Graduate Program
Biological Sciences
Degree Name
Master of Science (MS)
Semester of Degree Completion
Spring 2024
Thesis Director
Gary A. Bulla
Thesis Committee Member
Sanghoon Kang
Thesis Committee Member
Yordan S. Yordanov
Abstract
Despite all cells within an organism sharing the same genetic information, they develop into different tissues, each with a distinct gene expression profile required for tissue specialization. Understanding gene expression and the processes that regulate the expression, is crucial for understanding cell differentiation and tissue specialization, as these procedures are governed by unique gene expression and regulation patterns.
Whole genome microarray analyses identified several candidate genes that may be coding important transcription factors that play role in fibroblast identity such as Prrx1, Snai2, Shox2, Twist1, c-Fos, Msx1, Pdx1 (Idx1) and Pura (Pur1). The goal of this project is to determine if two of those candidate genes, Msx1 and Pura, can act to reprogram hepatoma cells to disrupt liver function in cultured hepatoma cells and activate fibroblast function. Our strategy was to transfect rat hepatoma cells (Fg14) or human hepatoma cells (HepG2) with mouse Msx1 and human hepatoma cells with mouse Pura (Pur1) gene expression plasmids, which contains the Neo gene, using lipofection and select for G418-resistant clones. Quantitative Polymerase Chain Reaction (qPCR) analysis confirmed successful overexpression of these genes in transfected cells while comparing to non-transfected cells. The same method was used to compare expression of introduced genes as well as liver and fibroblast marker genes to non-transfected parental cells by using gene specific primers. GAPDH was used to normalize signals and the DDCt method to calculate fold differences in expression between samples.
The overexpression of Msx1 and Pura genes resulted in variable effects on liver- and fibroblast- specific genes. Msx1 overexpression led to a modest reduction in the expression of liver genes and decreased expression of fibroblast genes. Specifically, Prrx1 and Col1a1 exhibited the strongest decreases, Snai2 and c-Fos displayed more modest reductions, while two other genes tested, Spp1 and Shox2, showed no or modest reduction in Msx1 transfectants. Unexpectedly, the expression of fibroblast-specific genes ranged from no effect to strong reduction. Pura overexpression in HepG2 cells led to modest reductions in liver-specific gene expression and significant repression of the fibroblast gene Cola1a. The expression levels of two other fibroblast genes, Prrx1 and Sema3, failed to be detected in either HepG2 cells or transfectants. These findings suggest that while the overexpression of Msx1 and Pura can influence gene expression, their potential to drive cell programming in hepatoma cells require more research. This indicates that more complex mechanisms are involved, possibly requiring more than a single driver for effective reprogramming.
Recommended Citation
Diler, Merve, "Reprogramming of Rat and Human Hepatoma Cells with Fibroblast-Specific Transcription Factors MSX1 and PURA" (2024). Masters Theses. 5043.
https://thekeep.eiu.edu/theses/5043
