MicroRNA‑204‑5p inhibits the osteogenic differentiation of ankylosing spondylitis fibroblasts by regulating the Notch2 signaling pathway

Zhao,Jianjun; Zhang,Yanyan; Liu,Bo

doi:10.3892/mmr.2020.11303

MicroRNA‑204‑5p inhibits the osteogenic differentiation of ankylosing spondylitis fibroblasts by regulating the Notch2 signaling pathway

Authors:
- Jianjun Zhao
- Yanyan Zhang
- Bo Liu
View Affiliations

Affiliations: Department of Joint Surgery and Traumatic Orthopedics, Shouguang People's Hospital, Shouguang, Shandong 262700, P.R. China, Department of General Surgery, Shouguang People's Hospital, Shouguang, Shandong 262700, P.R. China, Department of Trauma Orthopedics, The No. 4 Hospital of Jinan, Jinan, Shandong 250031, P.R. China
Published online on: July 6, 2020 https://doi.org/10.3892/mmr.2020.11303
Pages: 2537-2544
Copyright: © Zhao 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

Ankylosing spondylitis (AS) is a chronic inflammatory systemic disease and is difficult to detect in the early stages. The present study aimed to investigate the role of microRNA (miR)‑204‑5p in osteogenic differentiation of AS fibroblasts. Bone morphogenetic protein 2 (BMP‑2) was used to induce osteogenic differentiation. Cells were divided into the following groups: AS group, AS + BMP‑2 group, AS + BMP‑2 + miR‑negative control group, AS + BMP‑2 + miR‑204‑5p mimics group and AS + BMP‑2 + miR‑204‑5p mimics + pcDNA‑Notch2 group. The expression levels of miR‑204‑5p, Notch2, runt‑related transcription factor 2 (RUNX2) and osteocalcin were detected via reverse transcription‑quantitative PCR analysis. The binding site between Notch2 and miR‑204‑5p was predicted using TargetScan software and verified via the dual‑luciferase reporter assay. Alkaline phosphatase (ALP) activity was assessed via the ALP assay, while the mineralized nodules area was determined via the Alizarin Red S staining assay. The results demonstrated that Notch2 is a target gene of miR‑204‑5p. Furthermore, treatment with BMP‑2 significantly decreased miR‑204‑5p expression, and significantly increased ALP activity, the mineralized nodules area and the expression levels of Notch2, RUNX2 and osteocalcin in ligament fibroblasts (all P<0.05). Conversely, transfection with miR‑204‑5p mimics significantly increased miR‑204‑5p expression, and significantly decreased ALP activity, the mineralized nodules area and the expression levels of Notch2, RUNX2 and osteocalcin in ligament fibroblasts (all P<0.05). Notably, transfection with pcDNA‑Notch2 significantly reversed the inhibitory effects induced by miR‑204‑5p mimics on the osteogenic differentiation of ligament fibroblasts (all P<0.05). Furthermore, miR‑204‑5p inhibited the osteogenic differentiation of ligament fibroblasts in patients with AS by targeting Notch2. Thus, miR‑204‑5p may negatively regulate Notch2 expression and may be a potential therapeutic target for AS. Collectively, the results of the present study provide a theoretical basis for the effective treatment of patients with AS.

