miR-628-3p regulates osteoblast differentiation by targeting RUNX2: Possible role in atrophic non-union

Chen,Hua; Ji,Xinran; She,Fei; Gao,Yuan; Tang,Peifu

doi:10.3892/ijmm.2016.2839

miR-628-3p regulates osteoblast differentiation by targeting RUNX2: Possible role in atrophic non-union

Authors:
- Hua Chen
- Xinran Ji
- Fei She
- Yuan Gao
- Peifu Tang
View Affiliations

Affiliations: Department of Orthopaedic Surgery, The General Hospital of People's Liberation Army (301 Hospital), Wukesong, Beijing 100853, P.R. China, Department of Orthopaedic Surgery (304 Hospital), Haidian, Beijing 100048, P.R. China
Published online on: December 28, 2016 https://doi.org/10.3892/ijmm.2016.2839
Pages: 279-286
Copyright: © Chen 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

Atrophic non-union is a serious complication of fractures. The underlying biological mechanisms involved in its pathogenesis are not yet completely understood. MicroRNAs (miRNAs or miRs) are a type of endogenous small non-coding RNA, which participate in various physiological and pathophysiological processes. In this study, differentially expressed miRNAs were screened in patients with atrophic non-union. In total, 4 miRNAs (miR‑149*, miR‑221, miR‑628-3p and miR‑654-5p) were upregulated and 7 miRNAs (let-7b*, miR‑220b, miR‑513a-3p, miR‑551a, miR‑576-5p, miR‑1236 and kshv-miR‑K12-6-5p) were downregulated at the fracture sites in patients with atrophic non-union. Among the upregulated miRNAs, miR‑628-3p and miR‑654-5p expression was found to be persistently decreased during osteoblast differentiation, indicating their possible inhibitory effect on osteogenesis. Gain-of-function experiment demonstrated that miR‑628-3p, but not miR‑654-5p, attenuated osteoblast differentiation. Further, in silico analysis revealed that runt-related transcription factor 2 (RUNX2), the master transcript factor for osteoblast differentiation, was the target of miR-628-3p, which had two binding site-condense regions in the 3' untranslated region. The exact binding site of miR-628-3p was further identified with luciferase reporter assay. In addition, the overexpression of miR‑628-3p appeared to be associated with the suppression of RUNX2 expression at both the mRNA and protein level, suggesting that miR‑628-3p inhibits osteoblast differentiation via RUNX2. On the whole, the findings of this study provide evidence of the upregulation of miR‑628-3p in patients with atrophic non-union and that miR‑628-3p may exert an inhibitory effect on osteogenesis via the suppression of its target gene, RUNX2. The study provides valuable insight into the pathogenesis of atrophic non-union and suggests new potential therapeutic targets for the treatment of this disorder.

