Screening Of Compounds That Promote The Expression Of Oct4 And Study On Their Roles In Maintaining Stem Cells

Stem cell is a kind of primitive cell with high differentiation potential and self-renewal ability.Because of its unique properties,it plays an important role in disease treatment,regenerative medicine,and developmental biology.Although stem cells have been used in clinical therapy for a long time,stem cell therapy still faces problems such as insufficient cell numbers and easy differentiation in vitro culture,which affects the clinical application of stem cells.In view of the problems of unclear composition,unstable nature,and high price in current culture methods such as adding conditioned media,serum substitutes,and growth factors,it is necessary to screen low-priced and stable small molecule compounds to maintain the pluripotency of stem cells.Oct4 is the most important factor in the regulation of stem cell stemness,the change of its expression level directly affects stem cell stemness.Therefore,this study aims to screen small molecule compounds that can enhance stem cell stemness by promoting Oct4 gene expression,which can be used as medium additives for two-dimensional stem cell culture,and at the same time lay the foundation for the development of reprogramming inducers.In this study,the p EZX-MT01-Oct4-3'UTR luciferase reporter plasmid was used as the experimental material to screen small molecule compounds that can increase the expression of Oct4 at the post-transcriptional level.Then explore the biological activity of the compounds that promote the expression of Oct4 and find small molecule compounds that can promote stem cell stemness.Finally,the preliminary mechanism study of the obtained small molecule compounds was carried out.1.Preliminary screening of compounds with post-transcriptional regulation of Oct4expressionIn order to find compounds that can regulate the expression of Oct4 gene at the posttranscriptional level,cells transfected with p EZX-MT01-Oct4-3'UTR luciferase reporter plasmid were used as the screening model,and 627 listed drugs purchased in the laboratory were initially screened.The result of luciferase activity test showed that 66 kinds of drugs can obviously promote the activity of luciferase.It is suggested that these compounds may promote the expression of Oct4 at the post-transcriptional level.2.Rescreening of compounds with post-transcriptional regulation of Oct4 expressionIn order to find compounds that can really target the 3'UTR of Oct4 m RNA,p EZX-MT01-Oct4-3'UTR or p EZX-MT01 and p CMV-?-gal plasmids were co-transfected into the cells respectively,and then the target compounds obtained in the preliminary screening were added for stimulation.The results of luciferase activity detection showed that compounds A8-61,A8-66,2A1-20,2A2-52,2A3-6,and 2A2-11 can significantly enhance the activity of luciferase,indicating that they can specifically target Oct4 m RNA 3' UTR.3.The effect of the target compound on the expression of Oct4The expression level of Oct4 protein is the final determinant of stem cell stemness.Therefore,we selected embryonic cancer cell P19 as the target cell to detect the effect of the target compound on the expression of Oct4.Western Blot detection results showed that compounds 2A1-20,2A2-52,and 2A3-6 could not promote the expression of Oct4 protein in P19 cells,while A8-61,A8-66,and 2A2-11 could increase the expression of Oct4 protein in P19 cells,suggesting that these compounds may have a regulatory effect on the stemness of stem cells.4.Analysis of the non-toxic concentration of the target compoundTo investigate the effects of compounds A8-61,A8-66,and 2A2-11 on the stemness of P19 cells,we performed cell viability assays on P19 cells treated with the three compounds.Determine the concentration of the compound at IC10,and use the concentration lower than IC10 to study the effect of the compound on the stemness of P19 cells.MTT test results showed that compound A8-61 and 2A2-11 had no toxic effect on P19 cells within the experimental concentration range,compound A8-61 significantly promoted the proliferation of P19 cells at40?g/m L,and the IC value of compound A8-66 is 7.644+0.182?g/m L.5.The effect of target compound on dryness factors Oct4,Sox2 and NanogAccording to the results of the non-toxic concentration determination,we selected2.5?g/ml and 5?g/ml as the experimental concentrations to study the stemness of the three compounds on P19 cells.WB test result show that compound A8-61 and 2A2-11 can promote the expression of Oct4,Sox2 and Nanog at 2.5?g/ml and 5?g/ml,while compound A8-66 only promotes Oct4,Sox2 and Nanog at 5?g/ml.6.The effect of target compound on P19 cell self-renewalThe above results indicate that compounds A8-61,A8-66,and 2A2-11 can enhance the expression of pluripotency factors in P19 cells.Can the three compounds also promote the selfrenewal ability of P19 cells in varying degrees? To clarify this problem,we combined the plate clone culture method with the alkaline phosphatase staining method to explore the self-renewal ability of P19 cells.The results of alkaline phosphatase staining show that compared with the control group,compound A8-61 and 2A2-11 can promote the formation of P19 cell clones with2.5?g/ml and 5?g/ml,compound A8-66 only promoted the cloning ability of P19 cells with5?g/ml.7.Research on signal pathways of target compounds to maintain P19 cell stemnessTo better understand how the compound promotes the stemness of P19 cells,we studied the stemness-related signaling pathways on P19 cells treated with the compound.We used Western Blot to detect the changes of key factors pathways of PI3K/AKT and MEK/ERK.The results show that compound A8-61 can promote the phosphorylation of AKT at 2.5?g/ml and5?g/ml,suggesting that 2.5?g/m L and 5?g/m L A8-61 can promote the stemness of P19 cells by activating the PI3K/AKT signaling pathway.Compound 2A2-11 can promote the phosphorylation of PI3 K and AKT while inhibiting the phosphorylation of ERK.It is suggested that 2A2-11 promotes stem cell stemness by activating the PI3K/AKT signaling pathway and inhibiting the MEK/ERK signaling pathway that negatively regulates stemness.In summary,this study screened out three compounds that can promote the expression of Oct4 at the post-transcriptional level and promote the stemness of P19 cells from 627 kinds of marketed drugs,and preliminarily determined the mechanism of two of them to promote the stemness of P19 cells.This research provides a new additive for the large-scale expansion of stem cells in vitro,and lays the foundation for the development of reprogramming inducers.