Finding The New Factors And Signal Pathways Involved In Regulating ESCs Stemness Maintenance And Differentiation Into VECs By Chemical Small Molecules

BACKGROUND AND OBJECTIVEMTOR(mechanistic target of rapamycin)controls many cellular processes,including apoptosis,autophagy,translation,energy metabolism and inflammation.Rapamycin and its derivatives(rapalogs),inhibitors of MTOR,have been evaluated for their immunosuppressive and anti-proliferative properties and as anticancer agents.However,in some cases,inhibition of MTOR has negative effects on certain diseases.A possible avenue to solve the existing problems is the development of small chemical molecules that selectively modulate MTOR kinase.However,to our knowledge,we lack a chemical activator of MTOR.We previously synthesized a series of butyrolactone der:ivatives,and found 3-ben2yl-5-((2-nitrophenoxy)methyl)-dihydrofuran-2(3H)-one(3BDO)has a protective effect on neuronal cells via increasing the phosphorylation of RPS6KB1,a substrate of MTOR.We deduced that the 3BDO might activate the MTOR pathway.However,direct target and regulatory factors of 3BDO are not clear.Therefore,in this study we first plan to identify target of 3BDO and whether 3BDO could act as an activator of MTOR,and find out the key factors of downstream of MTOR by 3BDO.It has been reported that the 3'UTR-associated RNA(uaRNA),a kind of RNA derived from 3'UTR,can be independently expressed as developmentally regulated non-coding RNAs and may participate in regulating cell differentiation and embryonic development processes.Because of the unique position of uaRNAs in the transcript--overlapping protein-coding mRNAs--designing specific siRNA sequences for their knockdown or knockout is difficult.Therefore,it is found that many uaRNAs were significantly changed during embryonic development,but it is difficult to study the specific function and mechanism of action.To address this question,we need to find out the specialized technique and tool to regulate uaRNA.Based on the above studies,we found that 3BDO was targeted to FKBP1A and activated MTOR,and found out the selective downregulation molecule,uaRNA FLJ11812,after activation of MTOR by 3BDO.Therefore,3BDO is a powerful tool to study the function of uaRNA.Embryonic stem cells(ESCs)are pluripotent stem cells derived from the inner cell mass of a blastocyst,an early-stage embryo.They have been used as established models for investigating embryonic development and cell differentiation,especially in the human.In order to study the role of FLJ11812 in embryonic development and differentiation,we chose human embryonic stem cells(hESCs)as models to study the role of FLJ11812 on hESCs sternness maintenance and differentiation,and determine its molecular mechanism of action and role in human embryonic development.Because of pluripotency,ESCs are able to differentiate into more than 220 cell types in the adult body,including vascular endothelial cells.Vascular endothelial cells(VECs)are an important component of any organ and tissue regeneration.Vascular endothelial cells play an important role in thrombosis and hemostasis,angiogenesis,radiation injury,inflammation and immune response.In cell therapy and clinical applications,the lack of endothelial cells increased mortality of many diseases.Therefore,the clinical application needed develop new sources of endothelial cells.In our previous studies,we identified a novel benzoxazine derivative,6-amino-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine(ABO),could induce bone marrow stromal cells(BMSCs)differentiation to VECs by elevating Hmboxl expression.However,BMSCs have tendency of aging and limited proliferation when cultured in vitro,so they are very limited in clinical applications.Therefore,in this study we plan to investigate whether ABO could induce mESCs differentiation into VECs?To clarify which ABO enantiomers is more effective?To find out the key factor and signal pathway in differentiation of mESCs into VECs induced by ABO?Based on the backgrounds mentioned above,the objective of this study was to study the new factor and signal pathway of stemness maintenance,and investigate the key factors and molecular mechanisms of differentiation into vascular endothelial cells.For the study of these issues will provide new evidence to clarify the regulatory mechanisms of embryonic development,provide new ideas and theoretical basis for the clinical treatment of vascular diseases.STUDY CONTENTS1.To identify the target