MicroRNA-127-5p regulates osteopontin expression and osteopontin-mediated proliferation of human chondrocytes

The aim of this study was to determine the specific microRNA (miRNA) that regulates expression of osteopontin (OPN) in osteoarthritis (OA). The potential regulatory miRNAs for OPN messenger RNA (mRNA) were predicted by miRNA prediction programs. Among eight potential regulatory miRNAs, miR-220b, miR-513a-3p and miR-548n increased, while miR-181a, miR-181b, miR-181c, miR-181d and miR-127-5p decreased in OA patients. miRNA-127-5p mimics suppressed OPN production as well as the activity of a reporter construct containing the 3′-UTR of human OPN mRNA. In addition, mutation of miR-127-5p binding site in the 3′-UTR of OPN mRNA abolished miR-127-5p-mediated repression of reporter activity. Conversely, treatment with miR-127-5p inhibitor increased reporter activity and OPN production. Interestingly, miR-127-5p inhibited proliferation of chondrocytes through OPN. In conclusion, miRNA-127-5p is an important regulator of OPN in human chondrocytes and may contribute to the development of OA.

Osteoarthritis (OA) is regarded as the most prevalent chronic joint disease, and is characterized by a group of mechanical abnormalities, such as degradation of articular cartilage, thickening of subchondral bone, and synovial inflammation [1][2][3] . There is a growing knowledge and understanding on the pathogenesis of OA 3 . Osteopontin (OPN) is a 44~75 KD multifunctional phosphoprotein, and is associated with the pathogenesis of OA 4 . OPN regulates expression of various factors associating with the pathogenesis of OA, including matrix metalloprotease 13 (MMP13) 5 , hypoxia-inducible factor-2α 6 , ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs) 7 , tissue inhibitors of metalloproteinases (TIMPs) 8 , interlukine-6 and 8 9 , and even caveolin-1 10 .
Although the etiology of OA is complex, recent evidence has made it apparent that epigenetic changes, altered expression of regulatory RNA and its consequent in gene expression modifications could also participate in the pathogenesis of OA 11,12 . MicroRNA (miRNA) is small noncoding RNA with the length of about 20-25 nucleotides (nt), and is transcribed in the nucleus by RNA polymerase II or III. miRNA is involved in regulation of post-transcriptional gene expression by translational suppression or direct degradation of the mRNA via targeting of the coding genes through complementary base pairing between the miRNA and the 3′ -Untranslated region (UTR) of the messenger RNA (mRNA) target 13 .
Increasing investigations are evaluating the differential expression of miRNA in OA vs a normal condition. Early study has compared the miRNA profiling between OA patient-derived osteoarthritic cartilage and normal cartilage, and 16 microRNAs have been characterized as osteoarthritis gene signature 14 . Jones et al. have identified 17 differential expression miRNAs with more than 4-fold in OA cartilage, and 30 differential expression mRNAs with more than 4-fold in OA bone 14 . Further study has found 12 overexpressed miRNAs in the plasma of patients with primary OA by detecting the expression of 380 miRNAs in OA 15 . Subsequently, some specific miRNAs have shown important roles in OA; for example, miR-140 regulates ADAMTs-5 expression 16 , miR-27b regulates MMP13 expression 17 , and miR-146 is intensely expressed in low grade OA cartilage, and its expression Scientific RepoRts | 6:25032 | DOI: 10.1038/srep25032 is induced by stimulation of IL-1β , suggesting its involvement in OA pathogenesis 18 . These investigations are highlighting the importance of miRNA in the initiation and development of OA. However, the specific miRNA that regulates expression of OPN in OA is largely unknown. In this study, we have investigated the miRNA that targets expression of OPN.

