Author(s)
Laura R. Garcia-Rodriguez MD
Lamont Jones MD
Kang Chen MD
Indrani Datta MS
George Divine PhD
Maria Worsham PhD
Affiliation(s)
Henry Ford Hospital
Abstract:
Educational Objective: At the conclusion of this presentation, the participants should be able to explain the definition of a master regulator, role of a causal network, and impact of tributyrin on a keloid cell line and changes of gene expression. Objectives: We previously uncovered four master regulators through Ingenuity Pathway Analysis software s Causal Network Analysis (CAN) (QIAGEN, Redwood City, CA, USA) that were hypothesized to have a driver role in keloid methylation gene targets and influence keloid pathogenesis. The objective is to investigate the role of tributyrin as a master regulator and to assess its influence on modulating the expression patterns of four downstream statistically significant keloid genes in vitro. Study Design: Prospective cohort. Methods: Tributyrin (Sigma-Aldrich, St. Louis, MO, USA), was used in a methyl tetrazolium (MTT) cell proliferation assay (American Type Culture Collection, Manassas, VA, USA) to determine its IC50 dosage for in vitro studies using keloid cell lines. The expression levels of keloid specific downstream genes VAMP5, TNS1, GALNT3, PPP1R13-± in the tributyrin causal network will be assessed using quantitative reverse transcriptase PCR (qRTPCR) for verification based on the hypothetical experiment produced by CNA. Results: The MTT assay, concentrations of 5 mM, 2.5 mM, 1 mM, 0.5 mM, 0.25 mM, 0.1 mM for 24, 48 and 72 hours, demonstrated that the IC50 for tributyrin was at 48 hours with a concentration of 1 mM. We expect that the expression levels of the four gene targets of tributyrin will be consistent with the hypothetical experiment indicated in CNA, where VAMP5, GALNT3, PPP1R13-± are overexpressed and TNS1 is underexpressed. Conclusions: Tributyrin's influence in modulating the expression of the downstream keloid specific genes for would be one step in dissecting out mechanistically its role in keloid pathogenesis.