of 3BDO and whether it could act as an activator of MTOR.2.To find out the key factors downstream of MTOR after activation of MTOR.3.To study whether TIA1 involved in processing of 3'UTR-associated RNA,FLJ11812.4.To investigate whether FLJ11812 involved in the regulation of human embryonic stem cell stemness maintenance and differentiation.5.To study the molecular mechanisms of FLJ11812 regulating stemness maintenance of human embryonic stem cells.6.To investigate whether mixed ABO,R-ABO and S-ABO induce mESCs differentiation into VECs.7.To study the role and signal pathway of Hmboxl in differentiation of mESCs into VECs.METHODS1.We used mouse embryonic stem cells,human embryonic stem cells and human umbilical vein endothelial cells as study models,used ABO and 3BDO as study tools,to do related research.2.Identification of 3BDO target:2.1 Molecular docking analysis of binding sites of 3BDO in FKBP1A.2.2 Western blot analysis of phosphorylation of MTOR substrates RPS6KB1 and EIF4EBP1 after treated with 3BDO in the cells overexpressed FKBP1A cDNA.2.3 Western blot and immunofluorescence assay understand the antagonism between 3BD0 and rapamycin by detection of autophagy.3.Analysis of the phosphorylation of TIAl:Immunoprecipitation and Western blot analysis of phosphorylation of Ser residues in TIA1 protein.4.Analysis of independent expression of uaRNA FLJ11812:Northern blot and quantitative real-time PCR assay confirm that FLJ11812 were independently expressed from TGFB2 3'UTR.5.Detect the role of TIA1 in processing of FLJ11812:5.1 Real-time PCR analysis of the mRNA levels of FLJ11812 and TGFB2 mRNA in cells with siRNA knockdown of TIA1.5.2 RNA-chromatin immunoprecipitation assay to determine whether TIA1 involved in the processing of FLJ11812.6.Detect the effects on FLJ11812 levels by 3BDO in hESCs:6.1 Quantitative real-time PCR analysis of levels of FLJ11812 and TGFB2 mRNA levels in hESCs after treated with 3BDO.6.2 Fluorescence in situ hybridization detected the level and intracellular distribution of FLJ11812 in hESCs after treated with 3BDO.7.Measurement of stemness of hESCs after inhibition of FLJ11812:Quantitative real-time PCR,Western blot and immunofluorescence assay detected the core transcription factors(Oct4,Sox2 and Nanog)mRNA and protein levels in hESCs after inhibition of FLJ11812.8.Quantitative real-time PCR analysis of markers for three germ layers in hESCs after inhibited FLJ11812 by 3BDO.9.Detection of FLJ11812 binding with miR-4459,and validation of miR-4459 targets:9.1 Luciferase activity assays measured the luciferase activities after Luc-FLJ11812-WT and Luc-FLJ11812-A4459 plasmids were transfected into HEK239 cells with scrambled miRNA or miR-4459.9.2 Dual-luciferase reporter system were used to determine the targets(CDC20B?LARP1 and ATG13)of miR-4459.10.Detection of protein levels of miR-4459 targets and core transcription factors after inhibition of FLJ11812 or transduction of miR-4459 mimics in hESCs:10.1 Western blot analysis of protein levels of miR-4459 targets and core transcription factors after inhibition of FLJ11812.10.2 Western blot analysis of protein levels of miR-4459 targets and Oct4 after transduction of miR-4459 mimics in hESCs11.Flow cytometry,Western blot and immunofluorescence detect cell cycle distribution and autophagy after inhibition of FLJ11812.12.Detection of differentiation of mouse embryonic stem cells into vascular endothelial cell induced by R-ABO:12.1 Western blot and immunofluorescence assay of endothelial progenitor cell markers CD 133 and mature endothelial cell-specific markers(Flk-1,CD31,VE-cadherin,F VIII and eNOS protein)expression levels.12.2Flow cytometry detected differentiation rate of embryonic stem cells into vascular endothelial cells induced by R-ABO.13.Using matrigel capillary-like tube formation assay and Alexa488-Ac-LDL take-up assay detected function of vascular endothelial cells induced by R-ABO.14.Detect the role of Hmboxl in differentiation of mouse embryonic stem cells into vascular endothelial cell induced by R-ABO.14.1GFP-shRNA-Hmbox1 was applied for knockdown of Hmbox1 expression in mESCs.14.2Western blot,Matregel capillary-like tube formation assay,Dil-Ac-LDL take-up assay detect the effects on differentiation of mESCs into VECs induced by R-ABO after knockdown the Hmbox1.RESULTS1.3BDO inhibited FLJ11812 levels via activation of MTOR pathway1.1 3BDO might form hydrogen bonds with TYR82A and ILE56A sites in FKBP1A,the 2 amino acid sites for rapamycin binding