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MicroRNA-125b regulates the expression of aggrecanase-1 (ADAMTS-4) in human osteoarthritic chondrocytes.

Matsukawa T, Sakai T, Yonezawa T, Hiraiwa H, Hamada T, Nakashima M, Ono Y, Ishizuka S, Nakahara H, Lotz MK, Asahara H, Ishiguro N - Arthritis Res. Ther. (2013)

Bottom Line: MiR-125b was expressed in both normal and OA chondrocytes, with significantly lower expression in OA chondrocytes than in normal chondrocytes.Furthermore, IL-1β-induced upregulation of ADAMTS-4 was suppressed by overexpression of miR-125b in human OA chondrocytes.In the luciferase reporter assay, mutation of the putative miR-125b binding site in the ADAMTS-4 3'UTR abrogated the suppressive effect of miR125.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Increased expression of aggrecanase-1 (ADAMTS-4) has emerged as an important factor in osteoarthritis (OA) and other joint diseases. This study aimed to determine whether the expression of ADAMTS-4 in human chondrocytes is regulated by miRNA.

Methods: MiRNA targets were identified using bioinformatics. Chondrocytes were isolated from knee cartilage and treated with interleukin-1 beta (IL-1β). Gene expression was quantified using TaqMan assays and protein production was determined by immunoblotting. Luciferase reporter assay was used to verify interaction between miRNA and target messenger RNA (mRNA).

Results: In silico analysis predicted putative target sequence of miR-125b on ADAMTS-4. MiR-125b was expressed in both normal and OA chondrocytes, with significantly lower expression in OA chondrocytes than in normal chondrocytes. Furthermore, IL-1β-induced upregulation of ADAMTS-4 was suppressed by overexpression of miR-125b in human OA chondrocytes. In the luciferase reporter assay, mutation of the putative miR-125b binding site in the ADAMTS-4 3'UTR abrogated the suppressive effect of miR125.

Conclusions: Our results indicate that miR-125b plays an important role in regulating the expression of ADAMTS-4 in human chondrocytes and this identifies miR-125b as a novel therapeutic target in OA.

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IL-1β induces ADAMTS-4 expression and suppresses miR-125b expression. (A) Relative expression of miR-125b in normal (n = 3) and osteoarthritic (OA) (n = 7) chondrocytes stimulated with IL-1β for 24 hours. (B) Kinetics of expression of mRNA of ADAMTS-4 and ADAMTS-5 in normal (n = 3) and OA (n = 7) chondrocytes treated with IL-1β for 6, 12 and 24 hours. The data are expressed as mean and SEM of three independent experiments, each of which was run in triplicate. ** = P <0.001 as measured using an unpaired Student's t test. ADAMTS-4, aggrecanase-1; SEM, standard error of the mean.
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Figure 2: IL-1β induces ADAMTS-4 expression and suppresses miR-125b expression. (A) Relative expression of miR-125b in normal (n = 3) and osteoarthritic (OA) (n = 7) chondrocytes stimulated with IL-1β for 24 hours. (B) Kinetics of expression of mRNA of ADAMTS-4 and ADAMTS-5 in normal (n = 3) and OA (n = 7) chondrocytes treated with IL-1β for 6, 12 and 24 hours. The data are expressed as mean and SEM of three independent experiments, each of which was run in triplicate. ** = P <0.001 as measured using an unpaired Student's t test. ADAMTS-4, aggrecanase-1; SEM, standard error of the mean.

Mentions: We examined whether IL-1β affects the expression of ADAMTS-4, ADAMTS-5 and miR-125b. IL-1β treatment suppressed miR-125b expression at 12 and 24 h in normal chondrocytes and at 6,12 and 24 h in OA chondrocytes (P <0.001) (Figure 2A). IL-1β treatment induced ADAMTS-4 expression in a time-dependent manner in both normal and OA chondrocytes (P <0.001). ADAMTS-5 mRNA expression was also increased in a time-dependent manner, but the magnitude of induction was much lower than for ADAMTS-4 mRNA (Figure 2B).


