Alexander Graden
Downregulating the Androgen Receptor in Pancreatic Cancer
Overview: Lowering the expression of a steroid hormone receptor, androgen receptor (AR), in the pancreatic cancer cell line COLO-357 with small interfering RNA (siRNA) that can degrade the mRNA of the AR before it has the opportunity to become a full protein and be expressed as an intracellular receptor. Abstract: Pancreatic cancer affects hundreds of thousands of people globally every year, and with a current survival rate of 7%, there is a high demand for new and effective treatments. Pancreatic cancer has been shown to express the androgen receptor (AR), a member of the steroid receptor protein family that mediates cell differentiation, growth, and survival. While in prostate cancer, AR overexpression and hyperactivation is an oncogenic factor, and thus a therapeutic target, the role of the AR in pancreatic cancer remains unknown and inconclusive. The goal of the present project is to determine the role of the AR in the pancreatic cancer cell model COLO 357 with respect to cell viability using the small interference RNA (siRNA) method. Three different plasmids with a pLKO.1 backbone, pLKO.1 empty vector, as well as two vectors coding for two different siRNAs targeting two different segments of the 3’ region of the AR mRNA, pLKO.1/462 siRNA and pLKO.1/730 siRNA, were amplified in Escherichia coli by heat shock transformation and liquid culture followed by plasmid isolation using silica-based column chromatography. An additional plasmid coding for an enhanced green fluorescence protein (pEGFP) under the cytomegalo virus (CMV) promoter was also amplified. COLO 357 cells were transfected with the plasmids using the lipofectamine 2000 reagent and were incubated for 72 hours. Fluorescence microscopic analysis of pEGFP transfected cells indicated a transfection efficiency of ~30-50%. RNA was isolated from the cells by silica-based column chromatography and subjected to AR-specific quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) to determine the expression level of the AR. Preliminary data showed AR repression by pLKO.1/730 siRNA and AR induction by pLKO.1/462 siRNA. Future studies include a repetition of these inconclusive results, as well as cell viability and cell death assays to determine the importance of the AR in pancreatic cancer cells.
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