The Mechanism Of MiR-29 Targeting LRP6-mediated Wnt/β-catenin Inhibition Of End Mt In Pulmonary Hypertension Of The Newborn

Objective:Pulmonary hypertension of the newborn(PHN)is a common pulmonary vascular disease of newborn.Its clinical manifestations are refractory hypoxemia,progressive increase of right ventricular pressure,right heart failure,and even death in severe cases.At present,there is no specific targeted prevention and treatment plan for this disease,so it is very urgent to explore its pathological mechanism.The pathological changes of pulmonary hypertension mainly include pulmonary vasoconstriction,abnormal vascular remodeling,pulmonary arteriole obstruction,namely neointimal formation.The increase of the number of cells expressing α-smooth muscle actin(α-SMA)is the main reason for the progression of pulmonary hypertension into irreversible vascular remodeling.In addition to the proliferation of middle model vascular smooth muscle cells and the differentiation of outer model fibroblasts,the phenotypic transformation of inner model vascular endothelial cells to mesenchymal like cells(End MT)is also an important source of α-SMA like cells.Therefore,the study on the regulation mechanism of End MT will become a breakthrough point for the targeted treatment of pulmonary hypertension in the future.miRNAs can be involved in a variety of cell functions and are important regulatory factors of End MT.Combined with our previous work and literature search,we found that miR-29 was greatly expressed in smooth muscle cells and endothelial cells of distal pulmonary arterioles.The pathological changes of the lung tissue of the miR-29 knockout mice showed that impaired maturation of smooth muscle cells in the distal pulmonary arterioles vascular wall with immature synthetic smooth muscle cells appeared.It is speculated that miR-29 may be involved in abnormal pulmonary vascular remodeling.Therefore,miR-29 family was selected as the target miRNA in this study to verify its differential expression in hypoxic PHN rat lung and human pulmonary artery endothelial cell lines and its correlation with End MT.Wnt signaling pathway plays an important role in lung development and lung disease.Previous studies of our group found that Wnt signaling pathway is an important regulatory mechanism of alveolar dysplasia induced by hyperoxygen in newborn BPD mice.Key proteins in the signaling pathway,such as Wnt3 a,Wnt5a and β-catenin,expressed at the vascular site,and preliminary experiments have confirmed that the expression of β-catenin is enhanced in the distal pulmonary vascular wall of pulmonary hypertension rats,suggesting that classical Wnt signaling pathway may also be involved in pulmonary vascular remodeling in pulmonary hypertension.In order to investigate the importance of the interaction between miRNAs and signaling pathways in pulmonary hypertension End MT regulation,we predicted the binding of miR-29 with important regulatory factors of classical Wnt signaling pathway through the Target website.A binding site was found between the 3’UTR of miR-29 and LRP6 protein in the Wnt pathway.LRP6 is an important Wnt ligand co-activated receptor in Wnt signaling pathway.As a transmembrane cell surface protein,LRP6 can bind to Wnt ligand and Frizzled protein,mediating the activation of typical Wnt/β-catenin signaling pathway.There were rare datas about the relationship between LRP6 and pulmonary disease,only a small number of studies were involved and most of them focused on regulating the proliferation,migration and transdifferentiation of lung tumor cells.Studies involving chronic lung injury had only been reported in a few BPD reports.Therefore,the role of LRP6 in pulmonary vascular endothelial cell dysfunction and vascular remodeling needs to be further explored.By observing the dynamic changes of LRP6 and Wnt/β-catenin in vivo and in vitro,we attempted to confirm that the activation of the above signaling pathways is an important regulatory mechanism promoting pulmonary vascular remodeling and the formation of neonatal pulmonary hypertension.By intervening the levels of miR-29 and LRP6 and other experimental methods such as Dual Luciferase assay,we attempted to confirm that miR-29 affected the Wnt/β-catenin signaling pathway by targeting LRP6 inhibition in hypoxic PHN,and realized the regulation of End MT eventually.Methods:1.The hypoxic PHN model was established in neonatal SD rats treated with 10% hypoxia for 48 hours.On the 0th,7th,14 th,and 21 st days of the experiment,10 rats were randomly selected from PHN and the control group,and the lungs were separated.The morphology of lung tissue was observed by HE staining;qRT-PCR method was used to detect the dynamic changes of miR-29 a,miR-29 b and miR-29 c in lung tissue;qRT-PCR combined with Western-blot method was used to detect the dynamic changes of mRNA and protein levels of End MT-related indicators including Snail,CD31,α-SMA and signal transduction pathways LRP6,β-catenin genes in lung tissue;On the time of 21 days of the experiment,the right ventricular pressure was measured in the two groups to reflect the pulmonary artery pressure,and the right ventricular hypertrophy index was calculated.Immunofluorescence staining was used to clarify the localization of CD31 and α-SMA,and to observe the remodeling of distal pulmonary small blood vessels and the