MiR-1291 Regulates The Noncanonical Wnt Signaling Pathway Via The Wnt11 In Congenital Pulmonary Airway Malformation

Objective: Congenital pulmonary airway malformations(CPAM)are rare abnormalities of the development of the fetal lung.It is characterized by a polycystic mass in the lung tissue with hyperplasia of the bronchial structures and damage to the alveoli.It is considered a hamartomatous malformation by some researchers or a localized developmental arrest by others.The pathogenesis of CPAM is still unclear,and any mutation or abnormality of any gene or signaling pathway in the process of lung formation may lead to CPAM.The cause of CPAM should be a complex process produced by the interaction of multiple genes and several environmental factors.Some scholars have suggested that the airway pattern is abnormal during the morphogenesis of lung branching,and there are defects in the early lung development,suggesting that it mainly occurs in the pseudoglandular stage.(6-16 weeks in gestation).However,the research progress of related pathogenic genes of CPAM is relatively slow.There are still many related susceptibility genes that have not been identified.The Wnt signaling protein family is involved in multiple developmental events during embryogenesis and in adult tissue homeostasis.The Wnt signaling pathway plays an important role in correcting the development of the lung,which is one of the more well-studied pathways.In the previous study,the members of our research group observed that Wnt2 B,ACSL5,Wnt7 B,BMP4,SOX10 and COL2A1 were expressed differently in CPAM.Wnt11 belongs to the Wnt signaling family,and the non-canonical Wnt11 signaling pathway mediated by it is expressed in both epithelial cells and stroma during the lung development.Some studies have demonstrated that Wnt11,the non-canonical Wnt ligand,plays a role in mediating smooth muscle-actin expression in airway smooth muscle cells in asthmatic patients.It can regulate actin remodeling and may be important in various processes of airway remodeling.Rho A is a ubiquitously expressed cytoplasmic protein that belongs to the family of small GTPases.Rho A,as a downstream molecule of Wnt5 and Wnt11,regulates convergence and extension movement by involving effector molecules Rho kinase and transparent protein.Rho A is one of the downstream effector molecules in the PCP pathway.It plays an important role in the development of airway epithelium,especially in establishing the respiratory tract and maintaining the differentiation of airway cilia along the airway axis.It also participates in regulating the establishment of the pulmonary vascular system.Micro RNAs(miRs)are non-coding single-stranded RNA molecules of about 20-22 nucleotides in length.miRs are able to repress gene expression at the posttranscriptional level through nucleotide base pairing between the miRcomplementary sequence and the 3’-untranslated regions(3’-UTR).The miR-1291 has multiple functions,including regulation of cellular drug distribution and chemosensitivity,regulation of cellular proliferation and metabolism by targeting specific gene sets.It has been reported that miR-1291 was involved in the regulation of various cancers.This study aimed to observe the changes of Wnt11,Rho A and miR-1291 contents by comparing CPAM tissue samples with surrounding normal tissues,and further through cell culture and other experimental methods to study the effect of miR-1291 overexpression or inhibition on cell proliferation,and the changes of Wnt11 and Rho A protein content were observed.To explore their possible roles in the pathogenesis of CPAM.Methods: 1.Part Ⅰ : The data sets of lesion tissue and normal lung tissue samples from patients with CPAM were obtained from the Gene Expression Omnibus(GEO),and the differences between groups were analyzed to determine the differentially expressed genes(DEG).Gene Ontology(GO)enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)/Reactant pathway database were used to determine the functional role of DEG.Using bioinformatics prediction software,Target Scan and miRBD,the miRNA with the highest possibility of combining with DEG was selected.2.Part II : The surgical specimens of CPAM and surrounding normal lung tissue were collected from the children with CPAM who underwent surgery in the Pediatric Surgery Department of Shengjing Hospital of China Medical University.We used immunohistochemistry to observe the differences in the expression of Wnt11 and Rho A,and we used Western Blot and q RT-PCR to detect the expression of Wnt11 and Rho A in the above-mentioned tissues.At the same time,we used q RT-PCT to detect the expression of miR-1291 in the