hsa-miR-15a exerts protective effects against osteoarthritis by targeting aggrecanase-2 (ADAMTS5) in human chondrocytes

Lu,Xin; Lin,Jin; Jin,Jin; Qian,Wenwei; Weng,Xisheng

doi:10.3892/ijmm.2015.2446

hsa-miR-15a exerts protective effects against osteoarthritis by targeting aggrecanase-2 (ADAMTS5) in human chondrocytes

Authors:
- Xin Lu
- Jin Lin
- Jin Jin
- Wenwei Qian
- Xisheng Weng
View Affiliations

Affiliations: Department of Orthopaedic Surgery, Peking Union Medical College Hospital, Beijing 100730, P.R. China
Published online on: December 23, 2015 https://doi.org/10.3892/ijmm.2015.2446
Pages: 509-516

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

The aim of the present study was to examine the expression levels and role of hsa-miR-15a in osteoarthritis (OA), as well as the associated mechanisms. The expression levels of hsa-miR-15a and A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 5 (ADAMTS5, also known as aggrecanase-2) were measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) in both OA and normal chondrocytes. hsa-miR‑21 mimics or antisense oligonucleotides (ASO) were co-transfected into the chondrocytes to examine the effects on the putative binding sites compared with the negative control (NC)-mimics or NC-ASO. The relative ADAMTS5 mRNA and protein levels were measured by RT-qPCR and western blot anlaysis, respectively. Moreover, after inhibiting the expression of hsa‑miR‑15a and ADAMTS5 by ASO and small interfering RNA (siRNA), respectively, the amounts of proteoglycan and collagen in the cellular matrix and medium were determined. Additionally, the expression levels of collagen II were measured by western blot analysis. hsa‑miR‑15a expression was downregulated, but ADAMTS5 expression was upregulated in the human OA chondrocytes compared to the normal chondrocytes. Luciferase reporter assay confirmed that the hsa‑miR‑15a binding site was in the ADAMTS5 gene 3'-untranslated region (3'-UTR), and ADAMTS5 was negatively regulated by hsa‑miR‑15a. The downregulation of hsa‑miR‑15a decreased the aggregation of proteoglycan and the collagen content, but increased the release of proteoglycan and collagen; total collagen production was significantly lower, and collagenase activity was markedly higher. The downregulation of ADAMTS5 increased the aggregation of proteoglycan and the collagen content, but decreased the release of proteoglycan and collagen, along with total collagen production. Moreover, collagenase activity was markedly lower. The findings of our study suggest that hsa‑miR‑15a exerts protective effects against OA by targeting ADAMTS5 in human chondrocytes.

