MicroRNAs Expression in Response to rhNGF in Epithelial Corneal Cells: Focus on Neurotrophin Signaling Pathway

Abstract

Purpose. Nerve growth factor efficacy was demonstrated for corneal lesions treatment, and recombinant human NGF (rhNGF) was approved for neurotrophic keratitis therapy. However, NGF-induced molecular responses in cornea are still largely unknown. We analyzed microRNAs expression in human epithelial corneal cells after time-dependent rhNGF treatment. Methods. Nearly 700 microRNAs were analyzed by qRT-PCR. MicroRNAs showing significant expression differences were examined by DIANA-miRpath v.3.0 to identify target genes and pathways. Immunoblots were performed to preliminarily assess the strength of the in silico results. Results. Twenty-one microRNAs (miR-26a-1-3p, miR-30d-3p, miR-27b-5p, miR-146a-5p, miR-362-5p, mir-550a-5p, mir-34a-3p, mir-1227-3p, mir-27a-5p, mir-222-5p, mir-151a-5p, miR-449a, let7c-5p, miR-337-5p, mir-29b-3p, miR-200b-3p, miR-141-3p, miR-671-3p, miR-324-5p, mir-411-3p, and mir-425-3p) were significantly regulated in response to rhNGF. In silico analysis evidenced interesting target genes and pathways, including that of neurotrophin, when analyzed in depth. Almost 80 unique target genes (e.g., PI3K, AKT, MAPK, KRAS, BRAF, RhoA, Cdc42, Rac1, Bax, Bcl2, FasL) were identified as being among those most involved in neurotrophin signaling and in controlling cell proliferation, growth, and apoptosis. AKT and RhoA immunoblots demonstrated congruence with microRNA expression, providing preliminary validation of in silico data. Conclusions. MicroRNA levels in response to rhNGF were for the first time analyzed in corneal cells. Novel insights about microRNAs, target genes, pathways modulation, and possible biological responses were provided. Importantly, given the putative role of microRNAs as biomarkers or therapeutic targets, our results make available data which might be potentially exploitable for clinical applications.

Chitosan/PLGA-based tissue engineered nerve grafts with SKP-SC-EVs enhance sciatic nerve regeneration in dogs through miR-30b-5p-mediated regulation of axon growth

“…The top 60 highly expressed miRNAs in SKP-SC-EVs were identified and key biomolecules involved in nerve regeneration were found. Based on existing literature reports, miR-30b-5p, miR-26a-5p, miR-20b-5p, miR-34a-5p, miR-22-3p, and miR-93-5p are involved in neuroinflammation, neuropathic sensitivity and pain progression [ [31] , [32] , [33] , [34] , [35] ]; miR-23a-3p, miR-103-3p, miR-26a-5p, miR-99a-5p, miR-25-3p, miR-29a-3p, miR-27a-5p, miR-222-3p, miR-34a-5p, miR-17-5p, and miR-22-3p play critical roles in the regulation of neural cell survival, proliferation, migration, apoptosis, sensitivity to radiation and chemotherapy, and the secretion of neurotrophic factors [ [36] , [37] , [38] , [39] , [40] , [41] , [42] ]; miR-26a-5p, miR-29a-3p, miR-204-5p, miR-92a, and miR-17-5p are vital in the processes of neuronal differentiation, neurite development and growth, as well as axonal regeneration and myelination [ [43] , [44] , [45] ]. The above-mentioned miRNAs were selected for further validation based on their critical roles in axonal outgrowth and regeneration.…”