The Effect And Mechanism Of MiR-327 On Myocardial Ischemia Reperfusion Injury In Rat Model By Targeting RP105

Objective To explore the effect and molecular mechanism of miR-327 on myocardial ischemia reperfusion injury in rat model using adenovirus vectors overexpressing or downregulating miR-327.Methods(1)Sixty healthy male SD rats were randomly divided into 5 groups,named Sham group(normal saline + sham operation),I/R group(normal saline + I/R),Ad-miR-327-i group(Ad-miR-327-RNAi + I/R),Ad-NC group(Ad-EGFP + I/R)and Ad-miR-327 group(Ad-miR-327 + I/R).Rats in the sham group and the I/R group were injected with normal saline,while animals in the Ad-miR-327-i,Ad-NC and Ad-miR-327 group were injected with corresponding adenovirus,followed by stable transfection for 3 days before MIRI.(2)Adenovirus transfection efficiency was observed under fluorescent microscope.(3)Rat MIRI model was established by ligation of LAD for 30 min and reperfusion for 3 h.Sham group underwent the same procedures without LAD ligation.The blood and myocardium samples were collected for further test.(4)Concentration of LDH and CK-MB were measured by automatic biochemical analyzer.(5)Myocardial infarction size was measured by TTC staining.(6)Morphological changes of myocardial tissue were evaluated by HE staining.(7)Gene expression of miR-327 and RP105 were detected by Real-time PCR.(8)Protein expression of RP105,TLR4,TLR2,MyD88 and NF-?B-p65 were detected by Western blot.(9)Contents of IL-6 and TNF-? were detected by ELISA assays.(10)The target relationship between miR-327 and RP105 were analyzed by dual-luciferase reporter gene assay.Results(1)Adenovirus vectors were successfully transfected into myocardium.Compared with Sham group and I/R group,green fluorescent protein was expressed obviously in Ad-miR-327-i,Ad-NC and Ad-miR-327 group.(2)Rat MIRI model was successfully established.Compared with Sham group,LAD ligation induced significant increase of ST segment and T wave,the serum myocardial enzymes(LDH and CK-MB)and myocardial infarction size were accelerated obviously in I/R group(All P<0.05).Meanwhile,abnormal morphological changes,including myocardial fibers disorder or rupture,cell edema,and inflammatory cell infiltration appeared in I/R group.(3)Down-regulation of miR-327 could reduce MIRI.Compared with Ad-NC group,serum myocardial enzymes(LDH and CK-MB)and myocardial infarction size were significantly decreased,combined with decline of cell edema and inflammatory cell infiltration in Ad-miR-327-i group,while miR-327 overexpression performed opposite effects.(4)Down-regulation of miR-327 promoted the expression of RP105.Compared with Sham group,MIRI induced great increase of miR-327 and decrease of RP105(All P<0.05).Furthermore,Ad-miR-327-i not only reduced miR-327 expression,but also enhanced RP105 content,while an opposite effect observed in Ad-miR-327 group(All P<0.05).(5)Down-regulation of miR-327 inhibited the activation of TLR4/TLR2-MyD88 signaling pathway induced by MIRI.Down-regulation of miR-327 inhibited the signal transduction via attenuating downstream molecules expression,including TLR4,TLR2,MyD88,NF-?B-p65?IL-6 and TNF-?.However,miR-327 overexpression performed a promotive effect on TLR4/TLR2-MyD88 activation(All P<0.05).(6)miR-327 can bind to the 3'UTR of RP105.Compared to the 3'UTR-NC+miR-327 group,the luciferase activity was significantly decreased in 3'UTR+miR-327 group by the dual luciferase reporter gene assay(P<0.05).Conclusion Down-regulation of miR-327 plays a protective role in MIRI through targeting RP105-TLR4/TLR2-MyD88 signaling pathway.