Silencing of Wnt5a prevents interleukin‑1β‑induced collagen type II degradation in rat chondrocytes

Shi,Shiping; Man,Zhentao; Li,Wei; Sun,Shui; Zhang,Wei

doi:10.3892/etm.2016.3788

Silencing of Wnt5a prevents interleukin‑1β‑induced collagen type II degradation in rat chondrocytes

Authors:
- Shiping Shi
- Zhentao Man
- Wei Li
- Shui Sun
- Wei Zhang
View Affiliations

Affiliations: Department of Joint Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, Shandong 250021, P.R. China
Published online on: October 11, 2016 https://doi.org/10.3892/etm.2016.3788
Pages: 3161-3166
Copyright: © Shi et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

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.

View Figures

View References

1	Ni GX, Li Z and Zhou YZ: The role of small leucine-rich proteoglycans in osteoarthritis pathogenesis. Osteoarthr Cartil. 22:896–903. 2014. View Article : Google Scholar : PubMed/NCBI
2	Hiligsmann M, Cooper C, Arden N, Boers M, Branco JC, Brandi M Luisa, Bruyère O, Guillemin F, Hochberg MC, Hunter DJ, et al: Health economics in the field of osteoarthritis: An expert's consensus paper from the European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis (ESCEO). Semin Arthritis Rheum. 43:303–313. 2013. View Article : Google Scholar : PubMed/NCBI
3	Cutolo M, Berenbaum F, Hochberg M, Punzi L and Reginster JY: Commentary on recent therapeutic guidelines for osteoarthritis. Semin Arthritis Rheum. 44:611–617. 2014. View Article : Google Scholar : PubMed/NCBI
4	Glyn-Jones S, Palmer AJ, Agricola R, Prince AJ, Vincent TL, Weinans H and Carr AJ: Osteoarthritis. Lancet. 386:376–387. 2015. View Article : Google Scholar : PubMed/NCBI
5	Evans CH and Huard J: Gene therapy approaches to regenerating the musculoskeletal system. Nat Rev Rheumatol. 11:234–242. 2015. View Article : Google Scholar : PubMed/NCBI
6	Bandara G, Robbins PD, Georgescu HI, Mueller GM, Glorioso JC and Evans CH: Gene transfer to synoviocytes: Prospects for gene treatment of arthritis. DNA Cell Biol. 11:227–231. 1992. View Article : Google Scholar : PubMed/NCBI
7	Evans CH, Ghivizzani SC and Robbins PD: Getting arthritis gene therapy into the clinic. Nat Rev Rheumatol. 7:244–249. 2011. View Article : Google Scholar : PubMed/NCBI
8	Ivkovic A, Pascher A, Hudetz D, Maticic D, Jelic M, Dickinson S, Loparic M, Haspl M, Windhager R and Pecina M: Articular cartilage repair by genetically modified bone marrow aspirate in sheep. Gene Ther. 17:779–789. 2010. View Article : Google Scholar : PubMed/NCBI
9	Haupt JL, Frisbie DD, McIlwraith CW, Robbins PD, Ghvizzani S, Evans CH and Nixon AJ: Dual transduction of insulin-like growth factor-I and interleukin-1 receptor antagonist protein controls cartilage degradation in an osteoarthritic culture model. J Orth Res. 23:118–126. 2005. View Article : Google Scholar
10	Shi Q, Zhang XL, Dai KR, Benderdour M and Fernandes JC: siRNA therapy for cancer and non-lethal diseases such as arthritis and osteoporosis. Expert Opin Biol Ther. 11:5–16. 2011. View Article : Google Scholar : PubMed/NCBI
11	Gao Y, Liu S, Huang J, Guo W, Chen J, Zhang L, Zhao B, Peng J, Wang A, Wang Y, et al: The ECM-cell interaction of cartilage extracellular matrix on chondrocytes. BioMed Res Int. 2014:6484592014. View Article : Google Scholar : PubMed/NCBI
12	Tchetina EV, Squires G and Poole AR: Increased type II collagen degradation and very early focal cartilage degeneration is associated with upregulation of chondrocyte differentiation related genes in early human articular cartilage lesions. J Rheumatol. 32:876–886. 2005.PubMed/NCBI
