title: Silencing of Wnt5a prevents interleukin-1β-induced collagen type II degradation in rat chondrocytes.
abstract: Osteoarthritis (OA) is a joint disease, and few treatments to date have been able to delay OA progression. The degradation of collagen type II (COL2) in the cartilage matrix is an important initiating factor for OA progression; the upregulation of Wnt5a protein activates COL2 degradation. In the present study, small interfering RNA of Wnt-5a was delivered by a lentiviral vector (LV-Wnt5a-RNAi) to silence Wnt-5a mRNA and prevent COL2 degradation. To determine the function of LV-Wnt5a-RNAi, the OA chondrocyte model (OA-like chondrocytes) were constructed using interleukin (IL)-1β. Detected using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Wnt-5a mRNA in the OA-like chondrocytes were upregulated in a time-dependent manner, indicating that OA-like chondrocytes were successfully constructed. The bioactivity of OA-like chondrocytes was determined using Live-Dead staining, and the result illustrated that the OA-like chondrocytes stimulated with IL-1β for 6 h remained viable, and these were used in Wnt5a silencing. The OA-like chondrocytes were divided into three groups: Group I, cultivated with common medium; group II, cultivated with common medium supplemented with empty lentiviral vector; group III, cultivated with common medium supplemented with LV-Wnt5a-RNAi. The efficiency of LV-Wnt5a-RNAi transfection was determined using fluorescence microscopy, the result of which indicated that LV-Wnt5a-RNAi could efficiently be transfected into the OA-like chondrocytes. The LV-Wnt5a-RNAi efficiency for the Wnt5a mRNA silencing was determined using RT-qPCR. The result illustrated that the mRNA of Wnt5a in group III was significantly lower in group I compared with that in group II (P<0.05), indicating that the LV-Wnt5a-RNAi could successfully silence Wnt5a mRNA. To further verify whether the silencing of Wnt5a mRNA could prevent COL2 degradation, western blotting and immunohistochemical analyses were performed. The results demonstrated that COL2 in group III was significantly higher compared with that in groups I and II (P<0.05), which illustrated that the silencing of Wnt5a mRNA could prevent COL2 degradation. In conclusion, LV-Wnt5a-RNAi was formed successfully and could efficiently silence Wnt5a mRNA expressed by OA-like chondrocytes. In addition, the silencing of Wnt5a mRNA could prevent the degradation of COL2 in OA-like chondrocytes, confirming that LV-Wnt5a-RNAi may be used as a novel tool for OA treatment.
authors.affiliation: Department of Joint Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China.
authors.forename: Shiping
authors.initials: S
authors.lastname: Shi
authors.affiliation: Department of Joint Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China.
authors.forename: Zhentao
authors.initials: Z
authors.lastname: Man
authors.affiliation: Department of Joint Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China.
authors.forename: Wei
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Joint Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China.
authors.forename: Shui
authors.initials: S
authors.lastname: Sun
authors.affiliation: Department of Joint Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China.
authors.forename: Wei
authors.initials: W
authors.lastname: Zhang
keywords: Wnt5a signaling pathway; collagen type II; gene silencing; osteoarthritis; small interfering RNA