View Figures

View References

1	Tzioupis C and Giannoudis PV: Prevalence of long-bone non-unions. Injury. 38(Suppl 2): S3–S9. 2007. View Article : Google Scholar : PubMed/NCBI
2	Einhorn TA and Gerstenfeld LC: Fracture healing: mechanisms and interventions. Nat Rev Rheumatol. 11:45–54. 2015. View Article : Google Scholar :
3	Dimitriou R, Kanakaris N, Soucacos PN and Giannoudis PV: Genetic predisposition to non-union: evidence today. Injury. 44(Suppl 1): S50–S53. 2013. View Article : Google Scholar : PubMed/NCBI
4	Copuroglu C, Calori GM and Giannoudis PV: Fracture non-union: Who is at risk? Injury. 44:1379–1382. 2013. View Article : Google Scholar : PubMed/NCBI
5	He L and Hannon GJ: MicroRNAs: Small RNAs with a big role in gene regulation. Nat Rev Genet. 5:522–531. 2004. View Article : Google Scholar : PubMed/NCBI
6	Calin GA and Croce CM: MicroRNA signatures in human cancers. Nat Rev Cancer. 6:857–866. 2006. View Article : Google Scholar : PubMed/NCBI
7	Lim LP, Glasner ME, Yekta S, Burge CB and Bartel DP: Vertebrate microRNA genes. Science. 299:15402003. View Article : Google Scholar : PubMed/NCBI
8	Kozomara A and Griffiths-Jones S: miRBase: Integrating microRNA annotation and deep-sequencing data. Nucleic Acids Res. 39:D152–D157. 2011. View Article : Google Scholar :
9	Friedländer MR, Lizano E, Houben AJ, Bezdan D, Báñez-Coronel M, Kudla G, Mateu-Huertas E, Kagerbauer B, González J, Chen KC, et al: Evidence for the biogenesis of more than 1,000 novel human microRNAs. Genome Biol. 15:R572014. View Article : Google Scholar : PubMed/NCBI
10	Xu N, Li Z, Yu Z, Yan F, Liu Y, Lu X and Yang W: MicroRNA-33b suppresses migration and invasion by targeting c-Myc in osteosarcoma cells. PLoS One. 9:e1153002014. View Article : Google Scholar : PubMed/NCBI
11	He C, Xiong J, Xu X, Lu W, Liu L, Xiao D and Wang D: Functional elucidation of MiR-34 in osteosarcoma cells and primary tumor samples. Biochem Biophys Res Commun. 388:35–40. 2009. View Article : Google Scholar : PubMed/NCBI
12	Zhuo W, Ge W, Meng G, Jia S, Zhou X and Liu J: MicroRNA-20a promotes the proliferation and cell cycle of human osteosarcoma cells by suppressing early growth response 2 expression. Mol Med Rep. 12:4989–4994. 2015.PubMed/NCBI
13	Mori F, Sacconi A, Canu V, Ganci F, Novello M, Anelli V, Covello R, Ferraresi V, Muti P, Biagini R, et al: miR-181c associates with tumor relapse of high grade osteosarcoma. Oncotarget. 6:13946–13961. 2015. View Article : Google Scholar : PubMed/NCBI
14	Salah Z, Arafeh R, Maximov V, Galasso M, Khawaled S, Abou-Sharieha S, Volinia S, Jones KB, Croce CM and Aqeilan RI: miR-27a and miR-27a* contribute to metastatic properties of osteosarcoma cells. Oncotarget. 6:4920–4935. 2015. View Article : Google Scholar : PubMed/NCBI
15	Mu Y, Zhang H, Che L and Li K: Clinical significance of microRNA-183/Ezrin axis in judging the prognosis of patients with osteosarcoma. Med Oncol. 31:8212014. View Article : Google Scholar
16	Kawano M, Tanaka K, Itonaga I, Ikeda S, Iwasaki T and Tsumura H: microRNA-93 promotes cell proliferation via targeting of PTEN in Osteosarcoma cells. J Exp Clin Cancer Res. 34:762015. View Article : Google Scholar : PubMed/NCBI
17	Qu F, Li CB, Yuan BT, Qi W, Li HL, Shen XZ, Zhao G, Wang JT and Liu YJ: MicroRNA-26a induces osteosarcoma cell growth and metastasis via the Wnt/β-catenin pathway. Oncol Lett. 11:1592–1596. 2016.PubMed/NCBI
18	Yuan J, Lang J, Liu C, Zhou K, Chen L and Liu Y: The expression and function of miRNA-451 in osteosarcoma. Med Oncol. 32:3242015. View Article : Google Scholar
