Mechanism Of MiR-106a-STAT3/IL-6R Pathway Regulating Limbal Stem Cell Activation

PURPOSECorneal epithelium is a nonkeratinized stratied squamous epithelium.The rapid turnover of corneal epithelium is maintained by the proper activation of STAT3 pathway in the limbal stem cells(LSCs)in the basal layer of limbal epithelium.The exactly machinism of STAT3 pathway regulates the corneal epithelial stem/progenitor cell activation are not fully understood.MicroRNAs(miRNAs)have emerged as key regulatory molecules in multiple normal and cancer stem cells.Previous and our studies have confirmed the expression of miR-106 a were increased in STAT3 inhibitor induced corneal epithelium,while the ectopic expression of miR-106 a restrained the corneal epithelial stem/progenitor cell activation and wound healing.Based on the above results,the effects of miR-106 a on corneal limbal stem cell proliferation and differentiation in vitro and in vivo,and corneal epithelial healing in normal and diabetic mice is to be further investigated in this study.The crosstalk of miR-106 a and STAT3/IL-6R pathway would provide a theoretical basis for precisely regulation of stem/progenitor cell activation during corneal epithelial wound healing.METHODSIn this study,model C57BL/6 mice and streptozotocin(STZ)induced type 1diabetic mice were used for vivo experiments,and mouse corneal epithelial stem/progenitor cell line(TKE2 cells)were cultured for vitro tests.To assess the effect of miR-106 in corneal epithelial wound healing,mouse corneal epithelium was scraped with or without subconjunctival injection of miR-106 a agomir or antagomir.Fluoresceinsodium staining and Image J software were used to calculated the residual epithelial defect of mouse corneal epithelium wound healing at different time points.TKE2 cells were culcured to determine the influence of miR-106 a on the colony-forming efficiency of corneal epithelial stem/progenitor cells.Immunofluorescence staining and Western blot were tested to reveal ?NP63 and Ki-67 levels in miR-106 a agomir or antagomir treated cells.TargetScan bioinformatic analysis was used to explore possible miR-106 a target genes,then RT-PCR andWestern blot were used to test the expression level of STAT3 and IL-6R.To further explore if the miR-106 a restrains stem/progenitor cell activation through IL-6R/STAT3 pathway,we first used IL-6R or STAT3 shRNA to transfect TKE2 cells,then miR-106 a antagomir was applied to treat the cells for 7 to 10 days to calculate the colony-forming efficiency.Mouse corneal epithelium was collected to analyze the mRNA levels of miR-106 a in wound healing period by using quantitative PCR.The effects of miR-106 a on the activation of wound healing was explored in the diabetic mouse model.RESULTSThe corneal epithelial wound healing was significantly impaired in the miR-106agomir-injected normal mice from 24 h to 72 h after corneal epithelial scrape.In vitro experiments,miR-106 a agomir significantly impaired the colony-forming efficiency of TKE2 cells when compared with vehicle or NTC control.Moreover,immunofluorescence staining and Western blot revealed both ?NP63 and Ki-67 levels were decreased in miR-106 a agomir-treated cells.TargetScan bioinformatic analysis showed that the 3'-UTR of IL-6R and STAT3 contain more than one conserved putative target sites for human and mice miR-106 a sites.To further explore if both IL-6R and STAT3 are the direct targets of miR-106 a,we firstly analyzed their expression changes in mouse TKE2 cells after the treatment with miR-106 agomir or antagomir.Quantitative RT-PCR and Western blot results showed that the m RNA and protein expressions of STAT3 and IL-6R were significantly decreased with miR-106 agomir treatment and increased with antagomir treatment.More interestingly,the phosphorylation levels of STAT3 in TKE2 cells were decreased with mi R-106 agomir treatment and increased with antagomir treatment,which was consistent with the changes of IL-6R mRNA and protein expression.In IL-6R or STAT3 shRNA transfected TKE2 cells,miR-106 a antagomir could effectively increase cells colony-forming efficiency,which proved the miR-106 a could regulate stem/progenitor cell activation through IL-6R/STAT3 pathway.MiR-106 a antagomir promoted corneal epithelial wound healing in normal mice.In normal mice,miR-106 a expression was decreased in the early phase,while increased in the late phase ofcorneal epithelial wound healing.Exogenous application of miR-106 antagomir partially recovered the corneal epithelial wound healing rate in diabetic mice.CONCLUSIONSThis study revealed that miR-106 a restrained corneal epithelial stem/progenitor cell activation through the fine-tuning of IL-6R-STAT3 signaling pathway,and the inhibition of miR-106 a promoted corneal epithelial wound healing in both normal and diabetic mice.