The Effect Mechanism Of MiR-140 In Pulmonary Arterial Hypertension Complicated With Congenital Heart Disease

Congenital heart disease is one of the most common congenital diseases and it affects about 1% new-borns all over the world. Pulmonary arterial hypertension is a common complication of congenital heart disease characterized by continuous rise of pressure of pulmonary vasculature. And it is the main cause of death during perioperative period. The major etiopathogenesis of pulmonary arterial hypertension complicated with congenital heart disease(PAH-CHD) is pulmonary vascular remodeling and increased resistance of dynamical type pulmonary vascular. At present, effective treatments are very lack for PAH-CHD. It is of great meaning to study the pathogenesis and regulatory mechanism for PAH-CHD and find out more efficacious and safe therapies.MiRNAs are strongly associated with heart development and the generation of heart disease. MiRNAs participate in the occurrence and development of pulmonary arterial hypertension. They regulate a variety of physiological processes, such as cell metabolism, proliferation and apoptosis. In addition, miRNAs regulate many signal pathways in cell. In this study, the expression of miR-140 and proteins related to Wnt pathway in the lung tissues of PAH-CHD patients was detected and their link with the structure of pulmonary vessels and apoptosis of cells in it was analyzed at first. Then the effect of miR-140 on proliferation, migration and phenotype of HPASMCs was studied and the co-relation of miR-140 and Wnt pathway was confirmed. Finally, the effect of Wnt pathway on proliferation, migration and phenotype of HPASMCs was studied. The results of this study laid foundation for the study of pathogenesis and regulatory mechanism for PAH-CHD. This research was divided into two parts:Part I: The co-relation of miR-140 and PAH-CHD Methods1. The mRNA and protein expression of miR-140 and Wnt-1 and β-catenin, which were related to Wnt pathway in lung tissues of PAH-CHD patients were detected by qRT-PCR and western blot. The relationship of miR-140 and the pressure of pulmonary arterial was analysed, too. Lung tissues of patients of congenital heart disease without pulmonary arterial hypertension were used as control.2. The morphological alterations of lung tissues of PAH-CHD patients were checked out by hematoxylin and eosin stain and the alterations of vascular wall thickness/vascular diameter(WT%) and vascular area/total vascular area(WA%) were measured. The relationship of miR-140 and WT%, WA% was analysed, too.3. Apoptosis of lung tissues of PAH-CHD patients was detected by TUNEL and the apoptotic index(AI) was evaluated. The expression alterations of bax, bcl-2 and survivin, which were related to cell apoptosis were detected by qRT-PCR and western blot. The relationship of mi R-140 and AI, bax, bcl-2 and survivin was analysed, too. Results1. When compared with patients without pulmonary arterial hypertension, the miR-140 expression was down-regulated and expression of Wnt-1 and β-catenin were up-regulated in lung tissues of PAH-CHD patients. Correlation analysis results showed that the expression of miR-140 was negatively correlated with pressure of pulmonary arterial and Wnt-1 and β-catenin expression.2. Pathological changes of lung tissues of patients without pulmonary arterial hypertension were unapparent, while lung tissues of patients with mild PAH-CHD had slightly thickened alveoli septum, broadening interlobular septa and mild congestion of alveolar lumen and small vascular. Lung tissues of patients with moderate and severe PAH-CHD had obvious alveolar expansion and broadening alveoli septum, local fibrosis, thickened blood vessel wall and narrowed lumen. The blood vessel wall of lung tissueserial and negatively correlated with thicken and WT% and WA% were increased as the pressure of pulmonary arterial increase. Correlation analysis showed that WT% and WA% were positively correlated with pressure of pulmonary arterial and negatively correlated with miR-140 expression.3. Apoptotic cells in pulmonary vessels were decreased and AI was declined in patients of PAH-CHD. Correlation analysis showed that AI was positively correlated with miR-140 expression and negatively correlated with WT%, WA% and pressure of pulmonary arterial. The result of imunohistochemistry showed that bax was highly expressed in pulmonary vessels of patients