19	Zhang C, Yao C, Li H, Wang G and He X: Serum levels of microRNA-133b and microRNA-206 expression predict prognosis in patients with osteosarcoma. Int J Clin Exp Pathol. 7:4194–4203. 2014.PubMed/NCBI
20	Zhang C, Yao C, Li H, Wang G and He X: Combined elevation of microRNA-196a and microRNA-196b in sera predicts unfavorable prognosis in patients with osteosarcomas. Int J Mol Sci. 15:6544–6555. 2014. View Article : Google Scholar : PubMed/NCBI
21	Liua XD, Caia F, Liu L, Zhang Y and Yang AL: microRNA-210 is involved in the regulation of postmenopausal osteoporosis through promotion of VEGF expression and osteoblast differentiation. Biol Chem. 396:339–347. 2015.
22	Jia J, Feng X, Xu W, Yang S, Zhang Q, Liu X, Feng Y and Dai Z: miR-17-5p modulates osteoblastic differentiation and cell proliferation by targeting SMAD7 in non-traumatic osteonecrosis. Exp Mol Med. 46:e1072014. View Article : Google Scholar : PubMed/NCBI
23	Zhao B, Yu Q, Li H, Guo X and He X: Characterization of microRNA expression profiles in patients with intervertebral disc degeneration. Int J Mol Med. 33:43–50. 2014.
24	Yu X, Li Z, Shen J, Wu WK, Liang J, Weng X and Qiu G: MicroRNA-10b promotes nucleus pulposus cell proliferation through RhoC-Akt pathway by targeting HOXD10 in intervetebral disc degeneration. PLoS One. 8:e830802013. View Article : Google Scholar :
25	Miyaki S, Sato T, Inoue A, Otsuki S, Ito Y, Yokoyama S, Kato Y, Takemoto F, Nakasa T, Yamashita S, et al: MicroRNA-140 plays dual roles in both cartilage development and homeostasis. Genes Dev. 24:1173–1185. 2010. View Article : Google Scholar : PubMed/NCBI
26	Nakasa T, Shibuya H, Nagata Y, Niimoto T and Ochi M: The inhibitory effect of microRNA-146a expression on bone destruction in collagen-induced arthritis. Arthritis Rheum. 63:1582–1590. 2011. View Article : Google Scholar : PubMed/NCBI
27	Lee YJ, Jeong JK, Seol JW, Xue M, Jackson C and Park SY: Activated protein C differentially regulates both viability and differentiation of osteoblasts mediated by bisphosphonates. Exp Mol Med. 45:e92013. View Article : Google Scholar : PubMed/NCBI
28	Brinker MR and Bailey DE Jr: Fracture healing in tibia fractures with an associated vascular injury. J Trauma. 42:11–19. 1997. View Article : Google Scholar : PubMed/NCBI
29	Santavirta S, Konttinen YT, Nordström D, Mäkelä A, Sorsa T, Hukkanen M and Rokkanen P: Immunologic studies of nonunited fractures. Acta Orthop Scand. 63:579–586. 1992. View Article : Google Scholar : PubMed/NCBI
30	Moghaddam A, Elleser C, Biglari B, Wentzensen A and Zimmermann G: Clinical application of BMP 7 in long bone non-unions. Arch Orthop Trauma Surg. 130:71–76. 2010. View Article : Google Scholar
31	Fayaz HC, Giannoudis PV, Vrahas MS, Smith RM, Moran C, Pape HC, Krettek C and Jupiter JB: The role of stem cells in fracture healing and nonunion. Int Orthop. 35:1587–1597. 2011. View Article : Google Scholar : PubMed/NCBI
32	Oskowitz AZ, Lu J, Penfornis P, Ylostalo J, McBride J, Flemington EK, Prockop DJ and Pochampally R: Human multipotent stromal cells from bone marrow and microRNA: Regulation of differentiation and leukemia inhibitory factor expression. Proc Natl Acad Sci USA. 105:18372–18377. 2008. View Article : Google Scholar : PubMed/NCBI
33	Wei J, Shi Y, Zheng L, Zhou B, Inose H, Wang J, Guo XE, Grosschedl R and Karsenty G: miR-34s inhibit osteoblast proliferation and differentiation in the mouse by targeting SATB2. J Cell Biol. 197:509–521. 2012. View Article : Google Scholar : PubMed/NCBI
34	Zeng Y, Qu X, Li H, Huang S, Wang S, Xu Q, Lin R, Han Q, Li J and Zhao RC: MicroRNA-100 regulates osteogenic differentiation of human adipose-derived mesenchymal stem cells by targeting BMPR2. FEBS Lett. 586:2375–2381. 2012. View Article : Google Scholar : PubMed/NCBI
