miRNA-140 inhibits C3H10T1/2 mesenchymal stem cell proliferation by targeting CXCL12 during transforming growth factor-β3-induced chondrogenic differentiation

Wa,Qingde; Liu,Yi; Huang,Shuai; He,Peiheng; Zuo,Jianwei; Li,Xing; Li,Ziqing; Dong,Liming; Peng,Jiachen; Wu,Shuhong; Chen,Fang; Cai,Dongfeng; Zou,Xuenong; Liao,Wenbo

doi:10.3892/mmr.2017.6720

miRNA-140 inhibits C3H10T1/2 mesenchymal stem cell proliferation by targeting CXCL12 during transforming growth factor-β3-induced chondrogenic differentiation

Authors:
- Qingde Wa
- Yi Liu
- Shuai Huang
- Peiheng He
- Jianwei Zuo
- Xing Li
- Ziqing Li
- Liming Dong
- Jiachen Peng
- Shuhong Wu
- Fang Chen
- Dongfeng Cai
- Xuenong Zou
- Wenbo Liao
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Affiliations: Department of Orthopedic Surgery, The First Affiliated Hospital of Zunyi Medical College, Zunyi, Guizhou 563003, P.R. China, Department of Orthopedic Surgery, The First Affiliated Hospital of Sun Yat‑sen University, Guangzhou, Guangdong 510080, P.R. China, Department of Sports Medicine, Shenzhen Hospital of Peking University, Shenzhen, Guangdong 518036, P.R. China
Published online on: June 8, 2017 https://doi.org/10.3892/mmr.2017.6720
Pages: 1389-1394

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Abstract

The aim of the present study was to investigate the role of microRNA (miRNA or miR)-140 in C3H10T1/2 mesenchymal stem cells (MSCs). Cluster analysis was used to evaluate the miRNA expression profile. The expression level of miRNA‑140 was validated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR). TargetScan and microRNA.org databases were used to predict target miRNAs and cartilage‑associated target genes. Binding sites between miR‑140 and the target gene were predicted by bioinformatics software. A dual‑luciferase reporter assay was performed to determine whether miR‑140 could target C‑X‑C motif chemokine ligand 12 (CXCL12). Following the promotion/inhibition of miR‑140, 1, 7 and 14 days following transforming growth factor‑β3 (TGF‑β3)‑induction, western blotting was utilized to evaluate CXCL12 protein levels. MTT assays and alcian blue staining were applied to assess C3H10T1/2 MSC viability and chondrogenic differentiation, respectively. In the TGF‑β3‑induced group, RT‑qPCR verified that the mRNA level of Mus musculus (mmu)‑miR‑140 was significantly elevated when compared with the control group. miR‑140 was predicted to recognize and interact with CXCL12‑3'UTR and the dual luciferase reporter assay further validated that miR‑140 targeted the predicted region of CXCL12. CXCL12 was markedly decreased following miR‑140 overexpression and visibly increased following miR‑140 inhibition. In addition, the level of CXCL12 expression declined as the duration of induction increased. Following the promotion/inhibition of miR‑140, at 1 and 7 days following TGF‑β3‑induction, C3H10T1/2 MSCs inhibited or promoted cell viability, respectively, when compared with the control groups. In addition, in pellets achieved by chondrogenic differentiation following the induction of C3H10T1/2 MSCs for 7 days, alcian blue staining revealed no significant difference in characteristic extracellular matrix glycosaminoglycans between the miR‑140 up and downregulated groups, and their respective control groups. The present study concludes that miRNA‑140 inhibition promoted C3H10T1/2 MSC viability however, not C3H10T1/2 MSC differentiation by targeting and reducing CXCL12 protein levels during the process of TGF‑β3‑induced chondrogenic differentiation. In conclusion, the present study provided a potential target for the treatment of cartilage defection.

