor group was significantly increased(P < 0.05).Conclusion: 1.PPARG is a validated target gene for miR-301b-3p in HEK293 T cells.2.Low expression of miR-301b-3p can increase the expression of PPARG in H9C2 cardiomyocytes.Part Ⅲ The role of low expression of miR-301b-3p targeting the PPARG/NF-κB signaling pathway in myocardial ischemia/reperfusion injury in ratsObjective: To investigate the role of low expression of miR-301b-3p targetingthe PPARG/NF-κB signal pathway in myocardial ischemia/reperfusion injury in rats.Methods: Forty eight SD rats were randomly divided into six groups(8 in each group): sham group,ischemia reperfusion(I/R)group,LV-negative control+I/R group,GW9662+I/R group,GW9662+LV-miR-301 b inh+I/R group,LV-miR-301 b inh+I/R group.In the sham group and the I/R group,rats were intramyocardially injected with the same volume of physiological saline 7 days before the establishment of MI/RI.In the LV-negative control+I/R group,rats were injected intramyocardially with lentivirus-negative control 7 days before the establishment of MI/RI.In the GW9662+I/R group,rats were intragastrically administered with GW9662 daily 3 days before the establishment of the MI/RI model.In the GW9662+LV-miR-301 b inh+I/R group,rats were injected intramyocardially with lentivirus-miR-301b-3p inhibitor 7 days before the establishment of MI/RI,and were intragastrically administered with GW9662 daily 3 days before the establishment of MI/RI.In the LV-miR-301 b inh+I/R group,rats were injected intramyocardially with lentivirus-miR-301b-3p inhibitor 7 days before the establishment of MI/RI.After 7 days,in the sham group,the left anterior descending artery of the SD rats was threaded but not ligated.In the other groups,the left anterior descending coronary artery of the SD rats was ligated for 1 hour and then reperfused for 6 hours.After the MI/RI model was completed,the hemodynamic parameters were measured by the cardiac function analyzer.Western blot was used to detect the protein expression of PPARG,NF-κB(nucleus NF-κB p65,n-NF-κB p65),TNF-α,IL-1β and IL-6in myocardial tissue.Results: The results of hemodynamic indexes showed compared with the sham group,the LVEDP of the I/R group,the LV-negative control+I/R group,theGW9662+I/R group,the GW9662+LV-miR-301 b inh+I/R group,and the LV-miR-301 b inh+I/R group were significantly increased(P < 0.05),while LVSP,+dp/dtmax,-dp/dtmax were significantly reduced(P < 0.05).Compared with the I/R group,there were no significant changes in LVEDP,LVSP,+dp/dtmax,and-dp/dtmax in the LV-negative control+I/R group and the GW9662+LV-miR-301 b inh+I/R group(P > 0.05).Compared with the LV-negative control+I/R group,there were no significant changes in LVEDP,LVSP,+dp/dtmax,and-dp/dtmax in the GW9662+LV-miR-301 b inh+I/R group(P > 0.05).Compared with I/R group and LV-negative control+I/R group,LVEDP was significantly decreased in the LV-miR-301 b inh+I/R group(P <0.05),while LVSP+dp/dtmax,-dp/dtmax were significantly increased(P < 0.05);and LVEDP was significantly increased in the GW9662+I/R group(P < 0.05),while LVSP,+dp/dtmax,and-dp/dtmax were significantly decreased(P < 0.05).The results of western blot showed that compared with sham group,the protein expression of PPARG in the I/R group,the LV-negative control+I/R group,the GW9662+I/R group,the GW9662+LV-miR-301 b inh+I/R group and the LV-miR-301 b inh+I/R group were significantly decreased(P < 0.05),while the protein expression of n-NF-κB p65,TNF-α,IL-1β,and IL-6 were significantly increased(P < 0.05).Compared with the I/R group,there were no significant change in the protein expression of PPARG,n-NF-κB p65,TNF-α,IL-1β,and IL-6 in the LV-negative control+I/R group and the GW9662+LV-miR-301 b inh+I/R group(P > 0.05).Compared with the LV-negative control+I/R group,there were no significant change in the protein expression of PPARG,n-NF-κB p65,TNF-α,IL-1β,and IL-6 in the GW9662+LV-miR-301 b inh+I/R group(P >0.05).Compared with I/R group and the LV-negative control+I/R group,the protein expression of PPARG in the LV-miR-301 b inh+I/R group wassignificantly increased(P < 0.05),while the protein expression of n-NF-κB p65,TNF-α,IL-1β and IL-6 were significantly decreased(P < 0.05);the protein expression of PPARG in the GW9662+I/R group was significantly decreased(P< 0.05),while the protein expression of n-NF-κB p65,TNF-α,IL-1β and IL-6were significantly increased(P < 0.05).Conclusion: Low expression of miR-301b-3p regulates NF-κB signaling pathway by targeting PPARG,attenuating inflammatory response,thereby attenuating myocardial ischemia/reperfusion injury in rats.