View Figures

View References

1	Blalock D, Miller A, Tilley M and Wang J: Joint instability and osteoarthritis. Clin Med Insights Arthritis Musculoskelet Disord. 8:15–23. 2015.PubMed/NCBI
2	Lee AS, Ellman MB, Yan D, Kroin JS, Cole BJ, van Wijnen AJ and Im HJ: A current review of molecular mechanisms regarding osteoarthritis and pain. Gene. 527:440–447. 2013. View Article : Google Scholar : PubMed/NCBI
3	Loeser RF, Goldring SR, Scanzello CR and Goldring MB: Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum. 64:1697–1707. 2012. View Article : Google Scholar : PubMed/NCBI
4	Pereira D, Peleteiro B, Araujo J, Branco J, Santos RA and Ramos E: The effect of osteoarthritis definition on prevalence and incidence estimates: a systematic review. Osteoarthritis and cartilage/OARS. Osteoarthritis Res Soc. 19:1270–1285. 2011. View Article : Google Scholar
5	Zhang Y, Jia J, Yang S, Liu X, Ye S and Tian H: MicroRNA-21 controls the development of osteoarthritis by targeting GDF-5 in chondrocytes. Exp Mol Med. 46:e792014. View Article : Google Scholar : PubMed/NCBI
6	Litwic A, Edwards MH, Dennison EM and Cooper C: Epidemiology and burden of osteoarthritis. Br Med Bull. 105:185–199. 2013. View Article : Google Scholar : PubMed/NCBI
7	Buckwalter JA, Saltzman C and Brown T: The impact of osteoarthritis: implications for research. Clin Orthop Relat Res. 427(Suppl): S6–S15. 2004. View Article : Google Scholar : PubMed/NCBI
8	Wieland HA, Michaelis M, Kirschbaum BJ and Rudolphi KA: Osteoarthritis - an untreatable disease? Nat Rev Drug Discov. 4:331–344. 2005. View Article : Google Scholar : PubMed/NCBI
9	Flower RJ: The development of COX2 inhibitors. Nat Rev Drug Discov. 2:179–191. 2003. View Article : Google Scholar : PubMed/NCBI
10	Goldring MB and Marcu KB: Epigenomic and microRNA-mediated regulation in cartilage development, homeostasis, and osteoarthritis. Trends Mol Med. 18:109–118. 2012. View Article : Google Scholar :
11	Wu C, Tian B, Qu X, Liu F, Tang T, Qin A, Zhu Z and Dai K: MicroRNAs play a role in chondrogenesis and osteoarthritis (Review). Int J Mol Med. 34:13–23. 2014.PubMed/NCBI
12	Le LT, Swingler TE and Clark IM: Review: The role of microRNAs in osteoarthritis and chondrogenesis. Arthritis Rheum. 65:1963–1974. 2013. View Article : Google Scholar : PubMed/NCBI
13	Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 116:281–297. 2004. View Article : Google Scholar : PubMed/NCBI
14	Sethupathy P and Collins FS: MicroRNA target site polymorphisms and human disease. Trends Genet. 24:489–497. 2008. View Article : Google Scholar : PubMed/NCBI
15	Djuranovic S, Nahvi A and Green R: miRNA-mediated gene silencing by translational repression followed by mRNA deadenylation and decay. Science. 336:237–240. 2012. View Article : Google Scholar : PubMed/NCBI
16	Seeliger C1, Karpinski K, Haug AT, Vester H, Schmitt A, Bauer JS and van Griensven M: Five freely circulating miRNAs and bone tissue miRNAs are associated with osteoporotic fractures. J Bone Miner Res. 29:1718–1728. 2014. View Article : Google Scholar : PubMed/NCBI
17	Iliopoulos D, Malizos KN, Oikonomou P and Tsezou A: Integrative microRNA and proteomic approaches identify novel osteoarthritis genes and their collaborative metabolic and inflammatory networks. PLoS One. 3:e37402008. View Article : Google Scholar : PubMed/NCBI
18	Swingler TE, Wheeler G, Carmont V, Elliott HR, Barter MJ, Abu-Elmagd M, Donell ST, Boot-Handford RP, Hajihosseini MK, Münsterberg A, et al: The expression and function of microRNAs in chondrogenesis and osteoarthritis. Arthritis Rheum. 64:1909–1919. 2012. View Article : Google Scholar
19	Yu C, Chen WP and Wang XH: MicroRNA in osteoarthritis. J Int Med Res. 39:1–9. 2011. View Article : Google Scholar : PubMed/NCBI
20	Dudek KA, Lafont JE, Martinez-Sanchez A and Murphy CL: Type II collagen expression is regulated by tissue-specific miR-675 in human articular chondrocytes. J Biol Chem. 285:24381–24387. 2010. View Article : Google Scholar : PubMed/NCBI
21	Tardif G, Hum D, Pelletier JP, Duval N and Martel-Pelletier J: Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes. BMC Musculoskelet Disord. 10:1482009. View Article : Google Scholar : PubMed/NCBI
22	Bondeson J, Wainwright S, Hughes C and Caterson B: The regulation of the ADAMTS4 and ADAMTS5 aggrecanases in osteoarthritis: a review. Clin Exp Rheumatol. 26:139–145. 2008.PubMed/NCBI
23	Little CB and Fosang AJ: Is cartilage matrix breakdown an appropriate therapeutic target in osteoarthritis - insights from studies of aggrecan and collagen proteolysis? Curr Drug Targets. 11:561–575. 2010. View Article : Google Scholar : PubMed/NCBI
