Abstract
Purpose:
To test the hypothesis that miR-205 plays a key role in stimulation of growth factors release, TGFA and EGF, in wounded corneal epithelial cell by targeting 3-UTR of KCNJ10, thereby inhibiting the K channel activity which leads to Ca2+ influx and activating EGFR/ERK phosphorylation.
Methods:
The expression level of miR-205 and KCNJ10 were examined by stem-loop qRT-PCR and western blot in cultured HCE cells. The wound-healing assay was used to examine the recovery rate of HCE cells. TGFA and EGF in the media, an index as growth factor secreted by HCE, were measured by ELISA. We used dual luciferase activity as a report gene to assess the effect of miR-205 on KCNJ10 expression. To study the role of miR-205 and KCNJ10 in regulation of HCE function, the HCE cells were transfected with miR-205 mimic or KCNJ10 siRNA. The patch-clamp technique was used to measure the K currents in HCE cells.
Results:
Mechanic scratching HCE cells stimulated the expression of miR-205 by 50% within 1 hour and decreased the expression of KCNJ10 within 24 hours. Application of miR-205 antagomer significantly delayed the healing process of HCE, an effect was partially revered by inhibiting K channels with BaCl2. Moreover, inhibition of kcnj10 channels with shRNA also partially restored the delayed healing by miR-205 antagomer. The possibility that increase in miR-205 in wounded HCE cells facilitates the cell regeneration by inhibiting inward rectifier K channels was also confirmed by the finding that miR-205 decreased luciferase report gene activity in cells transfected with 3’UTR of KCNJ10. Moreover, Western blot and patch-clamp experiments demonstrated that suppression of the endogenous mir-205 expression enhanced the expression in KCNJ10 but not KCNJ16 in plasma membrane and inward K currents in HCE cells by 150%. The possibility that inhibition of K channels by miR-205 may stimulate growth factor release through a depolarization-induced Ca2+ signaling is also suggested by the finding that inhibition of KCNJ10 by siRNA increased TGFA/EGF release into media through upregulation of EGFR/ERK pathway.
Conclusions:
We conclude that miR-205 is one of the important factors stimulating healing of wounded HCE cell by inhibiting KCNJ10 inwardly-rectifying K channels thereby enhancing TGFA/EGF release and facilitating the repair process.
Keywords: 482 cornea: epithelium •
654 proliferation •
765 wound healing