with FKBP1A.Phosphorylation of RPS6KB1 and EIF4EBP1 was significantly increased by 3BDO with vector alone but suppressed with FKBP1A overexpression.1.2 After pre-treated with 3BDO for 30 min,and then treated with rapamycin for 6 h,the levels of p-MTOR and p-RPS6KB1 were decreased with rapamycin;however,rapamycin failed to decrease the phosphorylation of MTOR and RPS6KB1 in the presence of 3BDO.3BDO suppressed the autophagy caused by rapamycin.1.3 The phosphorylation of Ser residues was decreased in cells treated with rapamycin,and 60 ?M 3BDO reversed the phosphorylation and increased the level.1.4 3BDO significantly inhibited3 'UTR-associated RNA,FLJ11812.1.5 TIA1 may be involved in the processing of FLJ11812.2.A 3'UTR-associated RNA,FLJ11812,maintains stemness of hESCs2.1 The levels of FLJ11812 were significantly depressed and the levels of TGFB2 mRNA were not affected by 3BDO.FLJ11812 level was selectively decreased with 3BDO treatment in hESCs.2.2 The mRNAs and proteins expression of core transcript factors(Oct4,Sox2 and Nanog)in hESCs were significantly decreased,after FLJ11812 was suppressed by 3BDO treatment,and trigger spontaneous differentiation into three germ layers.3.FLJ11812 bind with miR-4459,and miR-4459 target to CDC20B?LARP and ATG133.1 FLJ11812 cDNA(Luc-FLJ11812-WT)and mutational cDNA with the presumed miR-4459 recognition sequences deleted(Luc-FLJ11812-?4459)downstream of the luciferase reporter gene were transfected in HEK293 cells along with miR-4459 mimics.Luciferase activity was reduced by about 20%and 40%compared their control miRNA with 10 and 40 nM miR-4459 mimics transfection,respectively.FLJ11812 could bind with miR-4459.3.2 Luciferase reporters containing 3'UTR of LARP1(Luc-LARP1),CDC20B(Luc-CDC20B)and ATG13(Luc-ATG 13)were constructed for targets investigations.We found that miR-4459 mimics reduced the luciferase activities of the reporter vectors containing 3'UTR of LARP1,CDC20B and ATG13.4.FLJ11812 functioned as an endogenous sponge of miR-4459 and regulated the targets of miR-4459-CDC20B,LARP1 and ATG13-in hESCs4.1 The protein levels of CDC20B,LARP1 and ATG13,which are targets of miR-4459,were decreased in hESCs inhibited FLJ11812 by 3BDO,and meanwhile,the levels of core transcriptional regulators were also decreased.4.2 Inhibition of FLJ11812 could cause hESCs lose ESC-specific cell cycle and decreased autophagy level via inhibition of miR-4459 targets.5.R-ABO efficiently induced differentiation of mESCs into functional VECs5.1 We used different concentrations of R-ABO to treat mESCs,10 ?M R-ABO significantly induced CD31 protein expression.After mESCs were treated with R-ABO,the level of endothelial progenitor cell marker CD133 protein was increased at the sixth day and the eighth day.Otherwise,the levels of mature VECs specific markers VE-cadherin,F ?,CD31,eNOS,and Flk-1 were increased in R-ABO-treated mESCs at the tenth day.5.2 R-ABO effectively promoted the capillary-like structure formation and uptake capacity of acetylated low-density lipoprotein from plasma.The rate ofCD31-positive cells was 32.13%in the R-ABO treated group.6.Hmboxl played important role in differentiation of mESCs into functional VECs induced by R-ABO6.1 R-ABO instead of S-ABO promoted Hmboxl expression dramatically after treatment for 2 to 6 days.6.2 When Hmboxl was knocked down in mESCs by its shRNA,R-ABO failed to promote CD 133 expression,and could not promote the capillary-like structure formation and uptake capacity of acetylated low-density lipoprotein from plasma.6.3 After treated with R-ABO,the expression of FGF-2 was most significantly increased at the fourth day compared with the control group.FGF-2 could not be up-regulated by R-ABO if Hmbox1 was knocked down.CONCLUSIONS1.3BDO was targeted to FKBP1A and activated MTOR signal pathway.2.TIA1 may be involved in the processing of 3'UTR-associated RNA,FLJ11812.3.3BDO selectively decreased the level FLJ11812.4.FLJ111812 is an important regulator of hESC stemness.5.FLJ11812 acted as a competing endogenous RNA,bound with miR-4459 and regulated the expression of its targets(CDC20B,LARP1 and ATG13),thereby regulating the cell cycle,mRNA stability and autophagy level,finally controlled sternness maintenance of human embryonic stem cells.6.R-ABO could efficiently induce mouse embryonic stem cells into functional vascular endothelial cells.7.Hmboxl acted upstream of FGF-2 signal pathway in R-ABO-induced mouse embryonic stem cells differentiation into vascular endothelial cells.