| OBJECTIVE:To confirm the negative effects of miR-328 during inducing mouse embryonic stem cell(ESC)into insulin-inducing cell(IPC),and to elucidate the mechanism of how miR-328 regulates TGF-β2.Clarify the effects of overexpression of miR-328 on the ability of IPC to regulate blood glucose in diabetic mice,and provide new ideas and epigenetic regulation targets for the treatment of diabetic stem cells.METHODS:(1)A three-step model of mouse ESC induced into IPC in vitro was constructed.The morphology of cells in each stage of differentiation was observed by microscope.Immunofluorescence(IF)was used to identify the expression of Insulin+/C-peptide+ proteins.Flow cytometry(FCM)was performed to identify the proportion of Insulin+ cells.Quantitative Real-time PCR(qRT-PCR)was carried out to identify islet function gene expression;(2)Agomir miR-328 was constructed.After transfecting multilineage precursor cells(Multilineage progenitor cell,MPC)with miR-328 agomir,qRT-PCR detected the overexpression efficiency.On day 21,step 3,qRT-PCR detected expression levels of pancreatic genes both experimental group and control group,FCM detected ratio of Insulin+ cell and enzyme-linked immunosorbent assay(Enzyme Linked Immunoserbent Assay,ELISA)detected the level of insulin after being stimulated by glucose.The expression of Insulin and Nkx6.1 protein was detected by IF;(3)Genes related with differentiation were screened by mRNA sequencing after overexpressing miR-328.GO and KEGG were performed to analyzed gene function and enrichment pathway separately.qRT-PCR verified down-regulation of gene expression;(4)TGF β2 siRNA and adenovirus(adv)were constructed respectively.The binding site of miR-328 and TGF-β2 was predicted,and the dual luciferase reporter system was carried out to verify whether miR-328 binds to the 3’-UTR region of TGF-β2.Western blot(WB)detected of antagonising and rescuing levels of TGF-β2 protein to clear whether TGF-β2 is a downstream target gene of miR-328;(5)After transfecting MPC with Tgf-β2 siRNA,WB detected the expression levels of TGF-β2 protein both experimental group and control group.The expression of key gene mRNA was detected by performing in the early stage of islet development.The expression of Pdx1 protein was detected by IF.(6)The decellularized scaffold of rat pancreas was constucted by continuous perfusion by splenic artery.The morphology changes of pancreatic decellularized scaffold were observed by microscope.Ultrastructure,DNA content and GAG content were detected by SEM and qrt-pcr in separate,Collagen Ⅰ,Fibronectin,Laminin and Collagen Ⅱ protein was detected by IF.(7)Type 1 diabetes model mice was constructed by injecting streptozocin(STZ).Mouse pancreatic decellularization scaffold was transplanted into STZ-mice after inoculating miR-328 overexpression and control group IPCs respectively.The expression of Insulin,Somatostatin and Glucagon protein in the transplanted cells of the experimental group and the control group were detected by IF.Fasting tail vein blood glucose was also tested both experimental group and NC group.RESULTS:(1)Cells in different stages had different cell morphology by microscope.FCM confirmed that about 48.2%of cells expressed Insulin at the end of differentiation.qRT-PCR confirmed that IPC and ESC had significant differences in islet related functional gene expression.IF confirmed that IPC expressed both Insulin and C-peptide.(2)qRT-PCR showed that MPC was transfected with miR-328 agomir successfully.After 21 days of differentiation,the islet-related functional genes and key transcription factors of miR-328 overexpression group were down-regulated compared with the control group.FCM showed that ratio of Insulin+ cells decreased from 43.8%in the control group to 18.3%in the overexpressed group.ELISA showed that the amount of insulin released from the overexpressed group was significantly lower than that in the control group,and there was no difference in the response to different concentrations of glucose.IF showed the overexpression group the ratio of Insulin+/Nkx6.1+ cells was significantly lower than that of the control group;(3)mRNA sequencing showed that 18 genes down-regulated after overexpression of miR-328 and 65 genes were up-regulated.GO analysis showed that the differently expressed genes mainly involved in the cell process.metabolic process,single-organism process and developmental process,etc.,KEGG analysis shows that differently expressed genes involved in MAPK,TGF-β and other stem cell differentiation-related pathways.qRT-PCR confirmed that stem cell differentiation-related genes such as TGF-β2,UNCX,Cphx2 and other expressions were down-regulated and consistent with sequencing results;(4)Bioinformatic analysis predicted and dual luciferase reporter system confirmed miR-328 and TGF-β2 3’-UTR have a binding site,WB confirmed that overexpression of TGF-β2 can revert to the inhibition of miR-328,knockdown miR-328 can antagonize the interference of siRNA on TGF-β2 expression;(5)WB shows interference with MPC After TGF-β2 expression,the Pdx1 protein level of pancreatic development was down-regulated.qRT-PCR showed that the expression of TGF-β2 interfered with the down-regulation of key transcription factors in early islet differentiation.IF showed that the proportion of Pdx1+cells in TGF-β2 interference group was significantly higher than that in the control group.(6)Morphology detection showed that the rat pancreas was transparent after decellularization,and the scanning electron microscopy showed that the stent retained a relatively complete ultra-microscopic network after the cell was removed.The quantitative detection of DNA showed that the cell removal rate met the standard,GAG The content showed that the collagen component was more intact,and the IF results showed that most of the Collagen I,Fibronectin,Laminin and Collagen Ⅱ proteins remained in the acellular scaffold;(7)Fasting tail vein blood glucose showed STZ-type 1 diabetes in mice was constructed successfully,and the fasting blood glucose was stable more than 25mmol/L.The blood glucose of the diabetic mice in the transplanted control group returned to normal level and maintained for 4 days.The miR-328 overexpressing cell transplantation group could not control the blood glucose to normal level.The transplanted tissue section IF showed that a small amount of Insulin,Somatostatin,Glucagon protein was expressed in the miR-328 overexpression group,and the cell arrangement was disordered.Conclusion:(1)During three-step differentiation,mouse ESC can differentiate into IPC in vitro stably;(2)miR-328 plays a negative regulatory role in the differentiation of IPC in vitro and has significant inhibitory effects;(3)miR-328 affects the formation of early Psx1+ progenitor cells by targeting TGF-β2 expression,thereby reducing the differentiation efficiency of terminal Insulin+ cells;(4)Overexpression of miR-328 in IPC cells cannot reverse hyperglycemia in STZ diabetic mice,led to low expression of Insulin,Somatostatin,Glucagon protein and cannot form a tight islet-like structure. |
