| Objective: Pulmonary artery hypertension(PAH)is a serious cardiovascular disease characterized by continuously increasing pulmonary vascular resistance and pulmonary artery pressure.The disease presents a low incidence rate but a poor prognosis.In the absence of targeted therapy drugs,the 5-year survival rate of PAH patients is only about34%,so it is called "the cancer in cardiovascular disease".With the advent of targeted therapy drugs,the prognosis of PAH patients has been improved,and the 5-year survival rate has reached about 55%.The data is far from expectation.At present,the targeted drugs for PAH mainly act on the prostacyclin pathway,the endothelin pathway and the nitric oxide pathway.However,the etiology of PAH is multifaceted and not limited to the pathways mentioned above.Therefore,in order to improve the prognosis,it is necessary to explore new therapeutic targets to block the potential molecular pathways that cause PAH.And the key to achieving this goal lies in in-depth research on the pathogenesis of PAH.Pulmonary artery smooth muscle cells(PASMCs)are the main cells that constitute the pulmonary vascular walls.As the main effector cells,they not only control the relaxation and contraction of pulmonary arteries,but also engage in the process of pulmonary vascular remodeling.Currently,it is accepted that aberrant proliferation and apoptosis are the most critical pathogenic events that initiate PASMCs to participate in pulmonary vascular remodeling.As a result,investigating the mechanisms of PASMC proliferation and apoptosis has always been a priority in PAH research.Circular RNAs(circRNAs),a recently identified class of non-coding RNAs,are characterized by their closed loop structures without a 5’ cap or a 3’ Poly(A)tail.They can regulate various cellular biological processes.There are evidences that circRNAs play important roles in the etiology of PAH through regulating PASMC proliferation and apoptosis.However,the number of circRNAs identified to influence PASMC proliferation and apoptosis is limited,and more research is needed.In order to provide new theoretical foundations and experimental evidences for extending the therapeutic targets of PAH,this study investigated the changes in circRNA expression profile of pulmonary artery tissues during the pathological process of PAH,identified the circRNA that was crucial to the disease’s pathogenesis,and clarified its roles in PASMC proliferation and apoptosis.Methods: 1.In this work,male SD rats were used as the research objects.SU5416 and hypoxia were coupled to create an rat model of PAH.The model was determined by right ventricular systolic pressure,right ventricular hypertrophy index,and HE staining.2. Transcriptome sequencing was employed to identify the expression profile of circRNAs in the pulmonary artery tissues of the rats in each group.3.RT-q PCR was used to validate circRNAs that were differentially expressed in pulmonary arteries and had high expression abundance in PASMCs.4.PASMCs were induced to present the pathophysiological alterations in PAH by hypoxia or platelet derived growth factor BB(PDGF-BB).CCK8 was used to access the viability of PASMCs under each intervention,and the expression of circRNAs was measured by RT-q PCR.5.To control the expression of circ LTBP1 in PASMCs,overexpression plasmids and small interfering RNA were utilized.The impact of circ LTBP1 on PASMC proliferation and apoptosis was then determined using the Ed U assay and the flow cytometry.6.To investigate proliferation and apoptosis associated genes and signal pathways that circ LTBP1 could regulate,transcriptome sequencing was carried out in PASMCs overexpressing circ LTBP1.7.The nuclear plasma separation assay was performed to clarify the subcellular location of circ LTBP1 within PASMCs.8.Subsequently,the RNA antisense purification experiment and the dual luciferase reporter assay were performed to confirm the binding interaction between circ LTBP1 and mi R-149-5p.9.After transfecting PASMCs with mi R-149-5p mimics,cell proliferation and apoptosis,and the expression of IRF9 were detected.10. The expression of IRF9 was measured following circ LTBP1 overexpression or knockdown in PASMCs.11.In addition,rescue experiments were performed to verify whether mi R-149-5p mediated the effects of circ LTBP1 on PASMC proliferation,apoptosis,and IRF9 expression,and whether IRF9 mediated the effect of mi R-149-5p on PASMC proliferation.12.RT-q PCR and Western blot were used to measure the expression level of IRF9 in the pulmonary arteries of rats,in order to validate the expression changes of IRF9 in vivo under PAH condition.Results: 1.Compared with the control group,the right ventricular systolic pressure,the right ventricular hypertrophy index,the percentage of pulmonary artery wall thickness in the lumen radius,and the percentage of pulmonary artery wall area in the total vascular area significantly increased in the PAH group.2.Transcriptome sequencing suggested that the expression profile of circRNAs in pulmonary artery tissues of rats in PAH group changed significantly.3.RT-qPCR was used to verify the expression of the top 5upregulated and top 5 downregulated circRNAs in the transcriptome sequencing data.We found 6 differently expressed circRNAs.Among them,circ LTBP1 had the highest expression abundance in PASMCs.4.The expression of circ LTBP1 was markedly elevated in PASMCs induced by hypoxia and PDGF-BB.5.Overexpression of circ LTBP1 could promote PASMC proliferation and inhibit PASMC apoptosis,and vice versa.6.Transcriptome sequencing combined with gene set enrichment analysis(GSEA)revealed that circ LTBP1 could cause PASMCs to present the gene expression pattern that was observed in PAH.Moreover,the differentially expressed genes were mainly enriched in JAK/STAT pathway and MAPK pathway.RT-q PCR confirmed the upregulation of several genes belonging to the two pathways mentioned above,including FOS,IL6,and IRF9.They had been reported to be associated with PASMC proliferation and apoptosis.7.The nuclear plasma separation assay indicated that circ LTBP1 mainly located in the cytoplasm of PASMCs.8.The RNA antisense purification experiment revealed the binding of circ LTBP1 with mi R-149-5p,and the dual luciferase reporter assay further verified the direct binding interaction between circ LTBP1 and the seed sequence of mi R-149-5p.9.RT-q PCR showed that circ LTBP1 and mi R-149-5p could inhibit each other’s expression.In addition,circ LTBP1 also upregulated the expression of CD47,HDAC4,and LIF,which had been confirmed as target genes for mi R-149-5p,indirectly verifying that circ LTBP1 inhibited the activity of mi R-149-5p.10.Mi R-149-5p could inhibit PASMC proliferation,induce PASMC apoptosis,and cause changes in the expression of PCNA and CASP-3 protein.11.Mi Randa software predicted that mi R-149-5p could specifically bind to 3’-UTR of IRF9 m RNA.RT-q PCR and Western blot suggested that mi R-149-5p inhibited the expression of IRF9 in PASMCs.12.Rescue experiments indicated that mi R-149-5p mediated the effects of circ LTBP1 on PASMC proliferation,apoptosis,and IRF9 expression,moreover,IRF9 mediated the effect of mi R-149-5p on PASMC proliferation,suggesting the important roles of circ LTBP1/mi R-149-5p/IRF9 axis on PASMC proliferation and apoptosis.13.RT-q PCR and Western blot verified the upregulation of IRF9 expression in the pulmonary arteries of PAH rats,indicating that,in vivo,the circ LTBP1/mi R-149-5p/IRF9 axis still had regulatory effect under PAH condition.Conclusion: The expression of circ LTBP1,which could promote the pathogenesis of PAH by regulating the proliferation and apoptosis of PASMCs,significantly increased in the pulmonary artery tissues of PAH rats induced by hypoxia and SU5416.Circ LTBP1 could regulate proliferation,apoptosis and IRF9 expression of PASMCs via acting as a sponge of mi R-149-5p.Circ LTBP1/mi R-149-5p/IRF9 axis played important roles in regulating proliferation and apoptosis of PASMCs. |
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