MicroRNA-486 promotes a more catabolic phenotype in chondrocyte-like cells by targeting SIRT6 : possible involvement in cartilage degradation in osteoarthritis

Abstract

Aims: Osteoarthritis (OA) is characterized by persistent destruction of articular cartilage. It has been found that microRNAs (miRNAs) are closely related to the occurrence and development of OA. The purpose of the present study was to investigate the mechanism of miR-486 in the development and progression of OA.

Methods: The expression levels of miR-486 in cartilage were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN), matrix metalloproteinase (MMP)-13, and a disintegrin and metalloproteinase with thrombospondin motifs-4 (ADAMTS4) in SW1353 cells at both messenger RNA (mRNA) and protein levels was determined by qRT-PCR, western blot, and enzyme-linked immunosorbent assay (ELISA). Double luciferase reporter gene assay, qRT-PCR, and western blot assay were used to determine whether silencing information regulator 6 (SIRT6) was involved in miR-486 induction of chondrocyte-like cells to a more catabolic phenotype.

Results: Compared with osteonecrosis, the expression of miR-486 was significantly upregulated in cartilage from subjects with severe OA. In addition, overexpressed miR-486 promoted a catabolic phenotype in SW1353 cells by upregulating the expressions of ADAMTS4 and MMP-13 and down-regulating the expressions of COL2A1 and ACAN. Conversely, inhibition of miR-486 had the opposite effect. Furthermore, overexpression of miR-486 significantly inhibited the expression of SIRT6, confirming that SIRT6 is a direct target of miR-486. Moreover, SW1353 cells were transfected with small interfering RNA (si)-SIRT6 and it was found that SIRT6 was involved in and inhibited miR-486-induced changes to SW1353 gene expression.

Conclusion: Our results indicate that miR-486 promotes a catabolic phenotype in SW1353 cells in OA by targeting SIRT6. Our findings might provide a potential therapeutic target and theoretical basis for OA. Cite this article: Bone Joint Res 2021;10(7):459-466.

Keywords: Chondrocyte catabolic phenotype; Osteoarthritis; SIRT6; SW1353 cell line; miR-486.

Fig. 1

Analysis of the expression levels of microRNA 486 (miR-486) in osteonecrosis tissue and cartilage from subjects with severe osteoarthritis (OA) by quantitative real-time polymerase chain reaction (qRT-PCR). ***p < 0.001, independent-samples t-test.

Fig. 2

Relationship between microRNA 486 (miR-486) and extracellular matrix (ECM) metabolism of SW1353 cells. a) and b) Detection of the messenger RNA (mRNA) and protein expressions of miR-486, collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN), matrix metalloproteinase (MMP)-13, and ACANase 4 (ADAMTS4) by quantitative real-time polymerase chain reaction (qRT-PCR) (a) and Western blotting (b). c) Detection of the secretions of COL2A1, aggrecan, MMP-13, and ADAMTS4 by enzyme-linked immunosorbent assay (ELISA). Compared with the control group (con), **p < 0.01, independent-samples t-test. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Fig. 3

Silencing information regulator 6 (SIRT6) as a microRNA 486 (miR-486) target gene binding site. a) Schematic representation of miR-486 predicted binding sites in the three prime untranslated region (3'-UTR) of SIRT6. b) Determination of the luciferase activity of SW1353 cells by luciferase assay. c) and d) Detection of the messenger RNA (mRNA) and protein expression levels of SIRT6 after transfection of SW1353 cells with miR-486 mimic, miR-486 inhibitor, or its negative control by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. e) Determination of the mRNA expression of SIRT6 in cartilage from subjects with severe OA and osteonecrosis tissue by qRT-PCR. f) Correlation analysis of SIRT6 and miR-486 expression in human osteonecrosis tissue and cartilage from subjects with severe OA. Compared with the control group, **p < 0.05, ***p < 0.001, independent-samples t-test. con, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; mut, mutant; wt, wild type. hsa-miR-486, homo sapiens miR-486.

Fig. 4

Silencing information regulator 6 (SIRT6) as a target for microRNA 486 (miR-486)-induced chondrocyte extracellular matrix (ECM) metabolism. a) Detection of the messenger RNA (mRNA) expression level of SIRT6 in small interfering RNA-control (si-con) or si-SIRT6 transfected SW1353 cells by quantitative real-time polymerase chain reaction (qRT-PCR). b) Detection of the mRNA expression levels of collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN), matrix metalloproteinase (MMP)-13, and ACANase 4 (ADAMTS4) in miR-486 inhibitor or si-SIRT6 co-transfected SW1353 cells by qRT-PCR. c) Detection of the protein expression levels of COL2A1, aggrecan, MMP-13, and ADAMTS4 in miR-486 inhibitor or si-SIRT6 co-transfected SW1353 cells by western blot. Compared to the control group, **p < 0.001; compared to the miR-486 inhibitor group, ##p < 0.001. All statistical analyses were conducted with independent-samples t-test.