Expression Of MiR-145 In Cartilage Of Primary Knee OA And Its Biological Effects

Objective To explore the activity and expression of Mi R-145 in cartilage of patients with osteoarthritis, evaluate the correlation between mi R-145 and osteoarthritis and the biological impact on normal chondrocyte after mi R-145 interference.Methods The cartilage samples were obtained from 10 patients with normal knee and 20 patients with osteoarthritis. According to Man Kin score standard, the osteoarthritic cartilage samples were classified into three groups. The activity of mi R-145 was detected in both normal and degenerative cartilage by real-time PCR. The level of mi R-145 protein was detected in both normal and degenerative cartilage by western bolt. The expressions of MMP13, collagen X, RANK and ALP were evaluated by RT-PCR and Western Blot technique. The data were statistically analysed using SPSS 19.0.Chondrocytes were isolated from normal cartilage and cultivated in vitro. Dyeing with Toluidine blue and type II collagen identified the cells. According to human mi R-145 sequence, these RNAis were cotransfected into chondrocytes using Lipofectamine? 2000. The expression of mi R-145 was detected using Real-time PCR and Western blot. Real-time PCR and Western blot detected the expressions of relative protein.Results Mi R-145 was located in the cytoplasm of chondrocytes of normal cartilage and degenerative cartilage. The expression of mi R-145 in degenerative group was weaker than normal cartilage(P<0.05). The activity and expression of mi R-145 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.In order to investigate the biological effect of mi R-145 in chondrocyte, PCR and western blot technique was used. The activity and expression of MMP13, collagen X and RANK can be disturbed by mi R-145.OA-related genes were 732 and mi R-145-related genes were 192, while associated with cartilage and mi R-145 genes were 13 using microarray analyses. Plasmid transfected chondrocytes of key genes in mi R-145 overexpression TNFRSF11 B proteins were detected, the results show that with the increase of mi R-145 concentration, protein TNFRSF11 B reduced. TNFRSF11 B gene was detected in articular cartilage cells, which were induced by TNF-a. Results showed that TNFRSF11 B were highly expressed in degenenrative cartilage cells. RANK protein changed evidently and collagen X and TNFRSF11 B protein increased significantly when TNFRSF11 B were interferenced.Conclusions 1.Activity and expression of mi R-145 in osteoarthritis cartilage was significantly lower than normal cartilage, and positively correlated with degenerative severity of cartilage. 2. Interference of mi R-145 can increase the activity and expression of MMP13, collagen X and RANK significantly. 3. TNFRSF11 B was regulated by mi R-145. 4. TNFRSF11 B showed high expression in articular cartilage. 5. TNFRSF11 B interference can rescue the inhibition of collagen expression in chondrocyte.