Transdifferentiation effects and related mechanisms of nerve growth factor and internal limiting membrane on Müller cells


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Abstract

In this study, we investigated the pro-proliferative, pro-transdifferentiation effects and mechanisms of nerve growth factor (NGF), internal limiting membrane (ILM), and NGF+ILM on Müller cells. The Müller cells cultured with NGF, ILM or both were mediated with tyrosine kinase A (TrkA) (a high affinity receptor for NGF) inhibitor, phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) (an intracellular signaling pathway important in regulating the cell cycle) inhibitor, or LIN28 (a RNA-binding protein and a posttranscriptional regulator of genes involved in developmental timing and self-renewal in embryonic stem cells) siRNA. Immunofluorescence (IF), western blotting (WB) and quantitative real-time polymerase chain reaction (qRT-PCR) were performed to detect the expression of related genes. As a result, NGF, ILM and NGF+ILM promoted cell proliferation, increased the ratio of 5-bromo-2-deoxyuridine (BrdU)-positive cells, and were correlated with TrkA and PI3K/Akt signaling. NGF alone promoted cell dedifferentiation and redifferentiation mainly towards neurons rather than towards glial cells, related to TrkA and PI3K/Akt signals. The expression of p-Akt and cyclinD1 was increased by the intervention of NGF, ILM or NGF+ILM via TrkA and PI3K/Akt signals. NGF alone promoted the expression of paired box 6 (PAX6) (a transcription factor present during embryonic development), sex-determining region Y-box 2 (SOX2) (a transcription factor essential for the self-renewal, or pluripotency, of undifferentiated embryonic stem cells) and LIN28, and inhibited the expression of lethal-7 (Let-7b), Let-7d, Let-7i and miR-98 (microRNAs, key developmental regulators). The expression of Achaete-scute homolog 1 (Ascl1, also called MASH1) (a neurodevelopmental gene) and endogenous NGF (closely related to neurogenesis) was also promoted by exogenous NGF and related to TrkA and PI3K/Akt signaling. The down-regulation of LIN28 significantly antagonized the effects of NGF on the transdifferentiation of Müller cells. Over all, our results showed that NGF promoted the proliferation and transdifferentiation of Müller cells towards photoreceptor neurons--not towards glial cells, which was related to the LIN28/Let-7 pathway through TrkA and PI3K/Akt signals. Additionally, ILM promoted Müller cells to enter the cell cycle and enhanced cell proliferation, since NGF+ILM promoted the proliferation of Müller cell more significantly than NGF alone.HighlightsNGF promoted the proliferation of Müller cells and ILM acted additively.NGF promoted the transdifferentiation of Müller cells towards photoreceptor neurons.The pathway was related to the LIN28/Let-7 pathway through TrkA and PI3K/Akt signals.

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