Experimental Investigation Of MiR-34b-5p Targeting TGFBR2 To Promote The Differentiation Of HiPSCs Into Neurons

Objective Adding TGF-β signaling pathway inhibitors induce human induced pluripotent stem cells(hiPSCs)for 7 days in nerve direction to obtain Pax6 and Nestin positive neural precursor cells(NPCs);The miR-34b-5p differentially expressed between hiPSCs-derived NPCs and undifferentiated hiPSCs;through overexpression of miR-34b-5p,down-regulated TGFBR2 inhibits the activity of TGF-β signaling pathway and promotes the differentiation of hiPSCs into neuronal cells.Methods Hi PSCs were cultured in vitro by monolayer adherent method,and TGF-βand BMP signaling pathway inhibitors were added to induce hiPSCs into neural precursor cells.On the 7th day of differentiation induction,RT-q PCR and immunofluorescence were used to detect the expression of neural precursor cell marker Nestin and dorsal forebrain marker Pax6.High-throughput sequencing of micro RNAs was used to screen out differentially expressed micro RNAs from hiPSCs and hiPSCs-derived NPCs.Correlation analysis with key factors of the TGF-β signaling pathway resulted in miR-34b-5p that directly targeted TGFBR2.Construction of stable hiPSCs with miR-34b-5p overexpression and TGFBR2-RNAi,using untransfected hiPSCs as control cells,and a single layer adherent culture method using N2 / B27 as the basic medium for 7 days of neural precursor cells Induced differentiation,and the induction differentiation of dopaminergic neural precursor cells for 25 days.Using RT-q PCR and immunofluorescence,at two time points(day 0,day 7)induced differentiation detection Expression of pluripotent stem cell marker Oct4 and Nanog;expression of neural precursor cell marker Nestin and ventral floor marker Foxa2;Add SHH,FGF8 a,Chir99021 and other related cytokines that promote the differentiation of DA neuron precursor cells to induce hiPSCs to differentiate into DA neuron precursor cells and early DA neurons.RT-q PCR and immunofluorescence were used to detect the expression of dopaminergic neural precursor cell markers Lmx1 a and En1 on the 14 th day of differentiation;the neuronal markers Tuj1 expression and dopaminergic neuron marker TH expression on the 25 th day of induced differentiation.Results Hi PSCs can be transformed into Nestin and Pax6 positive NPCs on the 7th day of induction.Adding TGF-β pathway inhibitors can significantly increase Nestin and Pax6 positive NPCs(p <0.05);by high-throughput sequencing of samples at two time points,hiPSCs and NPCs,we obtained differentially expressed miR-34b-5p;dual-fluorescein target gene verification experiments confirmed that TGFBR2 is the target gene of miR-34b-5p;i PSCs overexpressing miR-34b-5p and i PSCs of TGFBR2-RNAi relative to the control i PSCs,under the undifferentiated state(D0),the expression of target gene TGFBR2 was significantly down-regulated(p <0.01);the expression of TGF-β downstream gene Lefty1 was significantly down-regulated(p <0.01);the expression of pluripotency gene Oct4 was significantly increased(P<0.05),and Nanog showed no significant difference(p >0.05);TGFBR2-RNAi i PSCs and i PSCs of miR-34b-5p overexpression group on the seventh day(D7)were significantly higher than the control i PSCs in the expression of Nestin(p <0.05);Continue to induce differentiation into DA neuron precursor cells.The results showed that: on the 14 th day of induced differentiation(D14),miR-34b-5p overexpressed i PSCs group had a significant increase in the expression of En1 and Lmx1 a relative to the control i PSCs.(P <0.05),but Tuj1 expression showed no significant difference(p >0.05);on the twenty-fifth day of induced differentiation(D25),the miR-34b-5p overexpression group significantly increased the expression of TH relative to the control i PSCs group(p <0.05).Conclusion Inhibition of TGF-β signaling pathway can promote the differentiation efficiency of hiPSCs into Nestin-positive neural precursor cells(NPCs),miR-34b-5p can inhibit the activity of TGF-β signaling pathway by targeting down-regulation of TGFBR2 expression,and promote i PSCs Induction of NPCs,and miR-34b-5p can also promote the differentiation of hiPSCs-derived NPCs into DA neuron precursor cells and early dopaminergic neurons.