Design and validation of primers for accurate analysis of role of chemokines CCL4 and CCL21 on Fas mediated NF-kB pathway

Jennifer Frost, Rohini Mehta, A. Birerdinc. A. Baranova

This is a collaborative project between

Molecular and Microbiology Department, College of Science,George Mason University, Fairfax, VA

Translational Reseach Institute, Inova Hospital, VA

The increased prevalence of obesity in the United States and worldwide has resulted in a number of conditions collectively termed metabolic syndrome such as type 2 diabetes, hypertension, cardiovascular disease, non-alcoholic steatohepatitis (NASH), and non-alcoholic fatty liver disease (NAFLD). It is believed that nearly one half of the adult population is obese (BMI>25) and that of those individuals, up to 74% have NAFLD. A number of studies have implicated numerous cytokines in the role of liver inflammation and progression to NAFLD including IL-6, TNFα, CCL3, CCL5, and CCL34. Studies in mice have shown Fas receptor ligation resulting in up-regulation of CXC chemokines in hepatic inflammation. Furthermore, this Fas signaling was found to be independent of the NFκB pathway. This same study also determined that Fas-mediated apoptosis, specifically in the liver, contributes to liver fibrogenesis and stellate cell activation. Gene expression studies from our laboratory have shown CXC chemokines, CCL21 and CCL4, to be correlated with advanced hepatic inflammation in obese patients with biopsy proven NAFLD. Interesting, while CCL21 was positively correlated with advanced inflammation, CCL4 was negatively correlated. In order to validate our hypothesis that CCL21 and CCL4 may play an important role in progression of NAFLD via Fas mediated NF-kB signaling, gene expression studies were carried out. A critical step in gene expression studies is design and validation of specific primers. Primers were designed using NCBI primer BLAST and were validated by PCR.