MicroRNA-125b regulates the expression of aggrecanase-1 (ADAMTS-4) in human osteoarthritic chondrocytes.

Matsukawa T, Sakai T, Yonezawa T, Hiraiwa H, Hamada T, Nakashima M, Ono Y, Ishizuka S, Nakahara H, Lotz MK, Asahara H, Ishiguro N - Arthritis Res. Ther. (2013)

IL-1β induces ADAMTS-4 expression and suppresses miR-125b expression. (A) Relative expression of miR-125b in normal (n = 3) and osteoarthritic (OA) (n = 7) chondrocytes stimulated with IL-1β for 24 hours. (B) Kinetics of expression of mRNA of ADAMTS-4 and ADAMTS-5 in normal (n = 3) and OA (n = 7) chondrocytes treated with IL-1β for 6, 12 and 24 hours. The data are expressed as mean and SEM of three independent experiments, each of which was run in triplicate. ** = P <0.001 as measured using an unpaired Student's t test. ADAMTS-4, aggrecanase-1; SEM, standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672767&req=5

Figure 2: IL-1β induces ADAMTS-4 expression and suppresses miR-125b expression. (A) Relative expression of miR-125b in normal (n = 3) and osteoarthritic (OA) (n = 7) chondrocytes stimulated with IL-1β for 24 hours. (B) Kinetics of expression of mRNA of ADAMTS-4 and ADAMTS-5 in normal (n = 3) and OA (n = 7) chondrocytes treated with IL-1β for 6, 12 and 24 hours. The data are expressed as mean and SEM of three independent experiments, each of which was run in triplicate. ** = P <0.001 as measured using an unpaired Student's t test. ADAMTS-4, aggrecanase-1; SEM, standard error of the mean.
Mentions: We examined whether IL-1β affects the expression of ADAMTS-4, ADAMTS-5 and miR-125b. IL-1β treatment suppressed miR-125b expression at 12 and 24 h in normal chondrocytes and at 6,12 and 24 h in OA chondrocytes (P <0.001) (Figure 2A). IL-1β treatment induced ADAMTS-4 expression in a time-dependent manner in both normal and OA chondrocytes (P <0.001). ADAMTS-5 mRNA expression was also increased in a time-dependent manner, but the magnitude of induction was much lower than for ADAMTS-4 mRNA (Figure 2B).

Bottom Line: MiR-125b was expressed in both normal and OA chondrocytes, with significantly lower expression in OA chondrocytes than in normal chondrocytes.Furthermore, IL-1β-induced upregulation of ADAMTS-4 was suppressed by overexpression of miR-125b in human OA chondrocytes.In the luciferase reporter assay, mutation of the putative miR-125b binding site in the ADAMTS-4 3'UTR abrogated the suppressive effect of miR125.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Increased expression of aggrecanase-1 (ADAMTS-4) has emerged as an important factor in osteoarthritis (OA) and other joint diseases. This study aimed to determine whether the expression of ADAMTS-4 in human chondrocytes is regulated by miRNA.

Methods: MiRNA targets were identified using bioinformatics. Chondrocytes were isolated from knee cartilage and treated with interleukin-1 beta (IL-1β). Gene expression was quantified using TaqMan assays and protein production was determined by immunoblotting. Luciferase reporter assay was used to verify interaction between miRNA and target messenger RNA (mRNA).

Results: In silico analysis predicted putative target sequence of miR-125b on ADAMTS-4. MiR-125b was expressed in both normal and OA chondrocytes, with significantly lower expression in OA chondrocytes than in normal chondrocytes. Furthermore, IL-1β-induced upregulation of ADAMTS-4 was suppressed by overexpression of miR-125b in human OA chondrocytes. In the luciferase reporter assay, mutation of the putative miR-125b binding site in the ADAMTS-4 3'UTR abrogated the suppressive effect of miR125.

Conclusions: Our results indicate that miR-125b plays an important role in regulating the expression of ADAMTS-4 in human chondrocytes and this identifies miR-125b as a novel therapeutic target in OA.

Show MeSH
Related in: MedlinePlus