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1	Redman SN, Oldfield SF and Archer CW: Current strategies for articular cartilage repair. Eur Cell Mater. 9:23–32. 2005. View Article : Google Scholar : PubMed/NCBI
2	Xian CJ and Foster BK: Repair of injured articular and growth plate cartilage using mesenchymal stem cells and chondrogenic gene therapy. Curr Stem Cell Res Ther. 1:213–229. 2006. View Article : Google Scholar : PubMed/NCBI
3	Ji YH, Ji JL, Sun FY, Zeng YY, He XH, Zhao JX, Yu Y, Yu SH and Wu W: Quantitative proteomics analysis of chondrogenic differentiation of C3H10T1/2 mesenchymal stem cells by iTRAQ labeling coupled with on-line two-dimensional LC/MS/MS. Mol Cell Proteomics. 9:550–564. 2010. View Article : Google Scholar : PubMed/NCBI
4	Tang QQ, Otto TC and Lane MD: Commitment of C3H10T1/2 pluripotent stem cells to the adipocyte lineage. Proc Natl Acad Sci USA. 101:9607–9611. 2004. View Article : Google Scholar : PubMed/NCBI
5	Son HE, Kim TH and Jang WG: Curculactones A and B induced the differentiation of C3H10T1/2 and MC3T3-E1 cells to osteoblasts. Bioorg Med Chem Lett. 27:1301–1303. 2017. View Article : Google Scholar : PubMed/NCBI
6	Wa Q, Gao M, Dai X, Yu T, Zhou Z, Xu D and Zou X: Induction of chondrogenic differentiation of mouse embryonic mesenchymal stem cells through an in vitro pellet model. Cell Biol Int. 39:657–665. 2015. View Article : Google Scholar : PubMed/NCBI
7	Megosh HB, Cox DN, Campbell C and Lin H: The role of PIWI and the miRNA machinery in Drosophila germline determination. Curr Biol. 16:1884–1894. 2006. View Article : Google Scholar : PubMed/NCBI
8	Park JK, Liu X, Strauss TJ, McKearin DM and Liu Q: The miRNA pathway intrinsically controls self-renewal of Drosophila germline stem cells. Curr Biol. 17:533–538. 2007. View Article : Google Scholar : PubMed/NCBI
9	Wang Y, Medvid R, Melton C, Jaenisch R and Blelloch R: DGCR8 is essential for microRNA biogenesis and silencing of embryonic stem cell self-renewal. Nat Genet. 39:380–385. 2007. View Article : Google Scholar : PubMed/NCBI
10	Luzi E, Marini F, Sala SC, Tognarini I, Galli G and Brandi ML: Osteogenic differentiation of human adipose tissue-derived stem cells is modulated by the miR-26a targeting of the SMAD1 transcription factor. J Bone Miner Res. 23:287–295. 2008. View Article : Google Scholar : PubMed/NCBI
11	Guerit D, Brondello JM, Chuchana P, Philipot D, Toupet K, Bony C, Jorgensen C and Noël D: FOXO3A regulation by miRNA-29a Controls chondrogenic differentiation of mesenchymal stem cells and cartilage formation. Stem Cells Dev. 23:1195–1205. 2014. View Article : Google Scholar : PubMed/NCBI
12	Guerit D, Philipot D, Chuchana P, Toupet K, Brondello JM, Mathieu M, Jorgensen C and Noël D: Sox9-regulated miRNA-574-3p inhibits chondrogenic differentiation of mesenchymal stem cells. PLoS One. 8:e625822013. View Article : Google Scholar : PubMed/NCBI
13	Huang C, Geng J, Wei X, Zhang R and Jiang S: MiR-144-3p regulates osteogenic differentiation and proliferation of murine mesenchymal stem cells by specifically targeting Smad4. FEBS Lett. 590:795–807. 2016. View Article : Google Scholar : PubMed/NCBI
14	Huang K, Fu J, Zhou W, Li W, Dong S, Yu S, Hu Z, Wang H and Xie Z: MicroRNA-125b regulates osteogenic differentiation of mesenchymal stem cells by targeting Cbfβ in vitro. Biochimie. 102:47–55. 2014. View Article : Google Scholar : PubMed/NCBI
