Construction Of Screening Model For Nanog Gene Expression Modulators And Screening Of Nanog Modulators From Traditional Chinese Medicine

Stem cells are a group of pluripotent cells that have broad application prospect in clinic duo to its developmental totipotency,especially on tissue and organ reconstruction.Therefore,how to maintain their undifferentiated state during in vitro amplification and how to induce their differentiation into specific cell types and further develop into demand tissue and organs become problems that should be solved quickly.Nanog is a transcription factor discovered in 2003 which specifically expressed in totipotent cells.It was found that Nanog played a crucial role in the process of pluripotency maintaining,early differentiation and adult cell dedifferentiation.In the present study,we choosed Nanog promoter as a target and established a Nanog promoter-driven luciferase reporter screening model,and screened the compounds that modulate the Nanog promoter activity by detecting luciferase activity,and preliminary clarified the possible molecular mechanisms by which the candidate compounds regulated Nanog gene expression.The results were shown as following.1.pcDNA3-Nanog eukaryotic expression vector was successfully constructed.We cloned the coding sequence of Nanog gene by RT-PCR,and ligated it into pMD18-T cloning vector,and then subcloned into pcDNA3.The recombinant eukaryotic expression vector pcDNA3-Nanog was proved by sequencing.2.The Nanog promoter-driven luciferase reporter vector was constructed.We amplyfied 1931 bp of Nanog gene 5 'non-coding sequence from the A549 cells genome by PCR,and inserted it into pGL3-basic vector to construct the luciferase reporter vector pGL3-NanogP of Nanog promoter.The luciferase activity result indicated that the sequence we amplyfied from Nanog gene 5 'non-coding region,had high promoter activity and can be used in subsequent works.3.Nanog gene expression regulator was screened.We screened more than 400 traditional Chinese medicine compounds by using luciferase reporter activity assay.After primary and secondary screening,we found that compound NA202 had significant promotion effect on Nanog promoter's activity,whereas NII 125 had obvious inhibition effect on Nanog promoter's activity,and we also verified that NA202 can increase,while NI125 can reduce the expression of Nanog protein in P19 cells.4.Effects of NA202,NA125 on intracellular signaling pathways.By luciferase reporter gene assay and western blotting analysis,we found that the compound NA202 and NI125 affected different signaling pathways.NI125 significantly inhibited the activities of NF-?B responsive luciferase reporter,and reduced the phosphorylation levels of ERK and I?B,wnereas NA202 increasea the pnospnoryiation level of I?B.I nese resuits suggest that the inhibition effect of NI125 on Nanog gene expression may partially associate with down regulation of ERK/MAPK and NF-?B signaing pathways,whereas NA202 may regulate Nanog expression via NF-?B signaling pathway.