Abstract
To investigate the influence of siRNA-COX-2 gene upon the growth inhibition and apoptosis of cartilage endplate chondrocytes and provide new methods and evidence for siRNA in gene therapy of cartilage endplate chondrocytes. According to the sequence of COX-2 mRNA, COX-2 siRNA was designed, synthesized, cloned into the GFP reporter pcDNA6.2GW/EmGFPmiR vector and transfected into Hep cell line. The integrity of inset fragment was detected by colony PCR (polymerase chain reaction) and sequencing analysis. The cultured cartilage endplate chondrocytes were divided into 4 groups: control group (untreated), negative siRNA group (treatment with 30 nmol/L negative control siRNA), siRNA1 group (treatment with 15 nmol/L COX-2 siRNA) and siRNA2 group (treatment with 30 nmol/L COX-2 siRNA). The biological activity of recombinants was identified with the interference efficiency of COX-2 siRNA recombinant by real-time PCR and Western blot. And the effects of COX-2 inhibitor on the growth of chondrocytes were detected by WST-8 and the mRNA expressions of survivin, bcl-2 and bax genes measured by real-time PCR. The sequences of inset fragment in 4 siRNA expressing recombinants were correct. After COX-2 transfection, the expression of COX-2 mRNA in chondrocytes was 51.3% ± 7.2% in the siRNA1 group and 35.4% ± 3.6% in the siRNA2 group. Western blot showed that the expression of COX-2 protein decreased, especially in siRNA2 group (P < 0.05). And the cell survival rate was 100.0% ± 8.3% in the control group, 84.9% ± 4.2% in the negative control siRNA group, 52.5% ± 6.7% in the siRNA1 group and 48.9% ± 5.4% in the siRNA2 group (P < 0.05). Meanwhile, the expressions of mRNA of survivin and bcl-2 decreased while the expression of bax mRNA increased in degenerative cartilage endplate chondrocytes transfected with COX-2 siRNA (P < 0.05). COX-2-targeting siRNA inhibits the expression of COX-2, suppresses the proliferation of chondrocytes and induces the cell apoptosis. These effects may be attributable to the up-regulation of survivin and bcl-2 and the down-regulation of bax.
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