Abstracts

#12 The Role of Endothelin-Receptor A in the Development of Pulmonary Hypertension in the miR-200b Knockout Mouse


Chelsea Day, Children Hospital Research Institute of Manitoba, University of Manitoba; Naghmeh Khoshgoo, Children Hospital Research Institute of Manitoba, University of Manitoba; Landon Falk, Children Hospital Research Institute of Manitoba, University of Manitoba; Nolan De Leon, Children Hospital Research Institute of Manitoba, University of Manitoba; Daywin Patel, Children Hospital Research Institute of Manitoba, University of Manitoba; Richard Keijzer, Children Hospital Research Institute of Manitoba, University of Manitoba


Introduction

Pulmonary hypertension (PH) is one of the leading causes of morbidity and mortality in patients with congenital diaphragmatic hernia (CDH). Higher expression of microRNA-200b (mir-200b) is associated with better outcomes in CDH patients. To study the role of mir-200b we created a mir-200b KO mouse and found that these mice present with PH, similar to CDH patients. The objective of this study was to determine if endothelin-receptor A (ETA), a known vasoconstrictor and potential mir-200b target, could contribute to the development of PH.


Methods

Lungs of 8-week-old mir-200b KO and WT mice were extracted and flash frozen. Complete RNA was then extracted from these tissues and DNAse treated. cDNA, made from RNA was used to run reverse transcriptase quantitative-PCR (RT-qPCR) to determine expression levels of ETA mRNA. For immunohistochemistry (IHC), lungs of 8-week-old mice were perfused with formalin before extraction, dehydrated and embedded in paraffin. IHC was used to look for differences in expression of ETA between KO and WT mice using antibody UMB-8 (Abcam).


Results

RT-qPCR showed that expression of ETA mRNA in the lungs of mir-200b KO mice was higher then that in the lungs of WT mice (standardized to endogenous TBP). Though it is not statistically significant (p=0.1109, n=6), the sample size is currently being increased. IHC allowed us to see higher levels of expression of ETA protein around the arteries in both mir-200b WT and KO mice. Though expression levels around the arteries of mir-200b KO mice appear to be higher compared to WT mice.


Conclusion

Our data suggest that expression of ETA is higher in the mir-200b KO mouse then the WT mouse and expression appears to be highest around the lung arteries. This shows that mir-200b most likely regulates ETA expression and that higher levels of ETA could be contributing to PH in the mir-200b KO mouse.