[RNA interference inhibits expression of cyclooxygenase-2 and matrix metalloproteinase-2 in rabbit corneal stromal cells].
OBJECTIVE
To research the effect of short hairpin RNA (shRNA) targeting cyclooxygenase-2 (COX-2) on the expression of COX-2 and MMP-2 in rabbit corneal stromal cells in vitro.


METHODS
It was a experimental study. Rabbit corneal stromal cells were cultured in vitro. The transfection efficiency of siRNA mediated by HiperFect Transfection Reagent was determined in rabbit corneal stromal cells in order to optimize the transfection condition. shRNAs (shRNA-1,2,3) specific for COX-2 and one negative control nonspecific shRNA were designed, then were transfected by HiperFect Transfection Reagent into both normal rabbit corneal stromal cells and those which were stimulated by IL-1alpha. 6, 12, 24, 48, 72 hours after IL-1alpha added, cells were collected for Real-Time PCR to detect the gene expression of COX-2 and MMP-2, then the results were compared with those of control group. The data of all groups at the same time was analyzed by one-factor analysis of variance, and the comparison of any two groups was carried out by q-test.


RESULTS
The siRNA mediated by HiperFect Transfection Reagent can be transfected efficiently into rabbit corneal stromal cells in vitro, and the maximum efficiency was 70%-80%. The expression of COX-2 and MMP-2 mRNA after IL-1alpha stimulated was much higher than that of blank group. shRNA-2 can significantly inhibit the expression of COX-2 and MMP-2 mRNA in corneal stromal cells stimulated with IL-1alpha. The level of COX-2 and MMP-2 was reduced 83.04% and 73.69% respectively, compared with the expression of single IL-1alpha stimulated group, the difference had statistical significance (q = 24.03, P = 0.00; q = 14.76, P =0.00). The difference of COX-2 mRNA among transfection groups of shRNA-1, shRNA-3, negative control shRNA and single IL-1alpha stimulated group had no statistical significance (F = 0.02, P =0.99). The difference of MMP-2 mRNA among those groups also had no statistical significance (F = 0.02, P = 0.98).


CONCLUSIONS
The RNA interference targeting COX-2 can effectively inhibit the expression of COX-2 and MMP-2 in IL-1alpha stimulated rabbit corneal stromal cells in vitro.
Zhao
Gui-Qiu
GQ
Department of Ophthalmology, the Affiliated Hospital of Qingdao University Medical College, Qingdao 266003, China. zhaoguiqiu@tom.com
Du
Zhao-Dong
ZD
Liang
Tao
T
Liu
Jian-Wei
JW
Zhang
Li-Na
LN
Zhang
Zhen-Hua
ZH
D000818:Animals; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000378:metabolism*; D051546:Cyclooxygenase 2 / Q000378:metabolism*; D053582:Interleukin-1alpha / Q000494:pharmacology; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering; D011817:Rabbits
D053582:Interleukin-1alpha; D034741:RNA, Small Interfering; D051546:Cyclooxygenase 2; D020778:Matrix Metalloproteinase 2