Zeinab Shirvani Farsani - Department of Cellular and Molecular Biology, Shahid Beheshti University G.C. , Tehran, Iran
Mehrdad Behmanesh - Department of Genetics, Tarbiat Modares University, Tehran, Iran

Background and Aim: Vitamin D regulates a number of importantbiological processes. It protects the adult stem cells or limits the expansionof cancer stem cells. Recent the experimental data have indicated themolecular function of vitamin D in vitro cell culture systems usingcancer cell lines. Vitamin D signaling is through the Vitamin D receptor(VDR). VDR is expressed in the kinds of cells and involved in apoptosis,cell cycle, proliferation, immunity, and inflammation. In this study, weinvestigated the effects of VDR knockdown on some gene expression inJurkat and U87-MG cell lines.Methods: In the present study Jurkat and U87-MG cell lines were used.These cell lines were maintained in RPMI-1640 medium. In orderto knock-down VDR, Jurkat T and U87-MG cells were transfectedwith pGFP-V-RS shRNA clones targeting human VDR transcripts anda scrambled non-targeting control. Following the transfection, theexpression of GFP was observed under a fluorescence microscope.Next, Total RNA was extracted from cells and cDNA was synthesized. Toanalyze the expression of IL-10, NF-KB, TGF-β1, TGF-β2, TGF-β R I andTGF-β R II mRNAs and Test knockdown efficiency, real-time PCR wasperformed at 24 h and 48 h after transfection. Results: Quantitative PCR showed that the VDR transcript expressionwas decreased in VDR shRNA-transfected groups. In Jurkat T cells thereduction of the VDR mRNA expression at 24 and 48 h after transfectionwas77 % and 71% down respectively and in U87-MG cells was 74% and76% respectively. Analysis of qRT-PCR showed that the expression of IL-10, NF-KB, TGF-β1, TGF-β R I and TGF-β R II in Jurkat T cells transfectedwith VDR-shRNA was significantly decreased compared to ScrambleshRNAcells and the untransfected cells, but no significant differencewas observed in these cells for TGF-β2. Additionally, the expression ofNF-KB, TGF-β1, TGF-β2, TGF-β R I and TGF-β R II in U87-MG cells aftertransfection with the VDR-shRNA, was significantly increased comparedwith Scramble-shRNA cells and the untransfected cells, but this differencewas not significant for IL-10.Conclusion: We have shown a fundamental role of vitamin D receptorin regulating TGF-β signaling and highlighting the potential function ofVDR to control some gene expression in cancer cell lines; Jurkat T andU87-MG. There are clearly still significant gaps in our understanding ofthe VDR roles in stem cells. In future investigations, it will be of scientificand clinical interest to investigate effects down-regulation VDR on theapoptosis, cell cycle and other biological processes in different stemcells.

RNAi; Jurkat T cells; Stem cells; TGF-β signaling; IL-10