[Effect on the proliferation of bovine corneal endothelial cells by small hairpin RNA interference targeting p27Kip1].
OBJECTIVE
To clarify the proliferation of bovine corneal endothelial cells (bCEC) by interference with the recombinant plasmid of short hairpin RNA (shRNA) against p27Kip1, a kind of cyclin-dependent kinase inhibitor (CKI).


METHODS
It was an experimental study. Three p27Kip1-shRNA template DNA sequences containing small hairpin structure were designed and synthesized as experimental groups. Plasmid expressing irrelevant shRNA with a random combination was used as negative shRNA. The products were inserted into the Pgensil-1 plasmid and the recombinant plasmid of Pgenesil-P1, Pgenesil-P2, Pgenesil-P3 and Pgenesil-HK were constructed. The recombinant plasmids were transfected into bCEC cells with liposome and a blank group. The expression of mRNA and protein of p27Kip1 was detected by RT-PCR and Western blot after stable transfection, and the plasmid with the best inhibitory effect was selected. The growth of the experimental group, Pgenesil-HK group and blank group were assessed by MTT. The influence of shRNA-p27Kip1 on bCEC cell cycle was deteceted by flow cytometry (FCM). All statistical analyses were performed using one-way ANOVA.


RESULTS
Restrictive enzyme digestion and sequence analysis showed that four recombinant plamids were constructed successfully and the aim sequence was obtained. The expression of p27Kip1 mRNA and p27Kip1 protein of Pgenesil-P1 group, Pgenesil-P2 group and Pgenesil-P3 group were all lower than that in the control group, including blank group and negative siRNA group. The inhibitive rate of mRNA reached 32.71%, 67.76% and 80.28% (F = 453.102, P = 0.000 in each group) and the inhibitive rate of protein reached 29.27%, 64.73% and 76.13% (F = 75.385, P = 0.000 in each group) compared with the blank group. As the lowest expression among the three positive shRNA group, Pgenesil-P3 was selected for the next steps. There was no significant difference between blank group and negative Pgenesil-HK of the expression of p27Kip1 protein (P = 0.356) and the express of p27Kip1 mRNA (P = 0.246). Compared with the control group and the blank group, the growth of the bCEC transfected by Pgenesil-P3 was significantly promoted with increased cell percent of S-phrase (F = 334.957, P = 0.000) and decreased cell percent of G1-phrase (F = 134.224, P = 0.000).


CONCLUSIONS
shRNA-p27Kip1 can down-regulate the expression of bCEC effectively and increase the growth of bCEC. shRNA-p27Kip1 RNA interference may be an effective method to promote the proliferation of CEC.
Huang
Yu-kan
YK
Department of Ophthalmology, Union Hospital Affiliated to Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.
Zhang
Ming-chang
MC
Wang
Yong
Y
Fan
Ke-shun
KS
Jiang
Dong-ling
DL
Zhang
Guang-hong
GH
Zhou
Yan-li
YL
D000818:Animals; D002417:Cattle; D049109:Cell Proliferation; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology*; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000235:genetics*; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D010957:Plasmids; D034622:RNA Interference; D034741:RNA, Small Interfering
D034741:RNA, Small Interfering; D050760:Cyclin-Dependent Kinase Inhibitor p27