Silencing of P2Y2 receptor delays Ap4A-corneal re-epithelialization process.
OBJECTIVE
There are no selective antagonists for the metabotropic nucleotide P2Y(2) receptor subtype. This implies that it is not possible to demonstrate the importance of such a receptor in the relevant process of corneal wound healing. Therefore, we have cloned and designed a small interference RNA (siRNA) against the rabbit P2Y(2) receptor (P2Y(2)-R) mRNA, which clearly demonstrates the importance of this receptor in the process of wound healing triggered by nucleotides and dinucleotides both in vitro and in vivo.


METHODS
Rabbit P2Y(2)-R cDNA was cloned using a combination of degenerate reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). To test the efficacy of synthesized siRNAs targeting P2Y(2)-R, immunocytochemistry, immunohistochemistry, and quantitative RT-PCR (qRT-PCR) assays were performed. Migration assays were performed both in vitro and in vivo by wounding the epithelium with a pipette tip and n-heptanol, respectively. These wounds were performed 72 h after siRNA transfection either in the presence or the absence of the P2Y(2) agonist, 100 muM Ap(4)A (diadenosine tetraphosphate).


RESULTS
The cloned receptor presents 93% homology compared to the human gene. Two siRNAs were designed and synthesized against a rabbit P2Y(2)-R sequence. After transfection (in vitro assays) or topical instillation (in vivo assays), we demonstrated P2Y(2)-R siRNA efficient transfection/delivery and its efficient gene silencing. Clear reduction of P2Y(2)-R expression was observed at both the mRNA and protein levels in corneas treated with siRNA. In vitro and in vivo migration analysis showed that the silencing process has concomitantly reduced the ability of corneal cells to close the wounds in the presence of the Ap(4)A. In addition, both synthesized siRNAs exert a delay effect on the Ap(4)A-induced migration rate in vitro. These results suggest the absence of non-specific (off-target) effects by our siRNA.


CONCLUSIONS
The application of P2Y(2)-R siRNA has demonstrated the role of this receptor in the accelerating effect of diadenosine tetraphosphate (Ap(4)A) on the corneal wound healing process.
Crooke
Almudena
A
Departamento de Bioquímica, E.U. Optica, Universidad Complutense de Madrid, Madrid, Spain.
Mediero
Aránzazu
A
Guzmán-Aránguez
Ana
A
Pintor
Jesús
J
D000595:Amino Acid Sequence; D000704:Analysis of Variance; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002465:Cell Movement; D003315:Cornea / Q000378:metabolism; D065306:Corneal Injuries; D015226:Dinucleoside Phosphates / Q000378:metabolism*; D019573:Epithelium, Corneal / Q000378:metabolism / Q000502:physiology*; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D016014:Linear Models; D008297:Male; D008969:Molecular Sequence Data; D034622:RNA Interference; D034741:RNA, Small Interfering; D011817:Rabbits; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014945:Wound Healing
D015226:Dinucleoside Phosphates; C546385:P2RY2 protein, human; D034741:RNA, Small Interfering; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; C020733:diadenosine tetraphosphate