occurrence of End MT.2.hPAECs were used as the research object.In vitro cell model of hypoxic pulmonary hypertension was simulated after 1% hypoxic treatment for 72 h,cells were collected for 24,48 and 72 h to study the dynamic changes of miR-29 a,miR-29 b and miR-29 c at the cellular level.qRT-PCR and Western-blot were used to detect the mRNA and protein levels of Snail,CD31,α-SMA,LRP6 and β-catenin in vitro.The differential expression of the above indicators was further verified by immunofluorescence staining,and the nuclear transfer of β-catenin and the co-location of CD31 and α-SMA were observed.3.According to the differential expression of PHN in animal and cell models,mir-29 a with the most significant down-regulation in the model and LRP6 with significantly increased expression were selected as the research targets.miR-29 was overexpressed and LRP6 was inhibited in cell models,respectively.qRT-PCR and Western-blot methods were used to detect the changes of End MT related indicators including Snail,CD31 and α-SMA in different cell groups of NC,NC+ hypoxia and miR-29a-3p mimic+hypoxia/si LRP6+ hypoxia after intervention.4.miR-29 a mimics and miR-29 a inhibitor were used to transfect hPAECs to establish miR-29 a overexpression and inhibition cell models,and qRT-PCR and Western-blot methods were used to detect the changes of LRP6 levels after miR-29 a intervention;Dual luciferase reporter was used to verify the binding of miR-29a-3p to LRP6;hPAECs were transfected with miR-29a-3p mimic,and the cells were divided into three groups including NC,NC+hypoxia,and miR-29a-3p mimic+hypoxia group.After transfected for 48-72 hours,the changes of LRP6,Wnt/β-catenin signaling pathway and End MT-related genes for example Snail,CD31 and α-SMA levels were observed.Results:1.The result of Western-blot and qRT-PCR showed that the expression of CD31 was decreased in the lung tissue of the PHN group and the cells of model group,the expression of α-SMA and Snail was increased.Co-localization of CD31 and α-SMA can be seen by Immunofluorescence double-stained in the inner wall of the distal pulmonary arterioles in PHN group and cells in the model group.2.miRNAs qRT-PCR method was used to confirm that miR-29 a,miR-29 b and miR-29 c were differentially down-regulated in hypoxic PHN lung tissue and endothelial cells in vivo/in vitro,among which miR-29 a had the most significant changes.The inhibition of the above-mentioned miR-29 family in PHN was time-correlated with the changes of CD31,α-SMA and Snail,that is,with the down-regulation of miR-29 levels,the expression of CD31 decreased,and the expression of Snail and α-SMA increased.3.Western-blot and qRT-PCR methods were used to detect the mRNA and protein levels of LRP6,β-catenin genes in the lung tissue of PHN group rats and hypoxic group in cell model.The Staining of those genes were enhanced after hypoxic injury in endothelial cells,and the nuclear expression of β-catenin was increased.4.After constructing the miR-29 a overexpression cell model by miR-29a-3p mimic transfection,the cells were treated with 1% hypoxia.Comparing with the NC group,it was found that CD31 was down-regulated and α-SMA and Snail were up-regulated;compared with the NC+hypoxia group,the changes of the above indicators were partially reversed in the miR-29a-3p mimic+hypoxia group,shown as CD31 level rebounded,Snail and α-SMA expression decreased.The LRP6 down-regulated cell model was constructed,and the results of Western-blot method showed that the trend of Snail,CD3 and α-SMA was the same as that of the miR-29 a overexpression group.5.The miR-29 a overexpression and miR-29 a inhibition cell models were successfully constructed,and it was found that the level of LRP6 was significantly decreased in the miR-29 a overexpression group compared with the NC group,while the level of LRP6 was significantly increased in the miR-29 a inhibition group compared with the NC group.The results of dual luciferase reporter gene assay showed that the luciferase activity was significantly increased in the co-transfected LRP6-mut+miR-29a-3p mimics group compared with the LRP6-wt+miR-29a-3p mimics group,suggesting that LRP6 can interact with miR-29a-3p by direct binding.6.The miR-29 a overexpression cell model was constructed.Compared with the NC group,the expression of LRP6 and β-catenin increased,CD31 was down-regulated and the levels of α-SMA and Snail were increased in the NC+hypoxia group;But compared with the NC+hypoxia groups,the expression of LRP6 was significantly decreased,and the expressions of β-catenin,Snail and α-SMA were also significantly decreased,and the level of CD31 was increased in the miR-29a-3p mimic+hypoxia group.Conclusion: 1.End MT exists in hypoxic PHN animals and cell models,and the pathological process is closely related to the down-regulation of miR-29 family,especially miR-29 a.miR-29 a can play the potential protective role for End MT in hypoxic PHN.2.LRP6,Wnt/β-catenin signaling pathway activating exists in hypoxic PHN animals and cell models,and LRP6 is involved in the occurrence of End MT in hypoxic PHN.3.miR-29a-3p can bind to LRP6 directly,which reduce the activity of LRP6 in PHN endothelial cells,impair the activation of Wnt/β-catenin signaling pathway,and inhibit the occurrence of End MT.It can become a new target method for the prevention and treatment for PHN in the future.