View Figures

View References

1	Robinson PC, Leo PJ, Pointon JJ, Harris J, Cremin K, Bradbury LA, Stebbings S, Harrison AA; Australian Osteoporosis Genetics Consortium; Wellcome Trust Case Control Consortium, ; et al: Exome-wide study of ankylosing spondylitis demonstrates additional shared genetic background with inflammatory bowel disease. NPJ Genom Med. 1:160082016. View Article : Google Scholar : PubMed/NCBI
2	Zhao J, Huang C, Huang H, Pan JK, Zeng LF, Luo MH, Liang GH, Yang WY and Liu J: Prevalence of ankylosing spondylitis in a Chinese population: A systematic review and meta-analysis. Rheumatol Int. 40:859–872. 2020. View Article : Google Scholar : PubMed/NCBI
3	Raychaudhuri SP and Deodhar A: The classification and diagnostic criteria of ankylosing spondylitis. J Autoimmun. 48:128–133. 2014. View Article : Google Scholar : PubMed/NCBI
4	Ahsan T, Erum U, Jabeen R and Khowaja D: Ankylosing Spondylitis: A rheumatology clinic experience. Pak J Med Sci. 32:365–368. 2016.PubMed/NCBI
5	Dean LE, Jones GT, MacDonald AG, Downham C, Sturrock RD and Macfarlane GJ: Global prevalence of ankylosing spondylitis. Rheumatology (Oxford). 53:650–657. 2014. View Article : Google Scholar : PubMed/NCBI
6	Jethwa H and Bowness P: The interleukin (IL)-23/IL-17 axis in ankylosing spondylitis: New advances and potentials for treatment. Clin Exp Immunol. 183:30–36. 2016. View Article : Google Scholar : PubMed/NCBI
7	Darby SC, Doll R, Gill SK and Smith PG: Long term mortality after a single treatment course with X-rays in patients treated for ankylosing spondylitis. Br J Cancer. 55:179–190. 1987. View Article : Google Scholar : PubMed/NCBI
8	Şilte Karamanlioğlu D, Aktas I, Ozkan FU, Kaysin M and Girgin N: Effectiveness of ultrasound treatment applied with exercise therapy on patients with ankylosing spondylitis: A double-blind, randomized, placebo-controlled trial. Rheumatol Int. 36:653–661. 2016. View Article : Google Scholar : PubMed/NCBI
9	Wang T, Wang D, Cong Y, Yin C, Li S and Chen X: Evaluating a posterior approach for surgical treatment of thoracolumbar pseudarthrosis in Ankylosing Spondylitis. Clin Spine Surg. 30:E13–E18. 2017. View Article : Google Scholar : PubMed/NCBI
10	Wang M, Wang L, Zhang X, Yang X, Li X, Xia Q, Chen M, Han R, Liu R, Xu S and Pan F: Overexpression of miR-31 in Peripheral Blood Mononuclear Cells (PBMC) from patients with Ankylosing Spondylitis. Med Sci Monit. 23:5488–5494. 2017. View Article : Google Scholar : PubMed/NCBI
11	Perez-Sanchez C, Font-Ugalde P, Ruiz-Limon P, Lopez-Pedrera C, Castro-Villegas MC, Abalos-Aguilera MC, Barbarroja N, Arias-de la Rosa I, Lopez-Montilla MD, Escudero-Contreras A, et al: Circulating microRNAs as potential biomarkers of disease activity and structural damage in Ankylosing Spondylitis patients. Hum Mol Genet. 27:875–890. 2018. View Article : Google Scholar : PubMed/NCBI
12	He H, Chen K, Wang F, Zhao L, Wan X, Wang L and Mo Z: miR-204-5p promotes the adipogenic differentiation of human adipose-derived mesenchymal stem cells by modulating DVL3 expression and suppressing Wnt/β-catenin signaling. Int J Mol Med. 35:1587–1595. 2015. View Article : Google Scholar : PubMed/NCBI
13	Wang Y, Niu ZY, Guo YJ, Wang LH, Lin FR and Zhang JY: IL-11 promotes the treatment efficacy of hematopoietic stem cell transplant therapy in aplastic anemia model mice through a NF-κB/microRNA-204/thrombopoietin regulatory axis. Exp Mol Med. 49:e4102017. View Article : Google Scholar : PubMed/NCBI
14	Roozbehkia M, Mahmoudi M, Aletaha S, Rezaei N, Fattahi MJ, Jafarnezhad-Ansariha F, Barati A and Mirshafiey A: The potent suppressive effect of β-d-mannuronic acid (M2000) on molecular expression of the TLR/NF-kB Signaling Pathway in ankylosing spondylitis patients. Int Immunopharmacol. 52:191–196. 2017. View Article : Google Scholar : PubMed/NCBI
15	Wang G, Cai J, Zhang J and Li C: Mechanism of triptolide in treating ankylosing spondylitis through the anti-ossification effect of the BMP/Smad signaling pathway. Mol Med Rep. 17:2731–2737. 2018.PubMed/NCBI
16	Zou Y, Yang X, Yuan S, Zhang P, Ye Y and Li Y: Downregulation of dickkopf-1 enhances the proliferation and osteogenic potential of fibroblasts isolated from ankylosing spondylitis patients via the Wnt/β-catenin signaling pathway in vitro. Connect Tissue Res. 57:200–211. 2016. View Article : Google Scholar : PubMed/NCBI
