MicroRNA-26a-5p Mitigates Pulmonary Vascular Remodeling By Inhibiting Autophagy

Background:Pulmonary arterial hypertension（PAH）is a kind of progressive and relatively rare pulmonary vascular disease,which eventually leads to right heart failure and even death.The etiology of PAH is complex,and hypoxia is one of the important exogenous stimuli of PAH.The pathological changes involved in the development of PAH mainly include inflammatory cell infiltration,the formation of microthrombus,continuous contraction of pulmonary vessels and pulmonary microvascular remodeling.It has been confirmed that pulmonary arterial remodeling caused by excessive migration and proliferation of pulmonary artery smooth muscle cells（PASMCs）is the main pathological mechanism of PAH.At present,the clinical treatment of PAH is limited,the disease progresses rapidly,and has the characteristics of severe condition,poor prognosis,and high mortality,which is called"the cancer of cardiovascular diseases".Previous literature reported that the 5-year survival rate of PAH patients was only 57%,mainly due to the fact that the molecular mechanism of pulmonary vascular remodeling in the process of PAH is not very clear.Therefore,there is an urgent need to explore the molecular mechanism of pulmonary vascular remodeling in order to provide a basis for exploring new strategies and targets for the treatment of PAH.Previous studies had revealed that a variety of miRNAs were participated in the occurrence and development of PAH.Our previous clinical screening experiment showed that the level of miR-26a-5p in circulating blood of patients with PAH was significantly decreased,and the expression of miR-26a-5p in pulmonary artery smooth muscle cells was also significantly decreased,and it was negatively correlated with pulmonary artery pressure.Therefore,an animal model of PAH was constructed,and the role of miR-26a-5p in the development of PAH was studied by cell biological methods.Objective:To confirm the relationship between miR-26a-5p and PAH through clinical,cytological and animal models,and to study the role and mechanism of miR-26a-5p in the proliferation,migration and autophagy of PASMCs,so as to provide new targets and new ideas for clinical treatment of PAH.Methods:[1]The correlation between miR-26a-5p and PAH was reconfirmed in clinical practice.The clinical data,serum and lung tissue samples of 28 patients with congenital heart disease（14 cases with PAH and 14 cases without PAH）who were hospitalized in the Department of Cardiovascular Medicine or Cardiovascular Surgery of Changhai Hospital from January 2018 to July 2021 and underwent right heart catheterization were collected,and the serum samples of 11 healthy controls were collected.The baseline data of patients were analyzed,and the expression of miR-26a-5p in serum and lung tissue was detected by RT-q PCR.[2]The role and mechanism of miR-26a-5p in the proliferation,migration and autophagy of PASMCs under hypoxic stimulation were investigated at the cytological level.（1）The expression level of miR-26a-5p was regulated by miR-26a-5p mimic or inhibitor at the cellular level,and the effect of miR-26a-5p on PASMCs proliferation was detected by Beyo Click TM EDU staining and CCK8 assay,respectively.Wound healing assay and Transwell migration assay were used to detect the effect of miR-26a-5p on the horizontal and vertical migration of PASMCs.Western blot was used to observe the expression levels of miR-26a-5p in different hypoxia time,different proliferation models and different cell lines.At the same time,the effect of miR-26a-5p on the expression of autophagy markers LC3 and P62 in PASMCs was detected,and the changes of autophagic flux were observed by laser confocal fluorescence microscopy and electron microscopy.（2）Study on the mechanism of miR-26a-5pFirst,HIF-1αsi RNA was transfected into h PASMCs.The expression level of miR-26a-5p was tested,and chromatin immunoprecipitation（CHIP）assay was used to explore whether HIF-1αcould bind to the promoter region of miR-26a-5p.Dual luciferase reporter assay was used to detect the effect of HIF-1αoverexpression or knockdown on miR-26a promoter activity.Secondly,the downstream target genes of miR-26a-5p were predicted by bioinformatics websites,and PFKFB3 and ULK1/2 were predicted to be the binding targets of miR-26a-5p,which were verified by dual luciferase assay.Then,miR-26a-5p mimic was used to verify its inhibitory effect on PFKFB3 and ULK1/2 protein expression.Finally,PFKFB3 si RNA was constructed and transfected into PASMCs to observe its regulatory effect on ULK1/2 phosphorylation and hypoxia-induced autophagy.Meanwhile,PFKFB3 overexpression plasmid was transfected into PASMCs to observe whether it could reverse the effect of miR-26a-5p mimic.