Construction Of Screening Model For MafA Gene Expression Modulators And Screening Of MafA Activators From Small Molecule Compounds

Diabetes is a chronic metabolic disease,and its basic feature is the increase in blood sugar levels in the human body.In recent years,diabetes patient population has increased year by year and has become a serious health problem worldwide.As the disease progresses,the fuction and number of islet βcells in the patient’s body would gradually decrease which leads to progressive aggravation of hyperglycemia.At present,the enhancement of βcell function or the augmentation of the number of functionalβcells is still the important means of treating diabetes,and it is also the main direction of research and development of diabetes drugs.Among them,beta cell replacement therapy is considered to be a direct and effective approach of treating diabetes,however it is severely constrained by limited beta cell sources.In recent years,the induction of isletβcells from stem cells has become an important way to getβcells that attracts the most attention,but the efficiency and quality of induction have yet to be improved,and the induction process needs to be optimized as well.Therefore,the development of new high-efficiency inducers and the establishment of simpler and more effective induction methods have become the research focus of this research field,and it is also the hope that the application of β-cell replacement therapy will benefit diabetic patients in the future.The developmental maturation and functional manifestations of pancreatic isletβ cells are tightly regulated by some sequential expression regulators,such as pancreatic and duodenal homeobox factor-1(Pdx1),and myofascial fibrosarcoma genes.The source A gene(v-maf musculoaponeurotic fibrosarcoma oncogene homologue A,MafA),homeobox protein 6.1(Nkx6.1),neurogenic differentiation factor 1(NeuroD1),and so on.In view of this,our laboratory intends to target Pdx1,MafA,Nkx6.1,and NeuroD1 to select effective small molecule compounds that stimulate the expression of these important regulatory factors,which may provide effective inducers of stem cell differentiation into mature βcells.Also,it may also be possible to obtain an active ingredient that modulates islet beta cell functional activity.This study aimed to establish screening models formodulators of expression of three important transcription factors,MafA,Nkx6.1 and NeuroD1.Of these,we mainly screened small compound stimulators of MafA expression,the important terminal differentiation marker of of isletβcells and conducted related research.The results obtained are as follows.First,the promoter regions of the three genes,MafA,Nkx6.1 and NeuroD1,were amplified by PCR and ligated into the pGL3-basic luciferase reporter vector to construct the recombinant luciferase reporter vector,pGL3-MafA,pGL3-Nkx6.1 and pGL3-NeuroD1.The three recombinant reporter vectors were further demonstrated to have highluciferase activities by luciferase activity assay,indicating that each of the promoter fragments we have get has good transcriptional activity,which can be used to screen corresponding gene expression regulators.Second,we applied the pGL3-MafA reporter vector to screen stimulator of MafA expression based on luciferase activities from more than 700 small moleculer compounds of the laboratory’s two small-molecule compound libraries.Through primary and secondary screening processes,No.95 and No.202 compound was identified to be able to enhance the transcriptional activity of Ma Fa promoter.Then compounds 95 and 202 were further confirmed to indeed promote the expression of the MafA gene at both mRNA and protein levels in the islet βcell line INS-1 cells by using RT-q PCR and Western Blot techniques.Third,we found that compounds 95 and 202 significantly activated ERK and AKT signaling pathways in INS-1 cells,and increased the levels of phosphorylated ERK and AKT.By using inhibitors of these two signaling pathways,we further demonstrated that compounds 95 and 202 promoted the expression of the MafA gene through activating the ERK and AKT pathways.Finally,we investigated the possible biological effects of compounds 95 and 202 as promoters of MafA expression by detecting islet βcell function,differentiation of islet βcell tumors,and induction of differentiation of mesenchymal stem cells into islet βlike cells.ELISA showed that compounds 95 and 202 could promote insulin secretion of pancreatic islet βcells under glucose stimulation.Anchorage-independent colony formation experiment showed that compounds 95 and 202 could significantly reduce the ability of clone formation of pancreatic tumor cell line RINmf5 cellsindicating that the transforming ability of compound-treated pancreatic tumor cells is decreased.In the process to induce mesenchymal stem cells differentiation into islet β-like cells,we found compounds 95 and 202 could significantly promote the formation of islet β-cell-like cell clusters accompanied with enhanced expression ofβ cell characteristic factors such as Pdx1,MafA and insulin when compounds were added in the final step of induction process.In summary,this study successfully constructed three models for screening expression regulators of the transcription factors MafA,Nkx6.1 and NeuroD1,which are important in islet β cell function and differentiation,and screened out small moleculer compounds that stimulate MafA expression,compounds 95 and 202.Then we revealed the molecular mechanism to stimulate the expression of MafA and biological effects of compounds 95 and 202,especially their roles in promoting the differentiation of mesenchymal stem cells intoβ-like cells,suggesting that they can be used for induction of stem cell differentiation into islet β cells in the future,which has important scientific significance and application value.