without PAH and with mild PAH. Pulmonary vessels of patients with moderate and severe PAH-CHD had low bax expression. The expression of bcl-2 and survivin were just the opposite with the expression of bax expression. Correlation analysis results showed that the expression of miR-140 was positively correlated with the expression of bax and negatively correlated with the expression of bcl-2 and survivin.Part II: The effect mechanism of miR-140 in HPASMCs Methods1. Primary HPASMCs were cultured in mionectic and normoxia incubator for 24 h or 48 h. The mRNA and protein expression of miR-140 and Wnt-1 in HPASMCs were detected by qRT-PCR and western blot.2. HPASMCs were transfected with mi R-140 mimic and inhibitor and cultured under mionectic condition. Proliferation of HPASMCs was detected by CCK-8 method and migration ability was detected by wound healing assay. SM-MHC, SM-α-actin, Calponin-1 and SM22α expression, which were related with smooth muscle cell contractive phenotype, were detected by western blot.3. HPASMCs which were transfected with miR-140 mimic and inhibitor were cultured under mionectic condition for 24 h. The mRNA and protein expression Wnt-1 in HPASMCs were detected by qRT-PCR and western blot.4. The recognition site of Wnt-1 for miR-140 was predicted by online tools, TargetScan,DIANA and miRanda. Interaction of miR-140 and Wnt-1 3’-UTR was verified by dual luciferase reporter assay.5. Wnt-1 over-expression or knock-down were conducted by transfecting recombinant plasmid or siRNA into HPASMCs. Proliferation of HPASMCs was detected by CCK-8 method and migration ability was detected by wound healing assay. SM-MHC、SM-α-actin、Calponin-1 and SM22α expression, which were related with smooth muscle cell contractive phenotype, were detected by western blot. Results1. The results of qRT-PCR and western blot showed that in HPASMCs, miR-140 expression was down-regulated under mionectic condition and the mRNA and protein expression of Wnt-1 were up-regulated.2. Under mionectic condition, overexpressing miR-140 by transfecting miR-140 mimic, proliferation of HPASMCs was declined and migration distance and numbers of migrated cells were reduced. Expression of SM-MHC, SM-α-actin, Calponin-1 and SM22α, which were related with smooth muscle cell contractive phenotype, were enhanced. When HPASMCs were transfected with miR-140 inhibitor to inhibit the expression of miR-140, proliferation and migration of HPASMCs were increased and expression of proteins related with smooth muscle cell contractive phenotype were declined.3. Under mionectic condition, overexpressing miR-140 by transfecting miR-140 mimic, mRNA and protein expression of Wnt-1 were reduced. When HPASMCs were transfected with miR-140 inhibitor to inhibit the expression of miR-140, mRNA and protein expression of Wnt-1 were enhanced.4. The recognition site of Wnt-1 3’-UTR for miR-140 was recognised by online tools, TargetScan,DIANA and miRanda. Dual luciferase reporter assay verified the interaction of miR-140 and Wnt-1 3’-UTR. These results confirmed that miR-140 targeted Wnt-1 directly.5. Under mionectic condition, overexpressing Wnt-1 by transfecting recombinant plasmid, proliferation of HPASMCs was declined and migration distance and numbers of migrated cells were increased. Expression of SM-MHC, SM-α-actin, Calponin-1 and SM22α, which were related with smooth muscle cell contractive phenotype, were declined. When HPASMCs were transfected with siRNA to knock down the expression of miR-140, proliferation and migration of HPASMCs were declined and expression of proteins related with smooth muscle cell contractive phenotype was increased. Conclusions1. MiR-140 expression was down-regulated and expression of Wnt-1 and β-catenin were up-regulated in lung tissues of PAH-CHD patients. The expression of miR-140 was negatively correlated with WT%, WA% and pressure of pulmonary arterial and positively correlated with AI.2. Under mionectic condition, miR-140 expression was down-regulated and Wnt-1 expression was up-regulated in HPASMCs. MiR-140 inhibited the expression of Wnt-1 by targeting Wnt-1 3’-UTR.3. Under mionectic condition, miR-140 inhibited the proliferation and migration of HPASMCs and facilitated the expression of contractile phenotype protein of HPASMCs.4. Under mionectic condition, Wnt-1 promoted the proliferation and migration of HPASMCs and inhibited the expression of contractile phenotype protein of HPASMCs.