Function And Mechanism Of P65/miR-455/PTEN Signaling Pathway In Vascular Negative Remodeling

Objective: One of the most crucial causes of organ and tissue ischemia is atherosclerosis(AS),and it is also the most the common pathological change in cardio-cerebrovascular and peripheral vascular diseases.Atherosclerosis obliterans(ASO)is one of the most common peripheral vascular diseases and the morbidity has been increasing in recent years.After the appearance of lower limbs atherosclerosis,the lumen shrinks which leads to tissue ischemia.Because the pathological process keeps deteriorating,the collateral circulation cannot compensate the ischemia.Ultimately,the limb goes necrosed.Although the open surgical and endovascular repair technique have been developed rapidly,it is still hard to gain satisfied prognosis.No matter what kind of surgery we choose to perform,postoperative restenosis caused by vascular remodeling is evitable as usual.Therefore,it is crucial to investigate the specific mechanism of vascular remodeling in order to improve the technique and meliorate the prognosis.As aporia and key point of clinical treatment,vascular negative remodeling is the challenge for all vascular surgeons.ECM accumulation,vascular smooth muscle cells(VSMCs)excessive proliferation,phenotypic transformation and migration are the main pathological process in negative vascular remodeling.It is important to explore the mechanism of VSMCs excessive proliferation which is the key point of solving the problem.Lots of studies indicated non-codingRNAs(ncRNAs),especially microRNAs(miRs),participate in VSMCs excessive proliferation and phenotypic transformation.Mi Rs are able to regulate transcription,translation and protein degradation.It has been reported that miR-455 level was higher in rat carotid artery balloon-injured model.And a couple of studies showed miR-455 was related with cell proliferation.The aim of this study is to explore the role of miR-455 on VSMC proliferation,phenotypic transformation and pathological vascular remodeling.Methods: In order to explore the relationship between miR-455 and pathological vascular remodeling,we harvest vascular tissue RNA from ASO patients and organ donors,rat balloon-injured carotid artery model and RNA from T/G HA-VSMC dealt with 20% FBS.We detected the miR-455 level in different groups.Then we overexpressed miR-455 in T/G HA-VSMC and evaluated the proliferation and phenotypic marker level.Bioinformatic program,including Target Scan and DIANA predicted miR-455 potential target gene,which was proved by dual-luciferase assays.To discuss the specific upstream mechanism,JAPSAR was used to predict transcription factor.Results was verified by dualluciferase assays and chromatin immunoprecipitation(ChIP).Then we transfected p65 cDNA into T/G HA-VSMC to evaluate cellular proliferation,phenotypic transformation level.At last,we transfected miR-455 agomir/antagomir/NC into rat balloon-injured carotid artery and evaluate miR-455,VSMC phenotypic marker and PTEN level,plus,vascular stenosis level.Cellular proliferation was evaluated by CCK-8 assay.Flow Cytometer(FCM)was used to detect cell cycle.Western Blot(WB)was used to evaluate protein expression level.RNA level was detected by real-time polymerase chain reaction(RT-qPCR).Results are presented as mean±standard deviation.Student's t test was used to compare the difference between two groups.One-way ANOVA analysis was used for multiple groups differences compare.Significance was established by P-value less than0.05.We used SPSS 23.03 and Graphpad Prism 8 to analyze the data.Results: RNA samples were harvested from T/G HA-VSMC,human and rat artery.RTqPCR results showed hsa-miR-455 level was higher in ASO patients' stenosis arteries,rat balloon-injured arteries and 20% FBS T/G HA-VSMC.CCK-8 assay results indicated hsamiR-455 overexpression was able to promote T/G HA-VSMC proliferation.WB results showed ?-SMA and PTEN level was lower after transfected with hsa-miR-455 mimics,while OPN was higher than before.Bioinformatic prediction indicated PTEN3'untranslated region(3'UTR)and hsa-miR-455 has potential binding sites,according to which,we used dual-luciferase assay to prove that.Co-transfection with hsa-miR-455 mimics and could inhibit PTEN 3'UTR wild-type plasmid luciferase activity but the mutant-type plasmid could not.Potential binding sites were predicted by bioinformatic software between p65 and hsa-miR-455 promoter.The results were proved by dualluciferase assays and ChIP.Overexpression of p65 in T/G HA-VSMC was able to increase hsa-miR-455 level,while PTEN and ?-SMA level was downregulated and OPN level was upregulated according to WB results.When co-transfecting p65 c DNA and hsa-miR-455 mimics,PTEN and ?-SMA level was lower than p65 overexpressed group.Flow cytometer(FCM)detected the percentage of S phase cells was significantly elevated after p65 cDNA/hsa-miR-455 mimics co-transfection.The results above proved that hsa-miR-455 could mediate T/G HA-VSMC cell cycle,phenotypic transformation and PTEN level,which were regulated by p65.After local transfection of rno-miR-455 agomir,immunohistochemical(IHC)results showed that PTEN and ?-SMA level was decreased and OPN level was increased.On contrary,local transfection of rno-miR-455 antagomir,IHC results showed that PTEN and ?-SMA level was increased and OPN level was decreased.Then evaluate the negative vascular remodeling regulated by rno-miR-455,results indicated rno-miR-455 antagomir could delay rat carotid artery vascular stenosis induced by balloon-injury.Conclusion: In stenosis artery and excessive proliferating VSMCs,miR-455 level is upregulated.Overexpression of hsa-miR-455 is able to induce T/G HA-VSMC proliferation and phenotypic transformation,and it can also target PTEN 3'UTR and downregulate its level.Hsa-miR-455 is the crucial mediator of p65 regulating VSMCs proliferation and phenotypic transformation,which have the synergistic action with p65 function.Local transfection rno-miR-455 agomir can downregulate PTEN level and regulate local vascular phenotypic transformation to ease stenosis caused by balloon-injury.