17	Xu W, Liang CG, Li YF, Ji YH, Qiu WJ and Tang XZ: Involvement of Notch1/Hes signaling pathway in ankylosing spondylitis. Int J Clin Exp Pathol. 8:2737–2745. 2015.PubMed/NCBI
18	Zhou Y, Tanzie C, Yan Z, Chen S, Duncan M, Gaudenz K, Li H, Seidel C, Lewis B, Moran A, et al: Notch2 regulates BMP signaling and epithelial morphogenesis in the ciliary body of the mouse eye. Proc Natl Acad Sci USA. 110:8966–8971. 2013. View Article : Google Scholar : PubMed/NCBI
19	Ruan ZB, Fu XL, Li W, Ye J, Wang RZ and Zhu L: Effect of notch1,2,3 genes silicing on NF-κB signaling pathway of macrophages in patients with atherosclerosis. Biomed Pharmacother. 84:666–673. 2016. View Article : Google Scholar : PubMed/NCBI
20	Smith DM, Cooper GM, Mooney MP, Marra KG and Losee JE: Bone morphogenetic protein 2 therapy for craniofacial surgery. J Craniofac Surg. 19:1244–1259. 2008. View Article : Google Scholar : PubMed/NCBI
21	Khosla S, Westendorf JJ and Oursler MJ: Building bone to reverse osteoporosis and repair fractures. J Clin Invest. 118:421–428. 2008. View Article : Google Scholar : PubMed/NCBI
22	Sun J, Li J, Li C and Yu Y: Role of bone morphogenetic protein-2 in osteogenic differentiation of mesenchymal stem cells. Mol Med Rep. 12:4230–4237. 2015. View Article : Google Scholar : PubMed/NCBI
23	van der Linden SM, Valkenburg HA, de Jongh BM and Cats A: The risk of developing ankylosing spondylitis in HLA-B27 positive individuals. A Comparison of Relatives of Spondylitis patients with the general population. Arthritis Rheum. 27:241–249. 1984. View Article : Google Scholar : PubMed/NCBI
24	Hupkes M, Sotoca AM, Hendriks JM, van Zoelen EJ and Dechering KJ: MicroRNA miR-378 promotes BMP2-induced osteogenic differentiation of mesenchymal progenitor cells. BMC Mol Biol. 15:12014. View Article : Google Scholar : PubMed/NCBI
25	Kanayama S, Kaito T, Kitaguchi K, Ishiguro H, Hashimoto K, Chijimatsu R, Otsuru S, Takenaka S, Makino T, Sakai Y, et al: ONO-1301 Enhances in vitro osteoblast differentiation and in vivo bone formation induced by bone morphogenetic protein. Spine (Phila Pa 1976). 43:E616–E624. 2018. View Article : Google Scholar : PubMed/NCBI
26	Jung JI, Park KY, Lee Y, Park M and Kim J: Vitamin C-linker-conjugated tripeptide AHK stimulates BMP-2-induced osteogenic differentiation of mouse myoblast C2C12 cells. Differentiation. 101:1–7. 2018. View Article : Google Scholar : PubMed/NCBI
27	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
28	Qin X, Jiang T, Liu S, Tan J, Wu H, Zheng L and Zhao J: Effect of metformin on ossification and inflammation of fibroblasts in ankylosing spondylitis: An in vitro study. J Cell Biochem. 119:1074–1082. 2018. View Article : Google Scholar : PubMed/NCBI
29	Zou YC, Yang XW, Yuan SG, Zhang P and Li YK: Celastrol inhibits prostaglandin E2-induced proliferation and osteogenic differentiation of fibroblasts isolated from ankylosing spondylitis hip tissues in vitro. Drug Des Devel Ther. 10:933–948. 2016.PubMed/NCBI
30	Zhou YY, Huang RY, Lin JH, Xu YY, He XH and He YT: Bushen-Qiangdu-Zhilv decoction inhibits osteogenic differentiation of rat fibroblasts by regulating connexin 43. Exp Ther Med. 12:347–353. 2016. View Article : Google Scholar : PubMed/NCBI
31	Yu F, Cui Y, Zhou X and Han J: Osteogenic differentiation of human ligament fibroblasts induced by conditioned medium of osteoclast-like cells. Biosci Trends. 5:46–51. 2011. View Article : Google Scholar : PubMed/NCBI
32	Shang G, Wang Y, Xu Y, Zhang S, Sun X, Guan H, Zhao X, Wang Y, Li Y and Zhao G: Long non-coding RNA TCONS_00041960 enhances osteogenesis and inhibits adipogenesis of rat bone marrow mesenchymal stem cell by targeting miR-204-5p and miR-125a-3p. J Cell Physiol. 233:6041–6051. 2018. View Article : Google Scholar : PubMed/NCBI
33	Zhang YY, Zhou JB, Zeng XW, Zhao FM and Zhan XQ: Effects of puerarin on proliferation of osteoblasts and Runx2-targeting miRNAs. Chinese Pharmacological Bulletin. 32:1457–1462. 2016.
