Research On The Regulatory Role Of MiR-27a-3p On Mechanism Of Aortic Dissection Vascular Remodeling Through Interfering Endothelial Cell's Function

Part I The expression and Distribution of miR-27a-3p in aortic dissectionObjective To observe the expression and distribution of miR-27a-3p in human aorta tissue and to investigate the relationship between miR-27a-3p and aortic dissection.Methods 1.The expression levels of miR-27a-3p,miR-27 b,miR-217,miR-183,miR-20 and miR-24 in human aorta tissue were detected by q RT-PCR 2.The expressions and distributions of miR-27a-3p were detected by fluorescence in situ hybridization.The co-localization between miR-27a-3p and endothelial cell was analyzed.Results 1.miR-27a-3p was significantly down-regulated in the tissue of aortic dissection when compared with the donor's aorta and the rest five miRNAs showed no obvious differences 2.miR-27a-3p and endothelial cell were co-localizated in the aortic intima.As the q RTPCR results,the expression level of miR-27a-3p in aortic dissection patient's aorta was significantly lower than the normal aorta from donors.Conclusions miR-27a-3p and endothelial cells were significantly down-regulated in the aorta from aortic dissection patients,which indicated that miR-27a-3p might take part in the pathological process of aortic dissection through regulating endothelial cell's function.Part II The research on the effect and mechanism of miR-27a-3p on endothelial cell functionObjective To investigat the regulatory role of miR-27a-3p on human umbilical vein endothelial cell(HUVEC).Screening the specific targets,signal pathway and identified the mechanism of the regulating process of miR-27a-3p.Methods 1.Constructing the lentivirus to interfere the expression level of miR-27a-3p in HUVEC.q RT-PCR and Western blotting were used to verified the interfering effect of lentivirus.The proliferation and apootosis of HUVEC was detected by CCK-8 and flow cytometry.2.The expression pattern of proteins related with apoptosis in HUVEC after the transfection of lentivirus was analyzed by protein chip.Bioinformatics analysis was used to screen the target protein and pathway.Dual-luciferase reporter gene assay was used to confirm the miR-27a-3p's direct regulatory effect on FADD.To detected FADD and the proteins which were screened by protein chip,q RT-PCR and Western blotting were used to verify the results.3.RNA interfering technology was used to construct the plasmid to down-regulate FADD.The apoptosis function of HUVEC was detected by flow cytometry when transfected by lentivirus and plasmid together.Results 1.The interfering effect of lentirus was obvious.The apoptosis of HUVEC was significantly promoted by down-rgulating miR-27a-3p and up-regulation of miR-27a-3p can suppress HUVEC's apoptosis function(P<0.05).The proliferation of HUVEC was not significantly affected by miR-27a-3p interfering.2.Caspase8,Caspase3,IGFBP-6,IGFBP-4,bcl-2,bax,TNF-alpha and IGF-II were proteins in apoptosis pathway and the results of protein chip showed significant differential expression in the group with miR-27a-3p suppressed(P < 0.05).Dualluciferase reporter gene assay confirmed the miR-27a-3p's direct surpress effect on FADD.The results of q RT-PCR experiment showed that FADD's expression wasimproved by down-regulating miR-27a-3p and up-regulation of miR-27a-3p can inhibit FADD's expression(P<0.05).The expression level of FADD,Caspase8 and Caspase3 in miR-27a-3p down-regulation group were significantly higher than the negative control group when detected by Western blotting and up-regulating miR-27a-3p showed the ppposite results(P<0.05).3.Compared with the control group with si RNA NC,the promotive effect of HUVEC's apoptosis with miR-27a-3p down-regulated was inhibited by FADD down-regulated(P<0.05).Conclusions FADD's expression was significantly directly surpressed by miR-27a-3p and the apoptosis function of HUVEC was further inhibited.Part III The research on the effect and mechanism of miR-27a-3p on the interaction between endothelial cell and vascular smooth muscle cellObjective To investigat the regulatory role of miR-27a-3p on the interaction between endothelial cell and vascular smooth muscle cell.Methods 1.Constructing the coculture model between HUVEC and HASMC via the Transwell.After transfecting with lentivirus,HUVEC was cocultured with HASMC.Flow cytometry was used to detect HASMC's apoptosis ability.scratch test and transwell exprement were to determine the migration ability of HASMC.The proteins related