Discussion
Many miRNAs are differentially expressed during OA 14,19-21 , including miR-9, miR-98, miR-146a, miR-483, miR-149, miR-582, miR-1227, miR-634, miR-576, miR-641, miR-27a and b, and miRNA-140. However, as far as we known, no study has reported the miRNA that regulates the expression of OPN in OA. OPN is known as early T cell activation gene-1 (Eta-1) 22,23 . OPN is secreted by many types of cells, including macrophages, lymphocytes, epithelial cells, vascular smooth muscle cells, and even chondrocytes as well as synoviocytes [24][25][26][27] . OPN is highly abundant in the extracellular fluids at sites of inflammation, extracellular matrix (ECM) of mineralised tissues and even in the bone 24,26,28 . In the bone, OPN regulates the interactions of cell-matrix and cell-cell, the transitions of cartilage-to-bone in fracture repair, the attachment of osteoclasts to the bone matrix 23,29,30 . Interestingly, mRNA expression and protein abundance of OPN are associated with the pathogenesis of OA. At the begin, a study found that mRNA expression of OPN isolated from human OA cartilage is higher than the normal cartilage 31 . Subsequently, increased abundance of OPN in the plasma, synovial fluid and articular cartilage in OA patients are found 3,32,33 , indicating expression of OPN is associated with progressive joint damage, and the severity and progression of OA.
In human breast cancer cell lines (MCF7, MCF10AT and MCF10DCIS.com), hsa-miR-299-5p has been reported to target OPN and regulate the expression of OPN 34 . miRNA 181a targets OPN and decreases OPN expression in hepatocellular cancer cell lines (Hep 3B and Hep G2) 35 , vascular smooth muscle cells 36 . Besides miRNA 181a, miR-220b, miR-513a-3p, miR-181b, miR-181c, miR-181d, miR-548n and miR-127-5p, are also predicted to target and regulate the expression of OPN. Further analysis found that expression of miR-220b, miR-513a-3p and miR-548n increase in OA patients compared to non-OA patients. miR-220b inhibits the autoimmune regulator (AIRE) gene translation through the 3′ UTR region of AIRE gene, which is responsible for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy 37 . miR-513a-3p has been reported to regulate expression of the luteinizing hormone/chorionic gonadotropin receptor (LHCGR), which is essential for normal male and female reproductive processes 38 . miRNA-548n regulates host antiviral response by direct targeting of Interferon (IFN)-λ 1 39 . Although the exact role of miR-220b, miR-513a-3p and miR-548n in the pathogenesis of OA is unknown, it is interesting to investigate the function of increased expression of miR-220b, miR-513a-3p and miR-548n in the establishment and development of OA.
As OPN increased in the pathogenesis of OA 32 , we focused on the down-expressed miRNA, including miR-181a, miR-181b, miR-181c, miR-181d and miR-127-5p. miR-miR181 family members have been reported to regulate the differentiation stages of chondrocyte and chondrocyte formation 40 . Hypertrophic mesenchymal stromal cells (MSC)-derived chondrocytes and non-hypertrophic articular chondrocytes show differential expression of miR-181a 40 . This compelling study is indicating miR181 family members have critical importance in the establishment and development of OA. Indeed, previous reports have shown miR181a directly target and regulate expression of OPN in hepatocellular cancer cell lines (Hep 3B and Hep G2) 35 , vascular smooth muscle cells 36 . Thus, this study mainly focused on the miR-127-5p. miR-127-5p targets the 3′ UTR of β -F1-ATPase mRNA (β -mRNA), which is a catalytic subunit of mitochondrial H(+ )-ATP synthase and functions in the provision of metabolic energy by oxidative phosphorylation 41 . Notably, miR-127-5p is an important regulator of MMP-13 in human chondrocytes and contributes to the development of OA 42 . This study also shows that miR-127-5p is down regulated in the OA patients, and miR-127-5p targets the 3′ UTR of OPN mRNA to down-regulate the expression of OPN. More importantly, we have shown miR-127-5p regulates proliferation of chondrocytes through targeting expression of OPN. Thus, it is fruitful to use miR-127-5p to manipulate the establishment and development of OA.
In conclusion, this study identified that miR-127-5p targets the 3′ UTR of OPN mRNA to down-regulate the expression of OPN. In OA, the down-expressed miR-127-5p allows the expression of OPN, which mediates the establishment and development of OA. As far as we known, this is the first study show miR-127-5p directly targets OPN to regulate expression of OPN in OA.

Materials and Methods
Cartilage acquisition and assessment. The study was approved by the institutional review board and ethics committee of Xiangya Hospital affiliated to Central South University, which conformed with the regulations of medical ethics. All experiments were conducted in accordance with the approved guidelines. The normal cartilage tissues from non-OA patients and degenerated cartilage tissues from OA patients were obtained in previous study [5][6][7]32 . The cartilage tissues were assessed with hematoxylin-eosin (HE) and safranin-O staining, and Cell isolation and culture conditions. The chondrocytes were isolated and cultured according to previous study [5][6][7] . Briefly, samples were minced into pieces of less than 1 mm 3 , followed by sequential digestion at 37 °C with 0.15% collagenase II (Invitrogen, Carlsbad, CA, USA) for 5-6 h with stirring every 20 min after 2 h. Chondrocytes were isolated after centrifugation and cultured in DMEM-F12 containing 10% fetal bovine serum (FBS) and antibiotics for 5-7 days before use. miRNA prediction. To predict the miRNA targeting in the 3′ UTR of OPN, five miRNA prediction programs, RNA22, TargetScan, miRDB, miRWalk and miRanda, were used to confirm the same target binding sites. MTT assay. Cell viability was assayed by using 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT). After treatment, 10 μ L MTT (5 mg/mL) was added into cultured medium in each well for 2-4 hours until purple precipitate is visible. After the removal of culture medium, 75 μ L dimethyl sulphoxide was added to each well, leaving the cells at room temperature in the dark for 2 hours. The absorbance at 570 nm was recorded.
Expression of mature miRNA was quantified using a TaqMan miRNA assay kit (Applied Biosystems). Purified miRNA was reverse transcribed using a TaqMan miRNA RT kit (Applied Biosystems) and miRNA-specific stem-loop RT primers (Applied Biosystems). Real-time PCR was performed using a StepOnePlus Real-time PCR System (Applied Biosystems) in a 10 μ L PCR mixture containing 2 μ L RT product, 5 μ LTaqMan Universal PCR Master Mix, 0.2 μ M TaqMan probe, and 10 μ M forward and reverse primers. RNU6B was used as an internal control for miRNA detection.
Statistical analyses. Data shown are the means ± the standard error of the mean (SEM). All statistical analyses for data were performed using SPSS 16.0 software (Chicago, IL, USA). Data were analyzed between two groups using the Student's t-test, while among more than two groups by the One-Way ANOVA method [48][49][50][51] . Differences of p < 0.05 were considered significant.