Inhibition of miR-205 impairs the wound-healing process in human corneal epithelial cells by targeting KIR4.1 (KCNJ10).
OBJECTIVE
The aim of the study was to test the hypotheses that injury stimulates the expression of miR-205, which in turn inhibits KCNJ10 channels by targeting its 3' UTR, thereby facilitating the wound-healing process in human corneal epithelial cells (HCECs).


METHODS
A stem-loop qRT-PCR was used to examine the miR-205 expression. BrdU cell proliferation assay and wound scratch assay were applied to measure the effect of miR-205 mimic or antagomer in HCECs. The patch-clamp technique, dual luciferase reporter assay, and Western blot analysis were employed to test whether miR-205 regulates KCNJ10, one of the target genes of miR-205. Both of the primary human and mouse corneal epithelial cells (pH/MCECs) were employed to further confirm the observations obtained in HCECs.


RESULTS
The scratch injury in pH/MCECs increased the expression of miR-205 and decreased the expression of KCNJ10 within 24 hours. The notion that miR-205 may target KCNJ10 was supported by dual luciferase reporter assay showing an inhibition effect of miR-205 on 3' UTR of KCNJ10. Application of miR-205 antagomer significantly delayed the regrowth in wounded HCECs. However, inhibition of KCNJ10 partially abolished the effect from miR-205 antagomer and restored the healing process. Moreover, overexpression miR-205 antagomer enhanced the protein expression of KCNJ10 but not KCNJ16. In addition, patch-clamp demonstrated that inhibition of endogenous miR-205 expression increased Ba²⁺-sensitive inwardly rectifying K⁺ channels. In addition, an electrophysiological study of pHCECs showed the presence of KCNJ10-like 20 pS K⁺ channels and scratch injury significantly decreased the Ba²⁺-sensitive inwardly rectifying K⁺ currents.


CONCLUSIONS
miR-205 stimulates wound healing by inhibiting its target gene KCNJ10.
Department of Pharmacology, New York Medical College, Valhalla, New York.
Daohong
D
Lin
Adna
A
Halilovic
Peng
P
Yue
Lars
L
Bellner
Kemeng
K
Wang
Lijun
L
Wang
Chengbiao
C
Zhang
D000818:Animals; D015153:Blotting, Western; D049109:Cell Proliferation; D002478:Cells, Cultured; D015536:Down-Regulation; D004847:Epithelial Cells / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000293:injuries*; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007633:Keratins / Q000378:metabolism; D051379:Mice; D035683:MicroRNAs / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology; D024661:Potassium Channels, Inwardly Rectifying / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014945:Wound Healing / Q000502:physiology*; D000098854:Kcnj10 Channel
D007633:Keratins; D035683:MicroRNAs; D024661:Potassium Channels, Inwardly Rectifying; D000098854:Kcnj10 Channel; C534425:MIR205, human
KCNJ10; miR-205; wound healing