Function Of Fat Mass And Obesity Associated Gene FTO In Regulation Of Flag-Ghrh MRNA Expression

RNA,as a communication bridge between genetic information DNA and protein molecules,plays a central role in the transcription of genes,post-transcriptional modification and gene expression,protein synthesis and other life activities.especially,post-transcriptional processing of RNA,peculiarly RNA methylation lays important foundation for the diverse biological function of cells.N6-methyladenosine,as the most common form of eukaryotic post-transcriptional modifications,apart from eukaryotic mRNA 5’cap structure,has critical effects on mRNA splicing,nuclear transportation,translation and degradation,and m~6A modification involved in Lipogenesis,Carcinogenesis and Stem Cell Regeneration.m~6A methylation is reversible which is carried out by methylase and demethylase.Fat Mass and Obesity Associated gene FTO is demethylase,it enables N6-methyladenosine to demethylate and affects the life activity of cells and organisms.In this study,the effects of m~6A modification on the transcription and translation of genes were verified by the cells with overexpressing Flag-Ghrh and knocking out FTO in vitro.The detailed investigations and results were shown below.1.Construction of vector with Flag-Ghrh overexpression.Ghrh is mainly secreted in the hypothalamus,it is difficult to detect its expression in vitro,which has brought great difficulties to the study.In order to better study its transcriptional regulation mechanism,we constructed the Flag-Ghrh protein.Objective:The aim of this part is to construct and validate the expression of Flag-Ghrh.Methods:We constructed the overexpression vector of Flag-Ghrh p ENTR-Flag-Ghrh and adenovirus p Ad-CMV-SP-Flag-Ghrh-P2A-GFP-SV40,using frameshift mutations,and we validate the expression of Flag-Ghrh by Enzyme Cutting,Western Blot and Cytopathic Effect.Results:We successfully built the expression vectors and adenovirus of Flag-Ghrh in vitro,which made the gene of Flag-Ghrh be expressed in 293A,He La and C2C12 cells.Conclusion:The overexpressed vector of Flag-Ghrh was constructed in vitro to prepare for the study of the interaction between FTO and Flag-Ghrh.2.Construction of viral vector for knocking out FTO gene.FTO is detected extensively in multiple tissues,especially in the hypothalamus.our previous results revealed that FTO plays critical roles in growth.Therefore,we speculated that the FTO gene may regulate gene expression of growth-related Ghrh.Objective:The aim of this part is to construct a viral vector for knocking out FTO gene.Methods:We construct lentivirus and adenovirus with FTO knockdown by shRNA and CRISPR/Cas9,and we validate that FTO was knocked out by immunofluorescence,T7E1 and Western Blot.Results:FTO was knocked out by lentivirus and adenovirus,and the effects of the two viruses were consistent.Conclusion:The viral vector was constructed,which laid foundation for the study of the roles of FTO.3.Verification the roles of FTO in the regulation of mRNA of Flag-Ghrh.FTO is m~6A demethylase.m~6A modifications in mRNA increased when we knocked down FTO gene utilizing si RNA.It indicated that FTO knockdown increased the level of m~6A methylation of eukaryotic mRNA,Thus enhancing the transcription and translation of mRNA.Objective:The aim of this part is to detect the variation of mRNA and protein of Flag-Ghrh after FTO gene was knocked out.Methods:First,Flag-Ghrh was overexpressed in He La cells,and then FTO was knocked out.Finally,the changes of mRNA and protein levels of Flag-Ghrh were detected by q-PCR and Western Blot.Results:Knocking out demethylase FTO increased the m~6A methylation level of mRNA,which increased mRNA and the protein expression level of Flag-Ghrh.Conclusion:The number of mRNA was more than the one of control group,whilst the protein expression levels of Flag-Ghrh were higher than the one of control group after FTO was knocked down.Results indicated that m~6A methylation in mRNA of Flag-Ghrh gene was enhanced after FTO gene was knocked down,thereby regulating the transcription and translation of Flag-Ghrh gene,which enables the level of mRNA and protein expression of Flag-Ghrh to enhance.These provide critical foundation for the study of m~6A modification in mRNA.