Mechanism Of Lnc-OIP5-AS1 In The Pathogenesis Of Aortic Dissection Through Competitively Binding With MiR-143-3p

Part I Screening and validation of aberrantly expressed lncRNAs and miRNAs in arterial tissue from patients with aortic dissectionSubject: Screening of lncRNAs and miRNAs that were significantly differentially expressed between aortic tissues from aortic dissection(AD)patients and normal donors and identification of a pair of ce RNA for subsequent phenotypic and mechanistic studies.Methods: Twenty aortic dissection tissues were obtained fresh from acute thoracic AD patients who had undergone emergency aortic replacement surgery(experimental group/AD group).Patients enrolled in this study were diagnosed by physical examinations and computed tomographic angiography of the aorta,and also confirmed to have no connective tissue diseases or family history of aortic diseases.Twenty normal aortic tissues were collected from heart donors(control group/Donor group).Six specimens from each of the two groups were selected for detection by using lncRNAs and miRNAs microarrays analysis to obtain differentially expressed lncRNAs and miRNAs.A significant number of differentially expressed lncRNAs and miRNAs were initially screened according to the criteria of fold change < 0.5 or > 1.8,p < 0.05.Next,we performed real-time quantitative PCR(q RT-PCR)on 20 aortic samples from each of the experimental and control groups to validate the results of microarrays and select the most significantly differentially expressed lncRNA/miRNA pair for subsequent studies.Finally,we validated the lncRNA/miRNA expression relationships in a cellular model of aortic dissection induced by angiotensin II(Ang II)and confirmed the binding of the specific lncRNAs and miRNAs at the subcellular level by fluorescence in situ hybridization(FISH),RNA pull-down,RNA binding protein immunoprecipitation(RIP)and dual-luciferase reporter gene assays.Results: Through the detection of 20 pairs of arterial tissue samples included in this study,we found that lncRNA OIP5-AS1 was significantly enhanced(P<0.05),and miR-143-3p was significantly reduced(P<0.05)in aortic tissue samples from AD patients compared to the aortic specimens of donors.Meanwhile,the Pearson correlation coefficient analysis revealed that a significant correlation between the expression levels of miR-143-3p and lnc-OIP5-AS1(r=-0.6062,P=0.0023).The consistent results were also found in the Ang II-induced cell model(P<0.05).When we overexpressed lnc-OIP5-AS1,the expression of miR-143-3p was significantly down-regulated in the cells;while when we knocked down lnc-OIP5-AS1,the expression level of miR-143-3p was significantly enhanced(P<0.05).Consistently,the expression of lnc-OIP5-AS1 was attenuated and enhanced after miR-143-3p we upregulated or downregulated,respectively(P<0.05).The results of fluorescence in situ hybridization experiments(FISH)showed the co-localization of lnc-OIP5-AS1 and miR-143-3p in the cytoplasm,a result that provided a spatial basis for their relationship of endogenous competitive combination.Subsequent results of RNA pulldown and RNA-binding protein immunoprecipitation(RIP)confirmed the associated binding of lnc-OIP5-AS1 to miR-143-3p.Finally,the competitive binding of lncOIP5-AS1 to miR-143-3p at the transcriptional level was confirmed by dualluciferase reporter gene experiments.Conclusion: LncRNA OIP5-AS1 was confirmed to be involved in the development of AD via binding miR-143-3p as an endogenous competitive RNA(ce RNA)through the determination of clinical samples and a series of validations in cellular models.Part ? The role of lncRNA OIP5-AS1 competitively binding miR-143-3p in the pathogenesis of aortic dissectionSubject: To investigate the effect of lncRNA OIP5-AS1 on the aortic wall through competitive binding of miR-143-3p during the development of aortic dissection(AD).Methods: The major cellular components within the human aortic wall,including aortic endothelial cells(HAECs),aortic smooth muscle cells(HASMCs),and aortic epicardial fibroblasts(HAAFs),were treated with Angiotensin II(Ang II)to establish an in vitro cellular model of AD.The influences of lnc-OIP5-AS1/miR-143-3p on the biological function of these cell types were investigated to measure the effect of the lnc-OIP5-AS1/miR-143-3p axis on the aortic wall injury during the development of AD.To explored the effect of lnc-OIP5-AS1/miR-143-3p on aortic