2022-07-052022-07-052013-04-12https://hdl.handle.net/20.500.12503/31407Purpose: Methylation of CpG islands in CRH gene (crh) promoter region plays an important role in crh regulation. MeCp2 binds to methylated CpG islands and act as a transcriptional repressor. Increased crh promoter activity and expression following mutations in MeCP2 suggests its role in crh regulation. In hypothalamus glucocorticoids down-regulate CRH gene expression as part of negative feedback mechanism. The molecular mechanism and role of different co-regulators in this process is poorly understood. The purpose of this study is to elucidate the role of MeCP2 in the dexamethasone- a synthetic glucocrticoid (Dex) induced GR mediated crh repression in rat hypothalamic cell line. Methods: We used small hairpin RNA (shRNA) induced MeCP2 knockdown to understand the importance of MeCP2 in GR mediated crh regulation. We first screened the shRNA plasmids against rat MeCP2 for knock down efficiency using western blotting. Scrambled sequence was used as negative control. The IVB- rat hypothalamic immortalized cell line was transfected with shRNA or scrambled DNA containing vector with GFP. The transfection efficiency was monitored by fluorescent imaging using EVOS digital microscope. The transfected cells were incubated in stripped serum media for 48hrs prior to treatment with Dex 10-7M for 2hrs. Total RNA was extracted using Tri reagent and cDNA was synthesized. qPCR was performed using gene specific primers for gapdh, crh and heteronuclear RNA (hnRNA) of crh (hncrh). ΔΔCT method was used for calculation of relative gene expression. Results: After 72 hrs, following the shRNA transfection Mecp2 protein expression was knocked down to significant level in one out of four plasmids as compared to scrambled control. This shRNA plasmid was used for further experiments. Following Dex 10-7M treatment for 2 hrs there was considerable decrease in CRH mRNA as well as hnRNA. This effect of Dex was lost following MeCP2 Knock down. Conclusions: From the preliminary data we conclude that MeCP2 plays an important role in crh regulation. Furthermore, presence of MeCP2 is necessary for the GR mediated down regulation of crh.MeCP2Corticotropin Releasing HormoneGlucocorticoid Receptorsposter