Identifying the role of non-coding RNAs in normal and abnormal lung development due to congenital diaphragmatic hernia
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Date
2019-04-01
Authors
Kahnamoui Zadeh, Shana
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Abstract
Introduction:
Congenital Diaphragmatic Hernia (CDH) is a diaphragmatic defect with severe respiratory distress. Our laboratory found that miRNA-200b plays a crucial role in the etiology of CDH. We established three specific objectives to identify the mRNA targets of miR-200b, profile the upstream regulators of miRNAs such as circRNAs, and perform a descriptive study on Yes-associated protein (YAP) in healthy and CDH rat lungs during development.
Methods:
MiRNA-mRNA pull-down assay was performed by precipitating the mRNA targets bound to the complex of miR-200b mimic, biotin, and streptavidin beads from E19 rat lungs’ primary cells. Microarray analysis was used to determine the expression profile of circRNAs from E15 and E21 rat’s lung samples. A novel in situ hybridization method, BaseScopeTM, was used to validate the circRNAs of interest (mmu_circRNA_31436 and rno_circRNA_007475). Immunofluorescence method was performed to visualize the localization of YAP and p-YAP during lung development in control and CDH rat models. Moreover, Western Blotting was performed to measure the quantity of YAP and p-YAP among samples.
Results:
The quality and quantity of transcripts targeted by miR-200b was low. This suggest that miRNA-mRNA pull-down assay for RNA capture failed outright. I found that the expression profile of circRNAs was significantly altered in the lungs of CDH rat models compared to those from control rats. Moreover, the results from BaseScopeTM have confirmed the microarray data, in which mmu_circRNA_31436 and rno_circRNA_007475 were significantly up-regulated and down-regulated in E21 CDH rat’s lungs compared to control lungs, respectively. I observed that YAP was mainly inactive in the mesenchymal cells and the airway epithelium of CDH rat lungs during all developmental stages. Western Blotting data showed that YAP was significantly down-regulated in E21 CDH rat lungs compared to healthy lungs.
Conclusion:
Finding the mRNA targets of miR-200b and the profile of its upstream regulators, circRNAs, could lead us to understand the underlying biology of miR-200b in the etiology of CDH. Moreover, dysregulation of non-coding RNAs can affect the normal function of transcription factors, such as YAP. Our findings suggest a disruption in the regulation of YAP, which can be involved in the pathogenesis of abnormal lung growth in CDH.
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Keywords
Normal lung development, Congenital Diaphragmatic Hernia, MiRNA-200b, CircularRNAs, Yes-Associated Protein
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APA