34	Zhao J, Wang C, Song Y and Fang B: Arsenic trioxide and microRNA-204 display contrary effects on regulating adipogenic and osteogenic differentiation of mesenchymal stem cells in aplastic anemia. Acta Biochim Biophys Sin (Shanghai). 46:885–893. 2014. View Article : Google Scholar : PubMed/NCBI
35	Lee H, Kim KR, Cho NH, Hong SR, Jeong H, Kwon SY, Park KH, An HJ, Kim TH, Kim I, et al: MicroRNA expression profiling and Notch1 and Notch2 expression in minimal deviation adenocarcinoma of uterine cervix. World J Surg Oncol. 12:3342014. View Article : Google Scholar : PubMed/NCBI
36	Cai B, Zheng Y, Ma S, Xing Q, Wang X, Yang B, Yin G and Guan F: BANCR contributes to the growth and invasion of melanoma by functioning as a competing endogenous RNA to upregulate Notch2 expression by sponging miR-204. Int J Oncol. 51:1941–1951. 2017. View Article : Google Scholar : PubMed/NCBI
37	Hayes AJ, Dowthwaite GP, Webster SV and Archer CW: The distribution of Notch receptors and their ligands during articular cartilage development. J Anat. 202:495–502. 2003. View Article : Google Scholar : PubMed/NCBI
38	Wei Y, Mou D, Lian J, Luo J and Tang M: Role of Notch signaling in BMP2-induced osteogenic differentiation of MEFs and its mechanism. Chin J Cell Biol. 40:478–489. 2018.
39	Wegman F, Bijenhof A, Schuijff L, Oner FC, Dhert WJ and Alblas J: Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo. Eur Cell Mater. 21:230–242. 2011. View Article : Google Scholar : PubMed/NCBI
40	Song R, Fullerton DA, Ao L, Zhao KS and Meng X: An epigenetic regulatory loop controls pro-osteogenic activation by TGF-β1 or bone morphogenetic protein 2 in human aortic valve interstitial cells. J Biol Chem. 292:8657–8666. 2017. View Article : Google Scholar : PubMed/NCBI
41	Yu C, Li L, Xie F, Guo S, Liu F, Dong N and Wang Y: LncRNA TUG1 sponges miR-204-5p to promote osteoblast differentiation through upregulating Runx2 in aortic valve calcification. Cardiovasc Res. 114:168–179. 2018. View Article : Google Scholar : PubMed/NCBI
42	Liu Z, Yao X, Yan G, Xu Y, Yan J, Zou W and Wang G: Mediator MED23 cooperates with RUNX2 to drive osteoblast differentiation and bone development. Nat Commun. 7:111492016. View Article : Google Scholar : PubMed/NCBI
43	Huang J, Song G, Yin Z, Fu Z and Ye Z: MiR-29a and messenger RNA expression of bone turnover markers in canonical Wnt pathway in patients with ankylosing spondylitis. Clin Lab. 63:955–960. 2017. View Article : Google Scholar : PubMed/NCBI
44	Yang LQ, Dong CJ and Zhu S: Osteogenesis-related factor Runx2 expression in necrotic femoral head tissue: Study protocol for a non-randomized, parallel-controlled trial. Chinese Journal of Tissue Engineering Research. 2016.
45	Zhou YY, Liu HX, Jiang N, Feng XH, Feng XY, Zhang HQ, Wu ZK, Liang HY, Jiang Q and Chen P: Elemene, the essential oil of Curcuma wenyujin, inhibits osteogenic differentiation in ankylosing spondylitis. Joint Bone Spine. 82:100–103. 2015. View Article : Google Scholar : PubMed/NCBI
46	Wang Y, Chen S, Deng C, Li F, Wang Y, Hu X, Shi F and Dong N: MicroRNA-204 targets Runx2 to Attenuate BMP-2-induced osteoblast differentiation of human aortic valve interstitial cells. J Cardiovasc Pharmacol. 66:63–71. 2015. View Article : Google Scholar : PubMed/NCBI
47	Franck H and Keck E: Serum osteocalcin and vitamin D metabolites in patients with ankylosing spondylitis. Ann Rheum Dis. 52:343–346. 1993. View Article : Google Scholar : PubMed/NCBI
48	Kwon SR, Lim MJ, Suh CH, Park SG, Hong YS, Yoon BY, Kim HA, Choi HJ and Park W: Dickkopf-1 level is lower in patients with ankylosing spondylitis than in healthy people and is not influenced by anti-tumor necrosis factor therapy. Rheumatol Int. 32:2523–2527. 2012. View Article : Google Scholar : PubMed/NCBI
49	Solmaz D, Bulbul H, Uslu S, Kozaci LD, Karaca N and Akar S: AB0157 serum level of the vascular endothelial growth factor is elevated in Ankylosing Spondylitis and Osteocalcin may be related with osteoproliferation. BMJ. 74 (Suppl 2):AB01572015.
50	Jeon MJ, Kim JA, Kwon SH, Kim SW, Park KS, Park SW, Kim SY and Shin CS: Activation of peroxisome proliferator-activated receptor-gamma inhibits the Runx2-mediated transcription of osteocalcin in osteoblasts. J Biol Chem. 278:23270–23277. 2003. View Article : Google Scholar : PubMed/NCBI