Publication Date

12-2009

Type of Culminating Activity

Thesis - Boise State University Access Only

Degree Title

Master of Science in Biology

Department

Biology

Major Advisor

Dr. Cheryl Jorcyk

Abstract

The pleiotropic cytokine Oncostatin M (OSM) belongs to the interleukin-6 family and is produced by a variety of cell types including T-lymphocytes, neutrophils, monocytes and macrophages. OSM produces both cancer progression and growth retardation depending on cell type and its microenvironment. Our lab has also shown OSM may increase aggressive characteristics in tumor cells including the induction of vascular endothelial growth factor (VEGF), cell detachment, and invasiveness in cancer cells. The resulting effects of OSM exposure increase metastasis and tumor progression in selected cell types. The nearly sevenfold increase in VEGF attributed to OSM is one of the most influential factors in OSM-related tumor progression and metastasis.

To elucidate the signaling pathways involved in OSM and the subsequent aggressive tumor qualities, we have implemented RNA interference (RNAi) to knockdown the expression of the unique receptor, OSM receptor beta (OSMRß). RNAi utilizes double-stranded RNA (dsRNA) binding proteins of the silencing pathway to target endogenous mRNA for degradation. dsRNA in the form of short-hairpin RNA (shRNA) was expressed from a vector stably transfected into mammary cancer cells. The three shRNAs for mouse OSMRß (mOSMRß) had no knockdown effect on the expression of mRNA, protein expression or VEGF secretion. Additional transfections and subcloning supported the absence of knockdown. Current research indicates that the insufficiency of the shRNAs could be due in part to ineffective shRNA selection when ix compared to rational design and a single nucleotide alteration previously not reported within the target region.

Two auxiliary studies were conducted, one on the electroporation of a vector over expressing OSM and a second on a novel method for analysis of RNA quality. The pilot electroporation study showed no effect when the over expressing vector was compared to controls. The trial did provide a foundation from which future electroporation protocols will be based. The RNA analysis gel demonstrated significant efficacy in reducing the degradation posed by RNA degrading enzymes, such as ribonuclease A (RNase A). With the addition of household bleach to a standard agarose electrophoresis gel, the activity of introduced RNase was dramatically diminished. The application of this simple step has been demonstrated to significantly reduce the amount of toxic reagents used in RNA quality analysis and moreover substantially reduce the cost, as compared to standard formaldehyde based gels. Though no knockdown was observed during the OSM study, the novel RNA gel was proven to be an effective strategy and a manuscript has been submitted for publication.

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