MiR-125a-5p Facilitates The Induction And Differentiation Of Dopaminergic Precursor Cells From HiPSCs Via Targeting APC And The Activating Of Classical WNT Signaling Pathway

Part ?: Induction of induced pluripotent stem cells into dopaminergic precursor neurons and screening of Micro RNA in vitroOBJECTIVE: To screen out micro RNAs with differential expression in the process of differentiation of hi PSCs into dopaminergic neural progenitor cells in vitro,and to screen miR-125a-5p by comparison analysis with classical WNT signaling pathway.Methods: Grouping: 1)Experimental group: LSB / SHH / FGF8 b /CHIR99021 + N2 / B27 induction group;2)Control group: simple N2 / B27 induction group.In vitro,hi PSCs induce differentiation towards dopaminergic neural precursor cells.Immunocytochemistry,real-time quantitative PCR(q RT-PCR)method was used to detect cells at different stages during the differentiation of hi PSCs into DA neuronal precursor cells: neural precursor cell markers Nestin,floor plate cell marker Foxa2,DA neuron precursor cell marker En1,neuronal marker Tuj1 and DA neuron marker TH.Extract the total RNA of three stages of hi PSCs,neural precursor cells,and DA neuron precursor cells in the experimental group for high-throughput sequencing of micro RNAs,screen out differentially expressed micro RNAs,and screen them for correlation with classical WNT signaling pathways to select appropriate micro RNA for research.Results: During the induction and differentiation process,the cells in the neural precursor cells(NPCs)stage(day 7 of induction and differentiation)were basically NESTIN + cells,and the expression of floor plate cell marker FOXA2 in the experimental group was significantly increased and had statistical significance(P<0.001).The expression of dopaminergic precursor cell markers EN1,LMX1 A and MSX1 on the 25 th day of induction differentiation was also significantly increased(P <0.01),the expression of TH in the early neuronal phase was significantly increased in the experimental group compared with the control(P <0.01).Micro RNAs high-throughput sequencing results showed that differentially expressed micro RNAs existed on day 0,day 7,and day 25 of induced differentiation.Correlation analysis of the differentially increased micro RNAs and the classical WNT signaling pathway was screened to miR-125a-5p can directly interact with the classical WNT signaling pathway by targeting APC.Conclusion: Hi PSCs can induce differentiation into dopaminergic neural precursor cells in vitro,and there are differential expression of micro RNAs in cells at different stages.Compared with the classical WNT signaling pathway,it was found that miR-125a-5p showed increasing expression on hi PSCs,hi PSCs-derived NPCs,and hi PSCs-derived DA neurons.Part ?: MiR-125a-5p promotes the differentiation of hi PSCs into DA progenitor neurons by regulating the classic WNT signaling pathwayObjective: By overexpressing miR-125a-5p,down-regulating the expression of its target gene APC,and further regulating the activity of the classical WNT signaling pathway,to achieve high efficient induction of differentiation of human induced pluripotent stem cells(hi PSCs)into dopaminergic(DA)neural precursor cells.Methods: Double luciferase reporter gene method to verify the target gene APC of miR-125a-5p;miR-125a-5p precursor miR-125 a lentiviral vector's construction and packaging,miR-125a-5p overexpression hi PSCs cell line construction,screening and establishment,and its differentiation into DA neuron precursor cells and DA neurons;immunocytochemistry,real-time quantitative PCR(q RT-PCR)and Western blot(WB)methods to detect miR-125a-5p overexpression of hi PSCs and control hi PSCs into DA neuron precursor cells and DA neuron neurons induced differentiation markers: neural precursor cell marker Nestin,floor plate cell marker Foxa2,DA neuronal precursor cell markers En1,DA neuron precursor cell sorting marker Corin,neuronal marker Tuj1 and DA neuron marker TH.Results: The dual luciferase reporter gene results showed that miR-125a-5p targeted regulation of the key factor APC of the classical WNT signaling pathway(P<0.01).Hi PSCs stably overexpressing miR-125a-5p can further induce differentiation into DA neuron precursor cells and DA neurons,and their induction differentiation efficiency is significantly higher than that of control hi PSCs induced differentiation group.The expression of floor plate cell marker FOXA2 was significantly increased on day 7(P <0.05),the expression of dopaminergic precursor cell markers EN1,CORIN was significantly increased on the 14 th day(P <0.01),the expression of TH,DAT and Nurr1 in neuron stage on the 25 th day of induction differentiation was significantly increased.The difference in the expression of TH and DAT was P <0.01,and the difference in the expression of Nurr1 was P <0.05,suggesting that miR-125a-5p hi PSCs have better potential to induce differentiation into DA neuronal precursor cells.Conclusion: MiR-125a-5p can down-regulate the expression of APC to activate the activity of classical WNT signaling pathway,and promote the differentiation of hi PSCs into DA neuron precursor cells and DA neurons.