Title

Effects of AJAP1 Silencing on Gene Expression Pathways Related to Cell Adhesion, Migration And Epithelial-mesenchymal Transition

Date of Award

8-2020

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Cathy Hatcher, PhD

Second Advisor

Ruth Borghaei, PhD

Third Advisor

Heather Montie, PhD

Abstract

Coronary vessel anomalies affect up to 1% of the population. The proepicardium (PE) is a source of progenitor cells for the epicardium and coronary vessels and it consists of a transitory cluster of epithelial-like mesothelial cells that migrate out of the PE toward the heart tube where they adhere to the myocardium to form the epicardium. Epicardial cells proliferate, undergo apoptosis or epithelial-mesenchymal transition (EMT) to become epicardial-derived cells (EPDCs) that give rise to a subset of coronary vascular cells. Tbx5epi-/- mice with a conditional deletion of Tbx5 transcription factor in the PE exhibit impaired epicardium and coronary vessel formation. These mice have reduced cardiac expression of the Ajap1 mRNA encoding adherens junction-associated protein 1 that acts as a cell adhesion molecule in epithelial cells. AJAP1 is expressed in the developing mouse heart in the PE, epicardium and coronary vessels, but its role in cardiovascular development is unknown.

Study Objective: Preliminary data showed that AJAP1 gene silencing in human mammary epithelial cells increased epithelial cell-to-matrix adhesion, decreased cell migration, increased cell apoptosis and increased mRNA expression of E-cadherin and β-catenin epithelial markers. Our project goal was to determine the molecular target(s) of AJAP1 modifying epithelial cell behaviors important for epicardium and coronary vascular formation during cardiovascular development by: (1) identifying changes in epithelial gene expression that mediate cell migration, adhesion and EMT as a result of AJAP1 gene silencing, and (2) identifying the impact of AJAP1 gene silencing on CTNNB1 gene expression (β-catenin) and its correlation with epithelial cell migration and cell-matrix adhesion.

Methods: Primary human mammary epithelial cells (HMEpiCs) retain characteristics of epithelial cells. We silenced endogenous AJAP1 expression in HMEpiCS with small interfering RNAs (siRNAs) and produced observable changes in HMEpiC behavior. mRNA transcripts isolated from control and AJAP1-gene-silenced HMEpiCs were assayed for differential gene expression on qPCR array plates. We conducted gene ontology (GO) analysis to query the list of targeted genes and classify them by biologic functions. Also, we cross-referenced our results to previous assays of HMEpiC function for genes that likely contribute to vessel formation.

Results: siRNA-mediated silencing of the AJAP1 gene in HMEpiCs led decrease in AJAP1 mRNA expression by >85%. A comparison of Epithelial-Mesenchymal Transition, Adherens Junction and Tight Junction qPCR arrays revealed genes with altered expression in AJAP1-silenced versus control HMEpiCs. GO analysis showed these genes are involved in biological processes including blood vessel development, cell migration, cell-cell adhesion, cytoskeleton organization and cell junction organization. This analysis correlates with our previous functional data. One of the genes with altered expression, CTNNB1, encodes b-catenin and was increased in all 3 qPCR arrays.

Conclusion: AJAP1 is important for epithelial cells to maintain expression of genes involved in biological processes such as blood vessel development, cell migration, cellcell adhesion, cytoskeleton organization and cell junction organization. β-catenin appears to be a prominent player in these biological processes that likely occurs through an AJAP1-β-catenin interaction. The results of this research project will provide insight into the role of AJAP1 in epithelial cells of the epicardium and its progeny, the coronary vessels, during cardiovascular development.

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