[3]To analyze the effect of miR-26a-5p overexpression on PAH model in ratsMale 8-week-old Sprague-Dawley rats were divided into 4 groups:control group（normoxia group,21%O₂）,normoxia+Ad-GFP group,hypoxia+Ad-GFP group,and hypoxia+Ad-miR-26a-5p group.The rats in hypoxia group were treated with 10%O₂.After anesthesia,the rats in the normoxia group were intermittently injected with 0.9%sodium chloride injection 200μl and Ad-GFP solution（final concentration 1×10⁹PFU/ml）200μl through the trachea,respectively.After hypoxia treatment,the rats were intratracheally injected with 200μl Ad-GFP solution（final concentration 1×10⁹PFU/ml）and 200μl Ad-miR-26a-5p solution（final concentration 1×10⁹PFU/ml）,respectively.After4 weeks of routine feeding,right ventricular hemodynamic parameters were measured,the thickness of pulmonary artery wall and right ventricular wall was observed by HE staining,and the m RNA and protein levels of PFKFB3 were detected by RT-q PCR.Western blot and immunofluorescence were used to detect the level of autophagy,and PCNA immunohistochemical staining was used to detect the proliferation of injured pulmonary artery smooth muscle cells.Results:[1]The serum level of miR-26a-5p in patients with congenital heart disease complicated with PAH was significantly lower than that in patients without PAH and healthy controls（P<0.05）.The expression level of miR-26a-5p in pulmonary artery tissue of patients with PAH was also significantly lower than that in patients without PAH（P<0.05）.Serum miR-26a-5p level was negatively correlated with mean pulmonary artery pressure（rs=-0.322,p<0.05）.[2]Studies at the cytological level（1）Mi R-26a-5p mimic could significantly restrain proliferation and migration of PASMCs under hypoxic stimulation.On the contrary,miR-26a-5p inhibitor could enhance the proliferation and migration of PASMCs.At the same time,the expression of miR-26a-5p was down-regulated in three proliferation models of PASMCs.Under hypoxia,miR-26a-5p mimic could increase the expression level of autophagy substrate p62 and reduce the LC3II/I ratio,while miR-26a-5p inhibitor had the opposite effect.（2）Chromatin co-precipitation assay showed that HIF-1αcould bind to the promoter region of miR-26a-5p and regulate the expression of miR-26a-5p.Dual luciferase reporter assay showed that overexpression of HIF-1αreduced the fluorescence activity of Mir-26a-5p 3’UTR,while knockdown of HIF-1αenhanced the fluorescence activity of Mir-26a-5p3’UTR,while the fluorescence activity of cells with mutation of miR-26a binding site did not change significantly.（3）PFKFB3 and ULK1/2 were predicted to be the binding targets of miR-26a-5p by bioinformatics websites,and the binding sites were found in the 3’untranslated region（3’UTR）of PFKFB3 and ULK1/2.HEK293T cells were overexpressed miR-26a-5p and simultaneously transfected with the WT 3’UTR of PFKFB3 or ULK1/2.Dual luciferase assay showed that the luciferase activity was significantly inhibited in the overexpressed PFKFB3 or ULK1/2 groups compared with the control group.However,mutation of PFKFB3 or ULK1/2 3’UTR did not change the luciferase activity.The expression of PFKFB3 protein was increased under hypoxia.Mi R-26a-5p mimic inhibited hypoxia-induced upregulation of PFKFB3 or ULK1/2 protein levels.At the same time,the phosphorylation level of ULK1 was enhanced under hypoxia.Knockdown of PFKFB3inhibited the phosphorylation of ULK1.Knockdown of PFKFB3 inhibited hypoxia-induced autophagy in PASMCs.Overexpression of PFKFB3 reversed the inhibitory effect of miR-26a-5p mimic on PASMCs proliferation and migration under hypoxia.[3]Studies in a rat model of pulmonary hypertensionUnder hypoxic stimulation,right ventricular systolic pressure（RVSP）,right ventricular weight/（left ventricular+ventricular septal）weight[RV weight/（LV+septal）weight,RV/（LV+Sep）],right ventricular thickness and the proportion of muscularized pulmonary artery walls were significantly increased.Transbronchial administration of Ad-miR-26a-5p significantly reduced RVSP,RV/（LV+Sep）,right ventricular thickness and the proportion of muscularized pulmonary artery wall.At the same time,it down-regulated PFKFB3 m RNA and protein levels in lung tissue,significantly inhibited the level of autophagy in injured lung tissue,and reduced the number of PCNA positive cells in pulmonary artery tissue.Conclusion:[1]The level of miR-26a-5p in PAH patients was negatively correlated with the severity of PAH;In the cytological study,miR-26a-5p could inhibit the proliferation,migration and autophagic flow of cells induced by hypoxia.[2]The expression of miR-26a-5p was regulated by HIF-1α.PFKFB3 and ULK1/2were the downstream target genes of miR-26a-5p.PFKFB3 is also an important regulator of ULK1 phosphorylation.[3]HIF-1α/miR-26a-5p/PFKFB3/ULK1/2 signaling pathway plays an important role in the proliferation and migration of PASMCs induced by hypoxia.Overexpression of miR-26a-5p can significantly down-regulate PFKFB3 expression,inhibit autophagy and proliferation,improve pulmonary artery remodeling,and reduce the progression of PAH induced by long-term hypoxia.Therefore,the study suggests that the development of PAH can be mitigated by regulating the expression level of miR-26a-5p in clinical practice,which may become a new target for the treatment of PAH.