with HASMC's migration were screening by protein chip.ELISA test was used to detect the expression pattern of GDF8 and MMP20.Results 1.The results of scratch test and transwell exprement showed that down-regulating HUVEC's miR-27a-3p can significantly promoted the migration ability of HASMC.However,up-regulating HUVEC's miR-27a-3p can inhibit HASMC's migration(P<0.05).miR-27a-3p didn't affect HASMC's apoptosis ability in this coculture model(P>0.05).2.The protein chip was used to detect the differentially expressed proteins in the supernatant of the coculture model.Seventy three proteins were found when use 1.2 times difference to screen the result.Combined with bioinformatics analysis result,GDF8 and MMP20 were chosed to do further research.ELISA experiments found that up-regulating miR-27a-3p in HUVEC can improve GDF8 and inhibit MMP20's expression.Up-regulation of HUVEC's miR-27a-3p showed the contrary results(P<0.05).Conclusions In the coculture model,miR-27a-3p can inhibited HASMC's migration via inducing GDF8 and inhibiting MMP20 which were secreted by HUVEC.Part IV The effect and mechanism of miR-27a-3p on the incidence rate and vascular remodeling of aortic dissectionObjective To observe the protective role of miR-27a-3p on the incidence rate and vascular remodeling of aortic dissection.Methods 1.Using FVB mice to making aortic dissection model by ?-aminopropionitrile and angiotensin II.Tail vein injection was used to interfere miR-27a-3p in vivo and q RTPCR and Western blotting were used to confirm the interfering effect.FADD,Caspase3,Caspase8,GDF8 and MMP20 were also detected by q RT-PCR and Western blotting experiments.The incidence rate of aortic dissection and the morphological changes of aorta were envalued by the general picture and HE staining.2.TUNEL staining combined with immunofluorescence of CD31 were used to envalue the endothelial cell's apoptosis in the aorta section.Meanwhile,immunofluorescence was also used to detected the expression pattern and distribution of GDF8 and MMP20.3.EVG staining and MASSON staining were used to invested the content of elastic fiber and collagenous fiber in the aortic media.Results 1.The incidence rate of aortic dissection in model group was 63.3%.Down-regulation of miR-27a-3p obviously increased the incidence rate of aortic dissection(93.3%)and miR-27a-3p up-regulaiong group's incidence rate of aortic dissection is 13.3%.With the analysis of HE staining,media thickness and the ratio beteen media thickness and lumen diameter(MT/LD)in model group was significantly lower than blank control group.Down-regulation of miR-27a-3p significantly weaken the media and upregulation miR-27a-3p reversed the effect.The m RNA level and protein level of FADD was obviously increased by down-regulating miR-27a-3p(P < 0.05).Upregulating miR-27a-3p showed the opposite effect(P < 0.05).The promotive expression of Caspase3 and Caspase8 in protein and m RNA level was induced by down-regulation of miR-27a-3p(P<0.05).Up-regulation of miR-27a-3p significantly inhibited these two protein's expression in protein level(P<0.05).However,there was no such effect of up-regulation of miR-27a-3p in m RNA level.GDF8 and MMP20 were also showed no such effect in m RNA level(P>0.05).2.The endothelial cell's apoptosis in aortic intima was significantly promoted by downregulating miR-27a-3p(P<0.05).Up-regulation of miR-27a-3p significantly inhibited endothelial cell's apoptosis(P<0.05).Meanwhile,although the apoptosis cell in aortic intima was increased in model group,there was no statistical significance(P>0.05).GDF8 was distributed in media of aorta and it was significantly increased in model group when compared with blank control group via immunofluorescence(P<0.05).Down-regulation of miR-27a-3p showed promotive effect on GDF8's expression when compared with negative control or model group(P<0.05).Up-regulating miR-27a-3p inhibited GDF8's expression(P<0.05).MMP20's expression showed the opposite result(P<0.05).3.Elastic fiber and collagenous fiber in the aortic media showed decreased pattern in model group when compared with blank control group(P<0.05).Down-regulation of miR-27a-3p showed promotive effect on elastic fiber and collagenous fiber's degradation when compared with negative control or model group(P <0.05).Upregulation of miR-27a-3p showed the inverted results(P<0.05).Conclusions miR-27a-3p appeared a protective role in aortic dissection via inhibiting endothelial cell's apoptosis and degradation of elastic fiber and collagenous fiber.