Yonsei Med J.  2019 Nov;60(11):1081-1092. 10.3349/ymj.2019.60.11.1081.

LncRNA MALAT1/MiR-145 Adjusts IL-1β-Induced Chondrocytes Viability and Cartilage Matrix Degradation by Regulating ADAMTS5 in Human Osteoarthritis

• Liu C
• Ren S
• Zhao S
• Wang Y

• KMID: 2460226
• DOI: http://doi.org/10.3349/ymj.2019.60.11.1081

Abstract

PURPOSE
Accumulating evidence suggests that microRNA-145 (miR-145) plays an important role in osteoarthritis (OA), which is a chronic progressive joint disease. Long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) promotes metastasis in cancers and functions as a sponge for miR-145. However, the role of MALAT1/miR-145 in OA pathogenesis has not yet been elucidated.
MATERIALS AND METHODS
The expression of MALAT1 and miR-145 was examined by quantitative real-time PCR; the interaction between miR-145, MALAT1 and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) 5 was verified by luciferase reporter assay. Correlations among MALAT1, miR-145, and ADAMTS5 were analyzed by Spearman rank analysis. Chondrocytes viability and cartilage extracellular matrix (ECM) degradation were investigated with cell viability assay and Western blotting analyzing expression of ADAMTS5, collagen type 2 alpha 1 (COL2A1), aggrecan (ACAN), and cartilage oligomeric matrix protein (COMP).
RESULTS
MALAT1 was upregulated, and miR-145 was downregulated in OA samples and IL-1β-induced chondrocytes. Mechanically, miR-145 could directly bind to MALAT1 and ADAMTS5. Moreover, miR-145 expression was negatively correlated with MALAT1 and ADAMTS5 expression in OA patients, whereas MALAT1 and ADAMTS5 expression was positively correlated. Functionally, overexpression of MALAT1 inhibited chondrocyte viability and promoted cartilage ECM degradation in IL-1β-induced chondrocytes. In support thereof, MALAT1 silencing and miR-145 upregulation exerted the opposite effect in IL-1β-induced chondrocytes. Moreover, the effect of MALAT1 was counteracted by miR-145 upregulation, and ADAMTS5 restoration could abate miR-145 effects.
CONCLUSION
An MALAT1/miR-145 axis contributes to ECM degradation in IL-1β-induced chondrocytes through targeting ADAMTS5, suggesting that MALAT1/miR-145/ADAMTS5 signaling may underlie human OA pathogenesis.

Keyword

MALAT1; miR-145; ADAMTS5; IL-1β; osteoarthritis

MeSH Terms

Adenocarcinoma
Aggrecans
Blotting, Western
Cartilage Oligomeric Matrix Protein
Cartilage*
Cell Survival
Chondrocytes*
Collagen
Extracellular Matrix
Humans*
Joint Diseases
Luciferases
Lung
Neoplasm Metastasis
Osteoarthritis*
Porifera
Real-Time Polymerase Chain Reaction
RNA, Long Noncoding*
Temefos
Thrombospondins
Up-Regulation
Aggrecans
Collagen
Luciferases
RNA, Long Noncoding
Temefos
Thrombospondins

Figure

• 

  Fig. 1 The roles of MALAT1 and miR-145 in osteoarthritis (OA) samples. (A and B) qRT-PCR showed increased MALAT1 and decreased miR-145 in OA samples, compared with that in normal cartilage tissues. (C) Spearman rank analysis revealed a negative correlation between MALAT1 and miR-145 expression. *p<0.05.

• 

  Fig. 2 MALAT1 attenuates miR-145 expression by target binding. (A) According to starBase, there was a conserved complementary binding site of miR-145 in MALAT1. The mutant of MALAT1 wild-type (MALAT1-WT) is presented and named as MALAT1-MUT. (B and C) Transfection of miR-145 mimics/inhibitors distinctly decreased/facilitated the luciferase activity of MALAT1-WT chondrocytes. Expression level of MALAT1 and miR-145 in chondrocytes transfected with (D and E) siRNAs against MALAT1 (si-MALAT1 #1 and 2) or (F and G) pcDNA3.1-MALAT1 (MALAT1) vector. Data represent means±SD. *p<0.05.

• 

  Fig. 3 MALAT1 modulates IL-1β-induced chondrocytes cell viability and cartilage ECM degradation via miR-145 in IL-1β-induced chondrocytes. (A and B) Chondrocytes were treated with different concentrations of IL-1β for 24 h, and expression of MALAT1 and miR-145 was detected using qRT-PCR. (C and D) Cell viability was measured by MTT assay in IL-1β-induced chondrocytes when transfected with si-MALAT1 #1 and MALAT1 vector only or combined with miR-145 mimics. (E) Expression of ECM-related proteins was examined by Western blotting in chondrocytes transfected with si-MALAT1 #1 and MALAT1 vector. (F and G) Quantitative analysis of ADAMTS5, COL2A1, ACAN, and COMP expressions using Image J. (H and I) Expression of ADAMTS5, COL2A1, ACAN, and COMP in IL-1β-induced chondrocytes transfected with MALAT1 vector only or combined with miR-145 mimics. All data are presented as means±SD. *p<0.05. ECM, extracellular matrix.

• 

  Fig. 4 Correlation analysis between expression levels of ADAMTS5, MALAT1, and miR-145. Spearman rank analysis revealed (A) a negative correlation between ADAMTS5 and miR-145 expression and (B) a positive correlation between ADAMTS5 and MALAT1 expression.

• 

  Fig. 5 miR-145 might sequester ADAMTS5 by target binding. (A) The databases TargetScan (http://www.targetscan.org/) and miRcode (http://www.mircode.org/) showed two conserved binding sites of miR-145 in the 3′ UTR of ADAMTS5. The wild-type of ADAMTS5 (ADAMTS5-WT) was mutated as MALAT1-MUT. (B, C) Relative luciferase activity of ADAMTS5-WT/MUT was determined by luciferase reporter assay in chondrocytes when co-transfected with miR-145 mimics or inhibitors. (D, E) Western blotting detected ADAMTS5 protein expression level after chondrocytes transfected with miR-145 mimics or inhibitors. (F, G) qRT-PCR detected ADAMTS5 mRNA expression level after chondrocytes transfected with miR-145 mimics or inhibitors. Data are presented as means±SD. *p<0.05.

• 

  Fig. 6 ADAMTS5 modulates IL-1β-induced chondrocyte viability and cartilage degradation mediated by MALAT1/miR-145. Chondrocytes were transfected with MALAT1 only or together with miR-145 mimics and miR-145 mimics only or combined with ADAMTS5 vector. After 10 ng/mL of IL-1β treatment, (A) cell viability was detected by MTT assay at 12 h, 24 h, 48 h, and 72 h, and (B) ECM-related protein expression was measured by Western blotting at 24 h. (C and D) Quantitative analysis of ADAMTS5, COL2A1, ACAN, and COMP expression was conducted by Image J. All data are presented as means±SD. *p<0.05.

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