Study On The Mechanism Of Circ-Sirt1/SIRT1 Inhibiting The Proliferation And Migration Of PASMCs Via TGF-β1/Smads Signaling In Hypoxia-Induced Pulmonary Hypertension

Background:Pulmonary hypertension(PH)is characterized by long-term hypoxia in the lung tissue leading to pulmonary vasoconstriction and pulmonary artery vascular remodeling with a progressive increase in pulmonary vascular resistance and pulmonary arterial pressure(PAP),leading to hemodynamic changes in pulmonary artery wall thickening,pulmonary artery lumen narrowing,and increased right ventricular afterload,which are common pathophysiological mechanisms of PH.If not diagnosed and treated as soon as possible,it can quickly lead to right heart failure and even death.Although the existing treatment methods can improve the quality of life of PH patients,all available therapies are still only used for palliative care.However,there is still a lack of effective prevention and treatment methods.Therefore,how to provide new molecular diagnoses and targeted therapy targets is still a topic that needs to be explored and continuously researched.Sirtuinl(SIRT1)is a class III histone deacetylase that is highly expressive in the vasculature and regulates various cell biological events including cell proliferation,migration,and apoptosis.Circular RNA(circRNA)has a variety of biological functions and is involved in the pathogenesis of lung and cardiovascular diseases.Circ-Sirtl is a circular RNA molecule formed by exons 2-7 of the parental gene SIRT1.SIRT1 can inhibit the proliferation of pulmonary artery smooth muscle cells(PASMCs),while the effect of circ-Sirtl on PASMCs is still unclear.Transforming growth factor-β1(TGF-β1)/Smads signaling pathway is a series of signaling processes mediated by TGF-β1.TGF-β1,Smad3,and Smad7 have been shown to play a crucial role in vascular remodeling.Whether circ-Sirtl is associated with TGF-β1/Smad3/Smad7 has not been reported,so we conducted the following studies:Objective:Study on the mechanism of circ-Sirtl/SIRTl inhibiting the proliferation and migration of PASMCs via TGF-β1/Smad3/Smad7 signaling in hypoxia-induced pulmonary hypertension.Methods:1.Preliminary detection of animal models:To explore the expression of circ-Sirtl,SIRT1,and TGF-β1/Smad3/Smad7 in pulmonary artery tissue of chronic hypoxic PH rats.A chronic hypoxic PH rat model was constructed.RVSP was measured by RHC,right ventricular hypertrophy index(RVHI)was calculated,and lung tissue sections were stained by HE and immunofluorescence.After successful modeling,RT-qPCR and western blotting were used to detect circ-Sirtl mRNA and SIRT1 mRNA and protein levels,and TGF-β1,Smad3,and Smad7 mRNA and protein levels in pulmonary artery tissues of rats.2.Mechanism research at the cellular level:To explore the role and mechanism of circ-Sirtl/SIRT1 in regulating the proliferation and migration of rPASMCs at the cellular level in vitro.2.1 The pulmonary artery tissue of SD rats was collected,and the intima and adventitia were scraped and separated into small pieces.The primary rPASMCs were cultured by tissue adherent method and identified by α-SMA immunofluorescence assay.2.2 Cell function tests:A plasmid overexpressive circ-Sirtl was constructed and transfected into rPASMCs.After hypoxia culture,RT-qPCR and western blotting were used to determine the changes in circ-Sirtl mRNA,SIRT1 mRNA and protein levels in rPASMCs in each group.The mRNA and protein expression changes of the TGF-β1/Smad3/Smad7 signaling pathway were detected.Flow cytometry,CCK-8 assay,and scratch test were applied to detect the changes in cell cycle,proliferation activity,and migration ability of rPASMCs.Immunofluorescence experiments was detected the expression of α-SMA and VCAM-1 in rPASMCs.2.3 Cell rescue tests:Constructed small interfering RNA si-SIRT1,transfect rPASMCs after plasmid overexpressing circ-Sirtl.After hypoxia culture,the circ-Sirtl mRNA levels and the mRNA and protein levels of SIRT1,TGF-β1,Smad3,and Smad7 in rPASMCs were detected.The changes in cell cycle,proliferation activity,and migration ability of rPASMCs were detected.Immunofluorescence experiments was detected α-SMA and VCAM-1 in rPASMCs.3.Animal Model Treatment Research:To explore the circ-Sirtl adenovirus vector for the treatment of PH model of rats induced by hypoxia.An adenovirus vector with high expression of circ-Sirtl was constructed and injected via the tail vein of rats in a hypoxia-induced pulmonary hypertension arterial model.After 40 days of hypoxia induction,RHC measured RVSP and analyzed RVHI.RT-qPCR and Western blotting were used to detect the expression levels of circ-Sirtl and SIRT1,detect the expression levels of the TGF-β1/Smad3/Smad7 signaling pathway,and detect the expression levels of VCAM-1,α-SMA,ICAM-1,PCNA,and vimentin in rat pulmonary artery tissue.The effect of high expression of circ-Sirtl on pulmonary artery remodeling in rats was observed by HE staining and immunofluorescence ofα-SMA,ICAM-1,PCNA,and vimentin.Results:1.Compared with the normoxia group,the RVSP and RVHI increased in hypoxia-induced rats.Western blotting and RT-PCR analysis revealed a downregulated expression of circ-Sirtl and SIRT1 in hypoxia-induced rat,while the mRNA and protein expression levels of Smad3,Smad7,and TGF-β1 were increased.2.1 Primary rPASMCs were successfully isolated and cultured.2.2 After overexpression of circ-Sirtl,it was noted that the levels of SIRT1 mRNA and protein were upregulated,and the cell cycle,proliferation activity,and migration ability of rPASMCs were inhibited.However,the expressions of TGF-β1,Smad3,and Smad7 were inhibited,and the expressions of VC AM-1 and α-SMA were also reduced.Overexpression of circ-Sirtl weakened PASMC cell viability and proliferation,and inhibited cell cycle progression and migration ability.2.3 Si-SIRT1 can significantly inhibit the increasing effect of circ-Sirtl on SIRT1.Similarly,si-SIRT1 can significantly restore the inhibitory effect of circ-Sirt1 on the levels of TGF-β1/Smad3/Smad7 as well as PASMC markers,VCAM-1 and α-SMA.Meanwhile,restore the inhibitory effect of circ-Sirtl on the cell proliferation,cell viability and migration of rPASMCs,and-SMA and VCAM-1 in rat PASMC were increased.3.The RVSP and RVHI of the rats in the Circ-Sirtl adenovirus group were significantly lower than those in hypoxia group.The expression of TGF-β1/Smad3/Smad7 decreased,as well as the expressions of VCAM-1,α-SMA,ICAM-1,PCNA,and vimentin.The immunofluorescence of α-SMA,ICAM-1,PCNA,and vimentin of the pulmonary artery tissue of the rats in the Circ-Sirtl adenovirus group decreased.Conclusion:1.Circ-Sirt1,SIRT1,and TGF-β1/Smad3/Smad7 signaling pathways may be closely related to PH.2.Circ-Sirt1 positively regulates the transcription process of SIRT1 mRNA,thereby inhibiting TGF-β1/Smad3/Smad7 to participate in the control of proliferation and migration of rat P ASMC,and participating in the progression of pulmonary hypertension.3.High expression of circ-Sirt1 can significantly reduce hypoxia-induced RVSP,RVH,and pulmonary artery remodeling in rats.High expression of circ-Sirt1 inhibited the expression of TGF-β1,Smad3,and Smad7 pathways in hypoxia-induced rat pulmonary artery tissue.