Notch Regulates The Proliferation Of Müller Glial Cells Through The Lin28/let-7 MiRNA Pathway

Background: Retinal degenerative diseases are one of the leading causes of irreversible visual impairment and blindness,for which there is no effective treatment.Because of their potential for self-renewal and multidirectional differentiation,stem cells are uniquely suited for research applications in retinal diseases.In the retina,Müller glial cells(MG)are considered to be endogenous stem cell donors for the treatment of degenerative diseases of the optic nerve.In mammals,MG cells have difficulty proliferating and differentiating,so how best to activate MG cells back into a proliferative state and how to increase MG cell proliferation and re-differentiation are urgent questions.Aims: Damage can stimulate MG cell proliferation,but damage leads to neuronal death,which is contrary to the aim of restoring the regenerative capacity of the mammalian retina.We sought to find a strategy for MG cell proliferation that did not cause further damage to the diseased retina.Methods: A model of N-methyl-D-aspartic acid(NMDA)neurotoxic injury was first established and the RNA expression levels of genes related to the Notch signalling pathway in the retina were examined by q PCR.The proliferation of MG cells was measured using a 5-ethynyl-2’-deoxyuridine(Ed U)cell proliferation assay.Molecular biology including PCR techniques,targeted mutagenesis and homologous recombination were used to construct overexpression of experimentally relevant genes and various reporter plasmids for wild-type and targeted mutations.Adeno-associated virus(AAV)technology is used to deliver the relevant genes in vivo.The reporter gene,Ch IP method,verifies that Notch directly activates Lin28 transcripts and has binding sites.The effect of the Notch intracellular domain(NICD)on Lin28-mediated let-7 miRNA expression and MG proliferation was verified in Lin28 knockout mice.Results: promoter region of Lin28 to activate its transcription,and NICD binds to the promoter region of Lin28 a and Lin28 b to activate its transcription.NICD has multiple binding sites to CSL in the promoter regions of Lin28 a and Lin28 b.let-7miRNA expression was inhibited by Lin28,while co-deletion of Lin28 a and Lin28 b neutralized the effect of NICD on let-7 miRNA expression and MG proliferation.In conclusion,Notch signaling can negatively regulate let-7 miRNAs to promote MG proliferation by activating Lin28.Conclusion: Notch/Lin28/let-7 miRNA formed a signaling axis,and Notch signaling promoted MG proliferation by activating Lin28 transcription and thereby repressing the downstream effector let-7 miRNA of Lin28.This suggests that the Notch/Lin28/let-7 signaling axis has a key role in regulating MG proliferation and neurogenic potential in the adult mammalian retina.