[Effect on the proliferation of bovine corneal endothelial cells by small hairpin RNA interference targeting p27Kip1].

ABSTRACT

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.