endothelial function,the effect of lnc-OIP5-AS1/miR-143-3p on the proliferative capacity of HAECs was determined by the CCK8 proliferation assay,and their effect on the apoptosis of HAECs was examined by the TUNEL apoptosis assay and the expression level of key genes related to apoptosis were detected by q RT-PCR and western blots.To investigate the influence of lnc-OIP5-AS1/miR-143-3p on aortic media function,the effects of lnc-OIP5-AS1/miR-143-3p on the proliferation ability,and motility of HASMCs were detected by CCK8 proliferation assay as well as Ed U proliferation assay and Transwell migration assay,respectively.Moreover,their role in regulating the expression level of MMP-2/9 and TIMP-1/2,the key genes related to the progression of AD,were also detected.Furthermore,the effects of lnc-OIP5-AS1/miR-143-3p on the level of inflammatory factors secreted by HAAFs were assessed by applying ELISA to examine the levels of inflammatory factors in the supernatant of the medium.Meanwhile,in this study,a certain concentration of Ang II was continuously pumped subcutaneously into different subgroups of C57BL/6N mice to construct an animal model for stimulating the development of AD.The subgroup without Ang II treatment was termed as a blank group(Blank group,n=20).For the experimental groups,the number of mice in each group was also 20.The overexpression animal models were established by injecting adenoviral vectors loaded with lncRNA overexpression plasmids or miRNA mimics(AAV9-pc DNA3.1-OIP5-AS1 group,AAV9-miR-mimics group)into the tail vein of C57BL/6N mice.In the same way,injecting adenoviral vectors loaded with small interfering RNA or Adenoviral vectorsloaded with small interfering RNAs or miRNA inhibitors(AAV9-si-OIP5-AS1 group,AAV9-miR-inhibitor group)into the tail vein to construct animal models with low gene expression,while a blank vector was injected as a control group(AAV9-NC group).The survival time of different groups of mice was recorded to plot the survival curve.The twenty-eighth day after treatment was used as the endpoint,regardless of whether the mice died naturally or were executed,the aortic tissue and plasma samples of mice from each group were collected and analyzed immediately.The expression levels of apoptosis-related genes in the intima and key genes for membrane phenotype transformation in the aorta media were measured by real-time quantitative PCR and immunoblotting(Western Blot).The levels of inflammatory factors in the plasma of different groups of mice were measured by ELISA.Animal models were used to explore the role of lnc-OIP5-AS1/miR-143-3p in the development of AD in vivo.Results: The results of biological function at the cellular level in vitro showed that miR-143-3p significantly promoted the proliferation and inhibited the apoptosis of HAECs compared with the control group,meanwhile promoting the expression levels of intracellular anti-apoptotic genes(Mcl-1,Bcl-2)and inhibiting the expression levels of pro-apoptotic genes(Bax,Caspase-9)(p<0.05).We also demonstrated that miR-143-3p significantly enhanced the proliferation and migration ability of HASMCs,while decreasing the expression levels of MMP-2/9 and upregulating the expression of TIMP-1/2(p<0.05).Moreover,miR-143-3p inhibited the secretion levels of various inflammatory factors(IL-6,IL-1?,and IL-17A)of HAAFs(p<0.05).However,lncRNA OIP5-AS1 had the opposite regulatory effect to miR-143-3p.When cells were co-transfected with both of them,lncRNA OIP5-AS1 significantly counteracted the regulatory effect of miR-143-3p on these cellular functions.The findings in animal studies showed that miR-143-3p significantly increased the survival time in Ang II-treated mice compared to controls,whereas lnc-OIP5-AS1 significantly reduced this survival time and also attenuated the effect of miR-143-3p on the life span of mice.The measurement of the expression levels of endotheliumapoptosis-related genes in mouse aortic tissues found that miR-143-3p reduced the expression level of Bax,Caspase-9 and promoted the expression of Mcl-1 and Bcl-2,while lnc-OIP5-AS1 exerted the opposite effect(p<0.05).The results of detection of the expression levels of key genes associated with aortic media injury revealed that miR-143-3p downregulated the expression of MMP-2/9 and upregulated the expression of TIMP-1/2,while lnc-OIP5-AS1 exerted the opposite effect(p<0.05).Meanwhile,the examination of inflammatory factors levels