| 1 ObjectivePrevious studies of our research team have found that 27 base repeats(termed as "27nt-miRNA")from the fourth intron of endothelial nitric oxide synthetase(eNOS)can inhibit the expression of eNOS gene and vascular NO production,also participate in the regulation of VSMC phenotype transformation.Therefore,we hypothesize that 27nt-miRNA may participate in the regulation of VSMC pivotal functions reasonably.This study aim to explore the regulation of endothelial 27nt-miRNA mainly on autophagy and apoptosis of human aortic VSMC preliminary.Then we predict and verify the potential target gene of 27nt-miRNA involve in regulating autophagy-apoptosis crosstalk of VSMC.In order to provide a theoretical basis and a potential therapeutic target for molecular drug therapy of atherosclerosis disease.2 Methods2.1 27nt-miRNA lentivirus vector transfection with VSMCThe VSMC was randomly divided into four groups:control group,pre-27ntmiRNA group,anti-27nt-miRNA group and negative control(miR-NC)group.The control group was cultured without any treatment,and the other three groups were transfected with pre-27nt-miRNA,anti-27nt-miRNA and miR-NC lentivirus respectively.After transfection,GFP green fluorescence was observed by flow cytometry,and the expression level of 27nt-miRNA after transfection was detected by RT-qPCR.2.2 Detection of the autophagy level and molecular regulation in VSMCThe VSMC was randomly divided into five groups:control group,EBSS group(autophagy model),pre-27nt-miRNA group,anti-27nt-miRNA group and miR-NC group.Except the control group,other groups were given EBSS(1×)for 6h to induce autophagy.The autophagosomes were observed by transmission electron microscopy(TEM).Autolysosomes were assessed by Lyso-tracker Red staining.The mRNA expressions of LC3B,Beclin-1 and p62 was detected by RTqPCR,The protein expression of LC3B,Beclin-1 and p62 was detected by Western blot as the same.2.3 Detection of apoptosis level and molecular regulation in VSMCThe VSMC was randomly splited into five groups:control group,EBSS group(apoptosis model),pre-27nt-miRNA group,anti-27nt-miRNA group and miR-NC group.Except the control group,the other groups were all given EBSS(1×)for 24h to induce apoptosis.CCK-8 method was used to assess the cell viability and proliferation.Flow cytometry was used to determine the cell cycle ratio(PI staining)and apoptosis rate(Annexin V-APC/7-AAD staining).RTqPCR and Western blot were used to evaluate the mRNA levels and protein relative expressions of Bcl-2,Bax and caspase-3.2.4 Prediction and validation of 27nt-miRNA potential target geneFirst,27nt-miRNA target genes were predicted in miRBase,miRDB and Targetscan 7.2 bioinformatics databases and screened in GO,KEGG and HRPD databases.If the databases aforementioned cannot be retrieved,potential target genes will be selected on literature research and preliminary experiments.Furthermore,the expression levels of the target gene was preliminares verified by RT-qPCR and Western blot under its inhibitor condition.In addition,According to the requirements of luciferase activity reporter gene system,we constructed gene reporter plasmids containing 3’UTR wild type(wt)and mutant(mut)of target gene,as well as 27nt-miRNA overexpression and negative control plasmids.Hence,we transfected miRNA-associated plasmids and then transfected 3’UTRwt,3’UTR-mut and pmirGLO report plasmids of the target gene into VSMC respectively.Cells were lysed after 48 h of transfection.The activities of Firefly luciferase(LCU1)and Renilla luciferase(LCU2)were detected respectively.The ratio of LCU1/LCU2 was calculated and analyzed.3 Results3.1 The expressions of 27nt-miRNA were regulated by lentivirus in VSMC.When VSMC was transfected with the infection index MOI=80,GFP green fluorescence was expressed in cells after transfection basically,also the proportion of green fluorescence was up to 90%after puromycin screening.RT-qPCR showed that the expression level of 27nt-miRNA in pre-27nt-miRNA group was nearly three times higher than that before transfection(P<0.05),while the expression level of anti-27nt-miRNA was significantly lower oppositely(P<0.05).There was no significant difference between the miR-NC group and the control group,indicated that the expression level of 27nt-miRNA was successfully regulated by transfecting