The Regulation Mechanism Of NFI To Radiation Sensitive Gene IERS And Analysis Of IER5 Protein Structure

Objective:The Immediate early gene encodes a class of polypeptide factors that play an important role in cell regulation and response to external stimuli.These genes are expressed differently in different cells.The early slow response genes have a delayed response to stimuli,resulting in relatively slow transcription and longer response times.IER5(immediate-early response gene 5)belongs to the family of early slow response genes.Some studies revealed that the transcription or translation of the IER5 gene was up-regulated in Hela cells after exposure to radiation.Although domestic and foreign scholars have some understanding of the biological function and characteristics of IER5 gene,there are few researches on the regulatory mechanism of IER5 gene by transcription factors,especially under radiation mediation.Combined with the previous research work of this project,it was found that NFI was likely to be a transcription factor of the transcriptional regulation of IER5 gene.At the same time,the radiotherapy effect of cervical cancer varies greatly among individuals,so how to increase the radiotherapy effect is a big challenge in clinic.The purpose of this study was to explore whether NFI was involved in the transcriptional regulation of IER5 gene by radiation and its transcriptional regulation mechanism,and to express and purify a large number of IER5 protein in vitro by insect baculovirus expression system and to predict the structure of IER5 protein by bioinformatics technology,which aims to provide the clues to the determination of protein structure by experiment and the theoretical basis for radiotherapy sensitization and drug development in clinic.Methods:(1)The key regions of IER5 promoter were found by double luciferin reporter gene assay.Bioinformatics was used to predict the binding site of NF1 on IER5 promoter.(2)The over-expression vector of NFIA,NFIB,NFIC and NFIX were built and the siRNAs were synthesized.The radiation and no radiation groups were detected in IER5 gene transcription activity,the relative levels of mRNA and relative protein expression,which was to determine the regulation of the certain NFI members on IER5 gene and radiation effects on IER5.(3)EMSA experiment was conducted to compare the binding of NFIC to the IER5 gene promoter in vitro between the irradiated group and the unirradiated group.(4)NFIC enrichment efficiency in the promoter region of IER5 gene were detected by ChIP-PCR and ChIP-qPCR respectively.(5)The recombinant transfer vector pFastBacl-IER5 was built.The recombinant transfer vector was digested by restriction enzyme EcoR Ⅰ and Hind Ⅲ,and further identified by agarose gel electrophoresis.(6)The recombinant shuttle plasmid rBacmid-IER5 was built by transforming pFastBacl-IER5into the competent e.coli DH10Bac.The universal primer M13 of shuttle plasmid Bacmid was used to identify the recombinant transfer vector.(7)The P2 recombinant baculovirus was transfected into Sf9 insect cells.The indirect immunoinfluscent assay was used to observe the expression of recombinant protein when obvious cell lesions appeared.(8)The cells infected with the P2 recombinant baculovirus were collected and proteins were extracted when obvious cell lesions appeared.SDS-PAGE was used to isolate the identify proteins.Meanwhile,the non-coomassie bright blue dye samples were transferred to NC film for Western Blot analysis.(9)The physical and chemical properties,secondary structure and tertiary structure of IER5 protein were predicted and analyzed by bioinformatics.Results:(1)It was found that the key region regulating the expression of IER5 gene in HeLa cells was located in the region of-249 bp～-63 bp,and the transcriptional activity of IER5 promoter was up-regulated by radiation.The sequences GATT of NFI binding site was mutated into ATCC by using site-specific mutation technique.The transcriptional activity of IER5 promoter decreased significantly after mutated NF1 site.(2)After the transfection of siNFIC into HeLa cells,the transcriptional activity,mRNA level and protein expression of IER5 were down-regulated to different degrees,but radiation could reverse the down-regulation effect.After transferring into the recombinant NFIC overexpression vector pcDNA3.1-NFIC,the transcriptional activity,mRNA level and protein expression of IER5 were up-regulated respectively,and the upregulation was further enhanced by radiation.(3)The results of EMSA experiment showed that NFIC could directly act on the IER5 gene promoter in vitro,but this effect was independent of radiation.(4)The results of ChIP experiment showed that NFIC could be significantly enriched to the IER5 gene promoter in vivo,but the enrichment efficiency was not affected by radiation.(5)The electrophoresis results show that there were two expected bandings at 1000 bp and 4500 bp,which implied that the recombinant baculovirus transfer vector pFastBacl-IER5 was successfully constructed.(6)The PCR identification results showed that there was 1 banding at about 3300 bp,indicating the successful preparation of recombinant shuttle plasmid rBacmid-IER5.(7)After the recombinant baculovirus infected Sf9 cells,the cells’ diameter gradually increased and the refractive index became stronger.The results of IFA showed green fluorescence in cells infected with recombinant virus,while control cells showed no fluorescence.(8)SDS-page and Western blot results showed that specific bandings were observed at 48kDa,while no characteristic bands were observed in control.(9)Bioinformatics analysis showed that amino acid residues with negative charges accounted for 12.84%,while those with positive charges accounted for 9.48%.The total molecular weight was 33703.69.Theoretical isoelectric point(PI)was 4.91.The instability coefficient was 61.1,which indicated IER5 protein was an unstable protein.IER5 protein was a hydrophilic protein.The secondary structure showed that there were six helix,no secure folding and rotation angle on the inside of IER5 protein,and most of the rest were in irregular curl state.Conclusions:Radiation could up-regulated the expression of IER5 in HeLa cells,and NFIC could up-regulated the expression of radiation sensitive gene IER5 by directly binding to the IER5 promoter.However,NFIC was not involved in the regulation of IER5 gene in radiation.IER5 protein was successfully expressed by baculovirus expression system in vitro.The tertiary structure of the protein was predicted by bioinformatics,and the model of tertiary structure was shown.