15	Liu Y, Zhang ZC, Qian SW, Zhang YY, Huang HY, Tang Y, Guo L, Li X and Tang QQ: MicroRNA-140 promotes adipocyte lineage commitment of C3H10T1/2 pluripotent stem cells via targeting osteopetrosis-associated transmembrane protein 1. J Biol Chem. 288:8222–8230. 2013. View Article : Google Scholar : PubMed/NCBI
16	Schrader AJ, Lechner O, Templin M, Dittmar KE, Machtens S, Mengel M, Probst-Kepper M, Franzke A, Wollensak T, Gatzlaff P, et al: CXCR4/CXCL12 expression and signalling in kidney cancer. Br J Cancer. 86:1250–1256. 2002. View Article : Google Scholar : PubMed/NCBI
17	Blanchet X, Langer M, Weber C, Koenen RR and von Hundelshausen P: Touch of chemokines. Front Immunol. 3:1752012. View Article : Google Scholar : PubMed/NCBI
18	Hosogane N, Huang Z, Rawlins BA, Liu X, Boachie-Adjei O, Boskey AL and Zhu W: Stromal derived factor-1 regulates bone morphogenetic protein 2-induced osteogenic differentiation of primary mesenchymal stem cells. Int J Biochem Cell Biol. 42:1132–1141. 2010. View Article : Google Scholar : PubMed/NCBI
19	Liu C, Weng Y, Yuan T, Zhang H, Bai H, Li B, Yang D, Zhang R, He F, Yan S, et al: CXCL12/CXCR4 signal axis plays an important role in mediating bone morphogenetic protein 9-induced osteogenic differentiation of mesenchymal stem cells. Int J Med Sci. 10:1181–1192. 2013. View Article : Google Scholar : PubMed/NCBI
20	Pfeffer S and Voinnet O: Viruses, microRNAs and cancer. Oncogene. 25:6211–6219. 2006. View Article : Google Scholar : PubMed/NCBI
21	Taft RJ, Pang KC, Mercer TR, Dinger M and Mattick JS: Non-coding RNAs: Regulators of disease. J Pathol. 220:126–139. 2010. View Article : Google Scholar : PubMed/NCBI
22	Miyaki S, Nakasa T, Otsuki S, Grogan SP, Higashiyama R, Inoue A, Kato Y, Sato T, Lotz MK and Asahara H: MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses. Arthritis Rheum. 60:2723–2730. 2009. View Article : Google Scholar : PubMed/NCBI
23	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 : PubMed/NCBI
24	Karakus S, Bagci B, Bagci G, Sancakdar E, Yildiz C, Akkar O and Cetin A: SDF-1/CXCL12 and CXCR4 gene variants, and elevated serum SDF-1 levels are associated with preeclampsia. Hypertens Pregnancy. 1-7:2016.(Epub ahead of print).
25	Nagasawa T, Hirota S, Tachibana K, Takakura N, Nishikawa S, Kitamura Y, Yoshida N, Kikutani H and Kishimoto T: Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature. 382:635–638. 1996. View Article : Google Scholar : PubMed/NCBI
26	Ratajczak MZ, Zuba-Surma E, Kucia M, Reca R, Wojakowski W and Ratajczak J: The pleiotropic effects of the SDF-1-CXCR4 axis in organogenesis, regeneration and tumorigenesis. Leukemia. 20:1915–1924. 2006. View Article : Google Scholar : PubMed/NCBI
27	Kanbe K, Takemura T, Takeuchi K, Chen Q, Takagishi K and Inoue K: Synovectomy reduces stromal-cell-derived factor-1 (SDF-1) which is involved in the destruction of cartilage in osteoarthritis and rheumatoid arthritis. J Bone Joint Surg Br. 86:296–300. 2004. View Article : Google Scholar : PubMed/NCBI
28	Björnsson S: Simultaneous preparation and quantitation of proteoglycans by precipitation with alcian blue. Anal Biochem. 210:282–291. 1993. View Article : Google Scholar : PubMed/NCBI