in the plasma of mice showed that miR-143-3p suppressed the secretion of IL-6,IL-1?,and IL-17 A,while lnc-OIP5-AS1 promoted their secretion and partially counteracted the impacts of miR-143-3p on these indicators(p<0.05).Conclusion: MiR-143-3p can attenuate aortic wall damage during AD progression,whereas lnc-OIP5-AS1 exacerbates aortic wall injury during this process and impairs the protective effect of miR-143-3p on the vessel wall.Part ? TUB is a downstream effector gene for the biological function of lnc-OIP5-AS1 and miR-143-3p in the progression of aortic dissectionSubject: To identify the downstream effect target genes of lnc-OIP5-AS1/miR-143-3p in the pathogenesis of aortic dissection(AD)and investigate its biological functions in AD progression.Methods: We searched the miRNA target gene prediction databases,PicTar,Target Scan,and miRanda,to predict the downstream target genes of miR-143-3p and the intersection of their predictions is used for subsequent validation.Subsequently,real-time quantitative PCR(q RT-PCR)in different cells(HAECs,HASMCs)was performed to validate the accuracy of the predictions and the candidate genes with the most significantly correlated with the level of miR-143-3p.Next,the expression levels of this candidate gene were detected in clinical aortic tissue samples to determine whether its expression was abnormal in AD tissues and whether its expression was correlated significantly with miR-143-3p.Then the dual-luciferase reporter gene assay was performed to confirm the binding of miR-143-3p to the candidate gene at the transcriptional level.Since the target gene of miR-143-3p had been identified,combined with our research conclusion in the first two parts,we could scientifically make the hypothesis that lncRNA OIP5-AS1 could competitively combine with miR-143-3p and indirectly regulate the level of this gene during the development of AD,which influences the course of disease progression as a downstream effector.To confirm whether this scientific hypothesis is true,we explored the biological function of this gene in the progression of AD and the functional relationship between it with lnc-OIP5-AS1 and miR-143-3p at the cellular and animal levels respectively.Results: Taking the intersection of the predicted results from the three databases,including Target Scan,miRanda,and Pic Tar,we obtained 10 predicted target genes for miR-143-3p.The results of subsequent q RT-PCR assays showed that the transfection of miR-143-3p mimics significantly downregulated the expression levels of only two genes in HASMCs and HAECs,MAPK1 and TUB(p<0.05;p<0.01),while when cells were transfected into the miR-143-3p inhibitor,only the expression of two genes,PGK1 and TUB,were significantly enhanced(p<0.05),compared to the control.Combined with these results,we selected TUB as a candidate target gene for miR-143-3p for subsequent validation in this study.When TUB expression levelsbetween 20 pairs of normal aortic tissue and aortic tissue samples from AD patients were examined,the results showed that TUB was significantly upregulated in aortic tissue from patients with AD,and Pearson correlation coefficient analysis revealed a significant negative correlation between the expression level of it and miR-143-3p(r=-0.408,P=0.0371),the results that suggested a targeting relationship between them at the RNA level This result.Moreover,subsequent results of immunoblotting(Western Blot)revealed the targeting relationship between them at the protein level.Specifically,a significant decrease in TUB protein levels in the miR-143-3p mimics group and a significant increase in TUB protein levels in the miR-143-3p inhibitor group were observed.Finally,the binding of miR-143-3p to TUB at the transcriptional level was confirmed by using a dual-luciferase reporter gene assay.In phenotypic experiments at the cellular and animal model levels,we found that the overexpression of TUB partially counteracted the effects of miR-143-3p on the cellular functions of HAECs,HASMCs,and HAAFs and the development of AD in mouse models.Meanwhile,silencing TUB partially neutralized the effects of lncRNA OIP5-AS1 on the cellular functions of HAECs,HASMCs,and HAAFs and the development of AD in a mouse model.Conclusion: lncRNA OIP5-AS1 exacerbates aorta intima,media,and adventitia injury during the occurrence and progression of AD through indirectly upregulating TUB via competitively sponging miR-143-3p.