| Objective:1.To determine the effect of SREBP1 on hiPSCs differentiation;2.To study the effect of SREBP1 on endothelial function;3.To clarify the molecular mechanism of SREBP1 to promote the function of hiPSC-ECs;4.To investigate the effect of SEBP1 on mature endothelial cells and its possible molecular mechanismMethods:SREBP1 expression was detected in PAD artery by qPCR and westernblot To further understand how SREBP1 correlated with endothelial cells of PAD artery,we collected arteries from PAD patients receiving amputation and performed HE staining and dual staining for CD31 and SREBP1,whose intimal thickness was measured by Image J.The expression of OCT4-a marker of pluripotency of hiPSCs,CD31 and KDR representing endothelial cells were evaluated by westernblot and qPCR on day 0,day 3 and day 6.A specific SREBP1 inhibitor called fatostatin was used to decrease the expression of SREBP1 in hiPSCs at the concentration of 1?mol and 2um and its effect was confirmed by westernblot.We evaluated the differentiation efficiency on day 7 using FACS and study the influence of SREBP1 on endothelial function using transwell assay for migration function and matrigel for tube formation function.TRANSFAC database was used to predict possible binding site of SREBP1 within miR-199b-5p promoter to clarify how SREBP1 reduced miR-199b-5p expression,which was confirmed by CHIP.Besides the production of ECs,we further investigate the role SREBP1 and its mediated miR-199b-5p and SCD1 pathway played in ECs function.We took HUVEC as example to investigate the effect shSREBP1 hiPSC-ECs had on common endothelial function under non-interactive co-culture condition.To validate shSREBP1 hiPSC-ECs regulate HUVEC function by miR-199b-5p pathway,we knocked down SREBP1 in hiPSC-ECs and inhibited the expression of miR-199b-5p in HUVECResults:1 HiPSCs changed their colony structure to typical cobblestone-like appearance of endothelial cells.Besides,as classic endothelial markers,12.6%of CD31+cells and 22.3%of CD34+cells suggested an effective differentiation protocol was constructed successfully.SREBP1 had the most remarkable change in some important lipid regulatory factors and was downregulating during differentiation.2?mol SREBP1 inhibitor enhanced the production of hiPSC-ECs more than lum SREBP1 inhibitor did2.Then SREBP1 depletion in hiPSC-ECs promoted migration and tube formation function of hiPSC-ECs.SREBP1 had the biggest change and was highly expressed in ASO arteries.Besides,SREBP1 expression was negatively associated with CD31 and was higher in endothelial cells of ASO patients.ASO arteries was separated into high SREBP1 expression and low SREBP1 expression equally.Its results revealed high SREBP1 expression was associated with thicker intima and may predict the progress of ASO3.In our study,miR-199b-5p was increased gradually in contrast to decreasing SREBP1 expression during hiPSC differentiation.Moreover,SREBP1 depletion in hiPSCs results in increased miR-199b-5p level Through analyzing the promoter region of miR-199b-5p gene,we found a potential SREBP1 binding site.Using ChIP assays,we demonstrated that SREBP1 was recurred to promoter region of miR-199b-5p not the control region4.HiPSC-ECs promoted the function of mature endothelial cells in consistent with other studies.Interestingly,mature endothelial cells co-culture with shSREBP1 hiPSC-ECs presented stronger migration and tube formation function compared to those with control hiPSC-ECs.Similar to the effect in hiPSC-ECs,ShSREBP1 hiPSC-ECs also inhibited NOTCH1 pathway in mature endothelial cellsConclusion:1.SREBP1 suppresses hiPSC-ECs function and hiPSCs differentiation by inhibiting mir-199b-5p pathway2.SREBP1 decreased mature endothelial cells function by inhibiting miR-199b-5p pathway... |
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