Expression Of GGCX In Cartilage Of Primary Knee OA And Its Biological Effects

Objective:To explore the activity and expression of GGCX in cartilage of patients with osteoarthritis, evaluate the correlation between GGCX and osteoarthritis and the biological impact on normal chondrocyte after GGCX RNA interference.Methods:The cartilage samples were obtained from20patients with normal knee and20patients with osteoarthritis. According to ManKin score standard, the osteoarthritic cartilage samples were classified into three groups. Immunohistochemical method was used to explore expression of GGCX in cartilage. The activity of GGCX mRNA was detected in both normal and degenerative cartilage by real-time PCR. The level of GGCX protein was detected in both normal and degenerative cartilage by western bolt. GGCX levels were determined in SF using magnetic particles-based chemiluminescence enzyme immunoassay(MPs-CLEIA). The data were statistically analysed using SPSS19.0.Chondrocytes were isolated from normal cartilage and cultivated in vitro. The cells were identified by dyeing with Toluidine blue and type Ⅱ collagen. According to human GGCX mRNA sequence, three pairs of RNAi were designed and synthesized. These RNAis were cotransfected into chondrocytes using LipofectamineTM2000. The expression of GGCX was detected by Real-time PCR and Western blot to find the RNAi which can disturb the expression of GGCX most significantly.The siRNA with the highest interference efficiency was choosed in the following experiments. The effect of the GGCX that were on the cell proliferation were evaluated by using MTT. The apoptosis rates were assessed in chondrocytes using Annexin V-FITC&PI and then compared with each other. The expressions of MMP13, collagen X and ALP were evaluated by RT-PCR and Western Blot technique. All data were analyzed using SPSS19.0.Results:GGCX was located in the cytoplasm of chondrocytes of normal cartilage and degenerative cartilage. Immunohistochemistry results showed that GGCX activity in degenerative cartilage was significantly lower than the normal group(P<0.05). The expression of GGCX in degenerative group was weaker than normal cartilage(P<0.05). The activity and expression of GGCX decreased negatively with degenerative grade. And a statistically significant difference was found in Mild degeneration group, Moderate degeneration group and Severe degeneration group when compared with each other(P<0.05). GGCX level in SF of OA was lower significantly than normal knee(P<0.05).In order to investigate the biological effect of GGCX in chondrocyte, RNAi technique was used. RNAi sequence was designed, synthesized and transfected successfully. The expression of GGCX can disturbed by all RNAis. Results showed that the siRNA2291can disturb the expression of GGCX most significantly compared with the other two RNAis. The activity and expression of MMP13, collagen X and ALP were increased more significantly in siRNA2291group than normal cultivation group and meaningless control group(P<0.05). The apoptosis rate was significantly higher in siRNA2291group when compared with the other two groups (P<0.05). The cell proliferation rate was increased apparently in siRNA2291group in48h and72h time spot, but no difference in Oh,12h and24h time spot.Conclusions:1. Activity and expression of GGCX in osteoarthritis cartilage was significantly lower than normal cartilage, And positively correlated with degenerative severity of cartilage.2. GGCX level in SF of OA was lower significantly than normal knee.3. Interference of GGCX can increase the activity and expression of MMP13, collagen X and ALP significantly.4. Interference of GGCX can increase the apoptosis rate.5. The cell proliferation rate was increased apparently in interference group in48h and72h time spot, but no difference in Oh,12h and24h time spot.