Inhibiting MicroRNA-222 Attenuates Pulmonary Arterial Hypertension And Pulmonary Vascular Remodeling

Background:With high morbidity and mortality,pulmonary hypertension poses a serious threat to human health,and its important pathological basis includes remodeling of distal pulmonary arteries,pulmonary vasoconstriction and in situ thrombosis.Existing drugs mainly target vasodilation and fail to improve adverse vascular remodeling,thus the development of new targets is particularly important.Micro RNAs play an important role in the development and progression of pulmonary hypertension.Inhibition of micro RNA-222(mi R-222)can alleviate the proliferation of pulmonary artery smooth muscle cells.However,whether downregulation of mi R-222 could prevent pulmonary hypertension remained unclear.Objective:To clarify the role and mechanism of inhibition of micro RNA-222 in attenuating pulmonary hypertension and pulmonary vascular remodeling.Methods:Firstly,at the animal level,based on mi R-222 knockout rats,we induced pulmonary hypertension model by monocrotaline injection and measured right ventricular systolic pressure by Powerlab.The right and left ventricles were separated and weighed to evaluate the functional roles of mi R-222 in pulmonary hypertension.Immunohistochemical stainings for PCNA andα-SMA were used to analyze pulmonary vascular remodeling.Next,in order to elucidate the function of mi R-222 at the cellular level,excessive proliferation of human pulmonary artery smooth muscle cells was induced by platelet-derived growth factor-bb(PDGF-bb).Using mi R-222 mimic or inhibitor to regulate the expression of mi R-222,the proliferation of pulmonary artery smooth muscle cells was determined by Ed U staining and the expression of cell cycle regulatory genes was detected by quantitative PCR to clarify the functional role of mi R-222 in pulmonary arterial smooth muscle cell proliferation.Furthermore,bioinformatics analysis was applied to predict the downstream target of mi R-222.Luciferase reporter gene assay and functional-rescue assay were performed to clarify whether VGLL4 acted as a target gene of mi R-222 to regulate the proliferation of pulmonary artery smooth muscle cells.Finally,luciferase reporter gene assay and quantitative PCR were performed to clarify whether VGLL4 repressed the transcription of downstream proliferation-related genes through inhibiting the formation of YAP-TEAD4 complex.Results : In the rat model of pulmonary hypertension,mi R-222 knockout reduced right ventricular systolic pressure and right ventricular hypertrophy,and inhibited pulmonary vascular remodeling.In human pulmonary artery smooth muscle cells,inhibition of mi R-222 attenuated PDGF-bb-induced cell proliferation,while overexpression of mi R-222 aggravated cell proliferation.Luciferase reporter gene experiment showed that VGLL4 was a target gene of mi R-222.Mi R-222 negatively regulated VGLL4 in human pulmonary arterial smooth muscle cells.Functional-rescue assays showed that knockdown of VGLL4 reversed the protective effect of mi R-222 inhibitor against pulmonary artery smooth muscle cell proliferation.Our results further identified that VGLL4 could compete with YAP for binding the transcription factor TEAD4 and inhibited the expression of TEAD4 downstream genes CTGF,CYR61,and CDX2.Conclusion : At animal level,mi R-222 knockout protects against pulmonary arterial hypertension and pulmonary arterial remodeling.Inhibition of mi R-222 attenuates the proliferation of human pulmonary artery smooth muscle cells.VGLL4 is a target gene of mi R-222 which mediates the anti-proliferative effect of inhibiting mi R-222 in pulmonary arterial smooth muscle cells.Inhibiting mi R-222 upregulates VGLL4,which competes with YAP for binding the transcription factor TEAD4,thus reducing its transcriptional regulation of downstream pro-proliferative genes CTGF,CYR61,and CDX2.Collectively,targeting the mi R-222/VGLL4 axis will be a potential strategy for prevention and treatment of pulmonary arterial hypertension and pulmonary vascular remodeling.