35	Wang X, Guo B, Li Q, Peng J, Yang Z, Wang A, Li D, Hou Z, Lv K, Kan G, et al: miR-214 targets ATF4 to inhibit bone formation. Nat Med. 19:93–100. 2013. View Article : Google Scholar
36	Yang L, Cheng P, Chen C, He HB, Xie GQ, Zhou HD, Xie H, Wu XP and Luo XH: miR-93/Sp7 function loop mediates osteoblast mineralization. J Bone Miner Res. 27:1598–1606. 2012. View Article : Google Scholar : PubMed/NCBI
37	Huang S, Wang S, Bian C, Yang Z, Zhou H, Zeng Y, Li H, Han Q and Zhao RC: Upregulation of miR-22 promotes osteogenic differentiation and inhibits adipogenic differentiation of human adipose tissue-derived mesenchymal stem cells by repressing HDAC6 protein expression. Stem Cells Dev. 21:2531–2540. 2012. View Article : Google Scholar : PubMed/NCBI
38	Li H, Xie H, Liu W, Hu R, Huang B, Tan YF, Xu K, Sheng ZF, Zhou HD, Wu XP and Luo XH: A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans. J Clin Invest. 119:3666–3677. 2009. View Article : Google Scholar : PubMed/NCBI
39	Cui L, Qi Y, Li H, Ge Y, Zhao K, Qi X, Guo X, Shi Z, Zhou M, Zhu B, et al: Serum microRNA expression profile distinguishes enterovirus 71 and coxsackievirus 16 infections in patients with hand-foot-and-mouth disease. PLoS One. 6:e270712011. View Article : Google Scholar : PubMed/NCBI
40	Li A, Yu J, Kim H, Wolfgang CL, Canto MI, Hruban RH and Goggins M: MicroRNA array analysis finds elevated serum miR-1290 accurately distinguishes patients with low-stage pancreatic cancer from healthy and disease controls. Clin Cancer Res. 19:3600–3610. 2013. View Article : Google Scholar : PubMed/NCBI
41	Stratz C, Nührenberg T, Fiebich BL, Amann M, Kumar A, Binder H, Hoffmann I, Valina C, Hochholzer W, Trenk D, et al: Controlled type II diabetes mellitus has no major influence on platelet micro-RNA expression. Results from micro-array profiling in a cohort of 60 patients. Thromb Haemost. 111:902–911. 2014. View Article : Google Scholar
42	Zhang Y, Xie RL, Croce CM, Stein JL, Lian JB, van Wijnen AJ and Stein GS: A program of microRNAs controls osteogenic lineage progression by targeting transcription factor Runx2. Proc Natl Acad Sci USA. 108:9863–9868. 2011. View Article : Google Scholar : PubMed/NCBI
43	Li Z, Hassan MQ, Volinia S, van Wijnen AJ, Stein JL, Croce CM, Lian JB and Stein GS: A microRNA signature for a BMP2-induced osteoblast lineage commitment program. Proc Natl Acad Sci USA. 105:13906–13911. 2008. View Article : Google Scholar : PubMed/NCBI
44	Li Z, Hassan MQ, Jafferji M, Aqeilan RI, Garzon R, Croce CM, van Wijnen AJ, Stein JL, Stein GS and Lian JB: Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. J Biol Chem. 284:15676–15684. 2009. View Article : Google Scholar : PubMed/NCBI
45	Hassan MQ, Gordon JA, Beloti MM, Croce CM, van Wijnen AJ, Stein JL, Stein GS and Lian JB: A network connecting Runx2, SATB2, and the miR-23a~27a~24-2 cluster regulates the osteoblast differentiation program. Proc Natl Acad Sci USA. 107:19879–19884. 2010. View Article : Google Scholar : PubMed/NCBI
46	Hassan MQ, Maeda Y, Taipaleenmaki H, Zhang W, Jafferji M, Gordon JA, Li Z, Croce CM, van Wijnen AJ, Stein JL, et al: miR-218 directs a Wnt signaling circuit to promote differentiation of osteoblasts and osteomimicry of metastatic cancer cells. J Biol Chem. 287:42084–42092. 2012. View Article : Google Scholar : PubMed/NCBI
47	Zuo B, Zhu J, Li J, Wang C, Zhao X, Cai G, Li Z, Peng J, Wang P, Shen C, et al: microRNA-103a functions as a mechno-sensitive microRNA to inhibit bone formation through targeting Runx2. J Bone Miner Res. 30:330–245. 2015. View Article : Google Scholar