above-mentioned tissues.3.Part III : MiR-1291 mimics were used to establish miR-1291 overexpressed human bronchial epithelial cells of human lung(BEAS-2B),and 48 hours after transfection,the cells were detected by Phalloidin staining,EDU staining,MTS,and flow cytometry.Dual luciferase reporter assay was applied to validate the interaction between Wnt11 and miR‐1291 in 293 T cells.At the same time,the expression changes of Wnt11 protein were detected by Western Blot for the BEAS-2B cell lines overexpressed and knocked down by miR-1291,respectively.Pc DNA3-Wnt11 were used to establish Wnt11 overexpressed BEAS-2B cells,and 48 hours after transfection,the cells were detected by Phalloidin staining,EDU staining,MTS,and flow cytometry.BEAS-2B cells were transfected using miR-1291 mimics and pc DNA3-Wnt11 vectors,and 48 hours after transfection,the cells were detected by Phalloidin staining,EDU staining,MTS.Using miR-1291 overexpression cell lines,Western Blot was used to detect the expression changes of Rho A proteins.Results: 1.The data set GSE179404 was obtained from GEO,and the differences between groups were analyzed.1347 genes were significantly upregulated or down-regulated.Through GOKEGG analysis,it is found that there are differences among positive regulation of cytokine production,regulation of actin filament-based process,Wnt signaling pathway,planar cell polarity pathway,Cell adhesion molecules,Proteoglycans in cancer,Antigen processing and presentation in CPAM.Based on the previous research conducted by our research group,Wnt11 related to Wnt signal pathway was selected as our research objective.According to the screening results of bioinformatics prediction software,it is speculated that the miRNA binding to Wnt11 is miR-1291.2.It was obtained that the pathological tissues of CPAM and normal patients from surgery,and we collected the general information such as age,gender,and Stocker type.The results of immunohistochemistry showed that the average optical density value of Wnt11 in CPAM tissue was significantly lower than that in surrounding normal tissue,and that of Rho A in CPAM tissue was significantly higher than that in surrounding normal tissue.The results of Western Blot and q RT-PCR showed that the expression of Wnt11 in CPAM tissue was lower than that in surrounding normal tissue,and the expression of Rho A in CPAM tissue was higher than that in surrounding normal tissue.The results of q RT-PCR showed that the expression of miR-1291 in CPAM tissue was significantly higher than that in surrounding normal tissue,and the level of miR-1291 in CPAM tissue was about 2.8 times higher than that in the surrounding normal tissue.3.The number of miR-1291 overexpressed cells were slightly smaller and the cytoskeleton morphology was abnormal by phalloidin staining.MTS and EDU assays showed that miR-1291 overexpressed inhibited BEAS-2B cells viability.Flow cytometric analysis showed that BEAS-2B cells transfected with miR-1291 mimics were arrested in the G1 phase,and decreased in S phase.Luciferase reporter assay showed that miR-1291 mimics significantly reduced the luciferase activity of WNT11‐Wt,but not of WNT11‐Mut.Moreover,miR-1291 mimics significantly decreased WNT11 expression in protein levels in BEAS-2B cells,and miR-1291 inhibitor increased WNT11 expression.The cytoskeleton morphology of Wnt11 overexpressed cells was abnormal by phalloidin staining.MTS assays showed that Wnt11 overexpressed promoted BEAS-2B cells viability.Flow cytometric analysis showed that BEAS-2B cells transfected with pc DNA3-Wnt11 were arrested in the G2 phase,and decreased in G1 phase.The cytoskeleton morphology of miR-1291 mimics and pc DNA3-Wnt11 group cells was abnormal by phalloidin staining.MTS and EDU assays showed that miR-1291 mimics and pc DNA3-Wnt11 group cells were promoted viability.And miR-1291 mimics significantly decreased Rho A expression in protein levels in BEAS-2B cells by Western Blot method.Conclusion: 1.Our results show that overexpression of miR-1291,decreased expression of Wnt11,and increased expression of Rho A in the lesion area of CPAM resulted in changes in cells and tissues in the lesion area,which may be one of the pathogenesis of CPAM.2.Functional assays indicated that miR-1291 mimics remarkably decreased BEAS-2B cells proliferation in vitro,and Wnt11 increased BEAS-2B cells proliferation.MiR‐1291 targets WNT11 in BEAS-2B cells.Overexpressed miR-1291 and Wnt11 increased BEAS-2B cells proliferation.The present study revealed that overexpressed mir-1291 suppressed the progression of BEAS-2B cells by targeting the WNT11 axis.The miR‐1291/WNT11 axis might be one of the pathogenesis of CPAM.