13	Li Z, Meng D, Li G, Xu J, Tian K and Li Y: Celecoxib combined with diacerein effectively alleviates osteoarthritis in rats via regulating JNK and p38MAPK signaling pathways. Inflammation. 38:1563–1572. 2015. View Article : Google Scholar : PubMed/NCBI
14	Mabey T and Honsawek S: Cytokines as biochemical markers for knee osteoarthritis. J Orthop. 6:95–105. 2015.
15	Ge X, Ma X, Meng J, Zhang C, Ma K and Zhou C: Role of Wnt-5A in interleukin-1beta-induced matrix metalloproteinase expression in rabbit temporomandibular joint condylar chondrocytes. Arthritis Rheum. 60:2714–2722. 2009. View Article : Google Scholar : PubMed/NCBI
16	Ryu JH and Chun JS: Opposing roles of WNT-5A and WNT-11 in interleukin-1beta regulation of type II collagen expression in articular chondrocytes. J Biol Chem. 281:22039–22047. 2006. View Article : Google Scholar : PubMed/NCBI
17	Yu DG, Ding HF, Mao YQ, Liu M, Yu B, Zhao X, Wang XQ, Li Y, Liu GW, Nie SB, et al: Strontium ranelate reduces cartilage degeneration and subchondral bone remodeling in rat osteoarthritis model. Acta Pharmacol Sin. 34:393–402. 2013. View Article : Google Scholar : PubMed/NCBI
18	Wang PY and Tsai WB: Modulation of the proliferation and matrix synthesis of chondrocytes by dynamic compression on genipin-crosslinked chitosan/collagen scaffolds. J Biomater Sci Polym Ed. 24:507–519. 2013. View Article : Google Scholar : PubMed/NCBI
19	Yu L, Weng Y, Shui X, Fang W, Zhang E and Pan J: Multipotent Adult Progenitor Cells from Bone Marrow Differentiate into Chondrocyte-Like Cells. J Arthroplasty. 30:1273–1276. 2015. View Article : Google Scholar : PubMed/NCBI
20	Tekari A, Luginbuehl R, Hofstetter W and Egli RJ: Chondrocytes expressing intracellular collagen type II enter the cell cycle and co-express collagen type I in monolayer culture. J Orthop Res. 32:1503–1511. 2014. View Article : Google Scholar : PubMed/NCBI
21	Chen WP and Wu LD: Chlorogenic acid suppresses interleukin-1β-induced inflammatory mediators in human chondrocytes. Int J Clin Exp Pathol. 7:8797–8801. 2014.PubMed/NCBI
22	Tao R, Wang S, Xia X, Wang Y, Cao Y, Huang Y, Xu X, Liu Z, Liu P, Tang X, et al: Pyrroloquinoline Quinone Slows Down the Progression of Osteoarthritis by Inhibiting Nitric Oxide Production and Metalloproteinase Synthesis. Inflammation. 38:1546–1555. 2015. View Article : Google Scholar : PubMed/NCBI
23	Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI
24	Xu HG, Zhang XH, Wang H, Liu P, Wang LT, Zuo CJ, Tong WX and Zhang XL: Intermittent Cyclic Mechanical Tension-Induced Calcification and downregulation of ankh gene expression of end plate chondrocytes. Spine. 37:1192–1197. 2012. View Article : Google Scholar : PubMed/NCBI
25	Lu J, Sun Y, Ge Q, Teng H and Jiang Q: Histone deacetylase 4 alters cartilage homeostasis in human osteoarthritis. BMC Musculoskelet Disord. 15:4382014. View Article : Google Scholar : PubMed/NCBI
26	Wu PC, Tsai CL, Gordon GM, Jeong S, Itakura T, Patel N, Shi S and Fini ME: Chondrogenesis in scleral stem/progenitor cells and its association with form-deprived myopia in mice. Mol Vision. 21:138–147. 2015.
27	Chen LX, Lin L, Wang HJ, Wei XL, Fu X, Zhang JY and Yu CL: Suppression of early experimental osteoarthritis by in vivo delivery of the adenoviral vector-mediated NF-kappaBp65-specific siRNA. Osteoarthritis Cartilage. 16:174–184. 2008. View Article : Google Scholar : PubMed/NCBI
28	Hammond SM, Bernstein E, Beach D and Hannon GJ: An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature. 404:293–296. 2000. View Article : Google Scholar : PubMed/NCBI