24	Glasson SS, Askew R, Sheppard B, Carito B, Blanchet T, Ma HL, Flannery CR, Peluso D, Kanki K, Yang Z, et al: Deletion of active ADAMTS5 prevents cartilage degradation in a murine model of osteoarthritis. Nature. 434:644–648. 2005. View Article : Google Scholar : PubMed/NCBI
25	Matsukawa T, Sakai T, Yonezawa T, Hiraiwa H, Hamada T, Nakashima M, Ono Y, Ishizuka S, Nakahara H, Lotz MK, et al: MicroRNA-125b regulates the expression of aggrecanase-1 (ADAMTS-4) in human osteoarthritic chondrocytes. Arthritis Res Ther. 15:R282013. View Article : Google Scholar : PubMed/NCBI
26	Altman R, Asch E, Bloch D, Bole G, Borenstein D, Brandt K, Christy W, Cooke TD, Greenwald R, Hochberg M, et al: Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association. Arthritis Rheum. 29:1039–1049. 1986. View Article : Google Scholar : PubMed/NCBI
27	Lafont JE, Talma S and Murphy CL: Hypoxia-inducible factor 2alpha is essential for hypoxic induction of the human articular chondrocyte phenotype. Arthritis Rheum. 56:3297–3306. 2007. View Article : Google Scholar : PubMed/NCBI
28	Zhang XJ, Ye H, Zeng CW, He B, Zhang H and Chen YQ: Dysregulation of miR-15a and miR-214 in human pancreatic cancer. J Hematol Oncol. 3:462010. View Article : Google Scholar : PubMed/NCBI
29	Pieper JS, van der Kraan PM, Hafmans T, Kamp J, Buma P, van Susante JL, van den Berg WB, Veerkamp JH and van Kuppevelt TH: Crosslinked type II collagen matrices: Preparation, characterization, and potential for cartilage engineering. Biomaterials. 23:3183–3192. 2002. View Article : Google Scholar : PubMed/NCBI
30	Papaioannou G, Lisse T and Kobayashi T: miRNAs in bone formation and homeostasis. microRNA in Regenerative Medicine. Sen CK: Elsevier Inc; London: pp. 350–380. 2015
31	Miyaki S and Asahara H: Macro view of microRNA function in osteoarthritis. Nat Rev Rheumatol. 8:543–552. 2012. View Article : Google Scholar : PubMed/NCBI
32	Beyer C, Zampetaki A, Lin NY, Kleyer A, Perricone C, Iagnocco A, Distler A, Langley SR, Gelse K, Sesselmann S, et al: Signature of circulating microRNAs in osteoarthritis. Ann Rheum Dis. 74:e182015. View Article : Google Scholar
33	Gendron C, Kashiwagi M, Lim NH, Enghild JJ, Thøgersen IB, Hughes C, Caterson B and Nagase H: Proteolytic activities of human ADAMTS-5: comparative studies with ADAMTS-4. J Biol Chem. 282:18294–18306. 2007. View Article : Google Scholar : PubMed/NCBI
34	Thirunavukkarasu K, Pei Y and Wei T: Characterization of the human ADAMTS-5 (aggrecanase-2) gene promoter. Mol Biol Rep. 34:225–231. 2007. View Article : Google Scholar : PubMed/NCBI
35	Abbaszade I, Liu RQ, Yang F, Rosenfeld SA, Ross OH, Link JR, Ellis DM, Tortorella MD, Pratta MA, Hollis JM, et al: Cloning and characterization of ADAMTS11, an aggrecanase from the ADAMTS family. J Biol Chem. 274:23443–23450. 1999. View Article : Google Scholar : PubMed/NCBI
36	Tortorella MD, Burn TC, Pratta MA, Abbaszade I, Hollis JM, Liu R, Rosenfeld SA, Copeland RA, Decicco CP, Wynn R, et al: Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins. Science. 284:1664–1666. 1999. View Article : Google Scholar : PubMed/NCBI
37	Verma P and Dalal K: ADAMTS-4 and ADAMTS-5: Key enzymes in osteoarthritis. J Cell Biochem. 112:3507–3514. 2011. View Article : Google Scholar : PubMed/NCBI
38	Song RHD, Tortorella MD, Malfait AM, Alston JT, Yang Z, Arner EC and Griggs DW: Aggrecan degradation in human articular cartilage explants is mediated by both ADAMTS-4 and ADAMTS-5. Arthritis Rheum. 56:575–585. 2007. View Article : Google Scholar : PubMed/NCBI
39	Stanton H, Rogerson FM, East CJ, Golub SB, Lawlor KE, Meeker CT, Little CB, Last K, Farmer PJ, Campbell IK, et al: ADAMTS5 is the major aggrecanase in mouse cartilage in vivo and in vitro. Nature. 434:648–652. 2005. View Article : Google Scholar : PubMed/NCBI
40	Majumdar MK, Askew R, Schelling S, Stedman N, Blanchet T, Hopkins B, Morris EA and Glasson SS: Double-knockout of ADAMTS-4 and ADAMTS-5 in mice results in physiologically normal animals and prevents the progression of osteoarthritis. Arthritis Rheum. 56:3670–3674. 2007. View Article : Google Scholar : PubMed/NCBI
41	Vonk LA, Kragten AH, Dhert WJ, Saris DB and Creemers LB: Overexpression of hsa-miR-148a promotes cartilage production and inhibits cartilage degradation by osteoarthritic chondrocytes. Osteoarthritis and cartilage/OARS. Osteoarthritis Res Soc. 22:145–153. 2014. View Article : Google Scholar