Title

Downregulation of Caveolin-1 on Acinar Formation of Mammary Epithelial Cells

Date of Award

2019

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Abigail Hielscher, PhD

Second Advisor

Charles A Daniels, MD, PhD

Third Advisor

Dennis Peffley, PhD, JD

Fourth Advisor

Richard White, PhD

Abstract

Altered expression of caveolin-1 (cav-1), the main structural protein of caveolae responsible for macromolecular transcytosis and regulation of signal transduction, has been shown to support breast tumorigenesis. We have previously shown that there is a link between the loss of cav-1 and abnormal ductal architecture as well as fibronectin expression and epithelial cell growth in murine mammary glands. This study was performed to evaluate the role of the downregulation of cav-1 on the development of mammary epithelial acini, the structural and functional component of mammary glands. To determine whether mammary epithelial cells form acinar structures on, on Matrigel® (a basement membrane extract), human mammary epithelial MCF10-2A were grown on the matrix and cultured for 1,4,7,10,14,18 and 21 days. At each time point, acinar growth/structure was assessed using immunofluorescence for the nuclear marker DAPI. The thickness of the resultant acinar structures were analyzed using the confocal microscope and measured using ImageJ along with the circumference of 20x DAPI stained acinar structures. Results from these analyses showed a progressive increase in both thickness and width of the structures. Cav-1 was downregulated in MCF10-2A cells with shRNA. Eight different transfection methods/reagents were utilized to optimize transfection efficiency. Results showed that the method using 3ul lipofectamine and 20ul cav-1 shRNA produced the most efficient results. Additional trials will be needed in order to confirm these findings.

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