Degree Name

Master of Science (MS)

Semester of Degree Completion

2018

Thesis Director

Gary A. Bulla

Abstract

Mammalian development involves a complex system of regulatory signals and reactions, leading to the creation of distinct tissues with specific structures and functions controlled by various gene expression profiles. Tissue-specific gene expression is by controlled specific combinations of transcription factors and cofactors that dictate cell differentiation via activation and repression of genes in mammalian cells.

Whole genome microarray analyses of fibroblasts have revealed candidate genes that may serve as master regulators of fibroblast identity, such as Prrx1 and Snai2, that activate downstream genes such as Bmp3, Twist, Shox2, Cfos, Slug, Sema3A, Spp1, and Col1a1. A previous study showed that Prrx1 and Snai2 play important roles in activating expression of fibroblast identity using somatic cell hybrids as model systems. Based on these results, we sought to examine the role of Prrx1 and Snai2 in affecting the phenotyope of liver-derived cells using hepatoma cells as a model system.

Expression plasmids containing Prrx1 and Snai2 expression cassettes were transfected separately into Fg14 rat hepatoma cells using a standard transfection technique, then G418-resistant clones were selected (pooled and individually) and monitored for over-expression of Prrx1 and Snai2. RNA was extracted and used to synthesize cDNA. Quantitative real-time polymerase chain reaction (qPCR) was carried out using specific primer pairs and signals normalized to GAPDH mRNA levels. In addition, we observed alteration morphology and cell migration using Scratch Assays.

qPCR analysis revealed that both Prrx1 and Snai2 were successfully over-expressed in pooled Fg14 transfectants and individual clones compared to non-transfected cells. Next we monitored expression of several genes known to be important in liver and fibroblast function. Results show that Snai2 activated several genes (Twist,Cfos,Shox2,and Bmp3) in Fg14 hepatoma cells. Although several other fibroblast genes (SEMA3A and Spp1) were relatively unaffected. Prrx1 overexpression resulted in activation of (Cfos,Twist,Shox2,Bmp3,and Col1a1) expression in Fg14 hepatoma, but (SEMA3A and Spp1) were unaffected.

Based on these findings we hypothesized that overexpression of fibroblast-specific transcription factor Prrx1 and Snai2 would result in loss of hepatoma phenotype. We therefore monitored expression of a panel of liver-specific genes using qPCR analysis. Results show that several liver related genes are repressed by these transcription factors, leading to partial loss of the hepatic phenotype. We observed that the cells re-acquired a spindle-shaped morphology and more significantly an enhanced migration capability, which is reminiscent of parental fibroblast cells. In conclusion, these results suggest that candidate core Prrx1 and Snai2 serve as a useful starting point for studying transcriptional control of cell identity and reprogramming of mammalian cell types.

Download

Share

COinS