lentiviral vector at different degrees.3.2 27nt-miRNA overexpression promoted EBSS-induced VSMC autophagy.In the autophagy-associated results,autophagosomes were observed in the control group by means of TEM occasionally,and the red fluorescence of autolysosomes was weak.While in the EBSS group,there were more autophagosomes,and the red fluorescence of autolysosomes was stronger than that in the control group conversely(P<0.05).Beyond that,there was no significant differences between the EBSS group and the miR-NC group.While in the pre-27nt-miRNA group,autophagosomes and the red fluorescence of autolysosomes were more than that in the miR-NC group(P<0.05).In addition,anti-27nt-miRNA group was weaker than the control group in above indexes(P<0.05).In the detection of gene transcription and protein expression levels,compared with the control group,the mRNA and protein expressiones of LC3B and Beclin-1 were increased and p62 was decreased in EBSS group(P<0.05),while the pre-27nt-miRNA group was opposite to EBSS group(P<0.05).There were differences between miR-NC group and anti-27nt-miRNA group(P<0.05).3.3 27nt-miRNA overexpression inhibited EBSS-induced VSMC apoptosis.In the apoptosis-associated detection,compared with the control group,cell activity of EBSS group decreased significantly,and the cell cycle was blocked in G1-phase,hence the apoptosis rate increased(P<0.05).There was no significant difference between the EBSS group and the miR-NC group.Compared with miRNC group,pre-27nt-miRNA overexpressed increased cell activity,cell cycle Sphase and G2-phase proportion,meanwhile significantly reduced apoptosis rate(P<0.05).Also,anti-27nt-miRNA group decreased cell activity,cell cycle arrest in G1-phase,and increased apoptosis rate(P<0.05).Furthermore,in the detection of gene transcription and protein expression levels,compared with the control group,the mRNA and protein expression of Bax and caspase-3 in EBSS group increased,however the Bcl-2 level decreased significantly(P<0.05).There were no prominent differences between EBSS group and miR-NC group.Compared with miR-NC group,Bax and caspase-3 mRNA and protein expression in pre27nt-miRNA group decreased,however Bcl-2 mRNA and protein expression increased significantly(P<0.05).In the aforementioned indexes,the mRNA and protein expression of Bcl-2 was lower than miR-NC group in anti-27nt-miRNA group,besides caspase-3 levels remarkable rised.3.4 27nt-miRNA regulated autophagy-apoptosis crosstalk of VSMC by targeting inhibit mTOR expression.It was found that 27nt-miRNA was not recorded in miRBase,miRDB,and TargetScan 7.2 databases,either in GO,KEGG and HRPD databases.Therefore,by thoroughly literature investigation and preliminary research,we came to a knowledge of that there was a potential binding site between 27nt-miRNA and 3’UTR of mTOR.Also,RT-qPCR and Western blot confirmed that the mRNA and phosphorylated protein expression of mTOR were significantly inhibited after overexpression of 27nt-miRNA(P<0.05).The result of the dual luciferase activity reporter gene system showed that overexpression of 27nt-miRNA could inhibit the luciferase activity of the vector carrying wild-type mTOR-3’UTR sequence.Beyond that,there had no effect on the luciferase activity of the vector carrying mTOR-3’UTR mutation sequence,which confirmed that 27nt-miRNA could combine with mTOR-3’UTR targetly.Taken together,these results suggest that 27nt-miRNA may play a regulatory role in autophagy-apoptosis crosstalk of VSMC by targeting mTOR phosphorylation.4 Conclusions4.1 27nt-miRNA overexpression could promote EBSS-induced VSMC autophagy,increase the production of autophagosomes and autolysosomes,while regulate autophagy by increasing the mRNA and protein expression levels of LC3B and Beclin-1 and decreasing p62 meanwhile.4.2 27nt-miRNA overexpression could inhibit EBSS-induced apoptosis of VSMC,enhance cell activity,weaken the arrest effect of G1-phase cell cycle,reduce the apoptosis rate,meanwhile,regulate apoptosis by inhibiting Bax and caspase-3 and activating Bcl-2 mRNA and protein expression.4.3 It can be preliminarily known through verification about 27nt-miRNA targets on mTOR-3’UTR seed region and regulates VSMC autophagy-apoptosis crosstalk by inhibiting mTOR signaling. |
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