Open AccessMicroRNA‐27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytes
Author(s) -
Akhtar Nahid,
Rasheed Zafar,
Ramamurthy Sangeetha,
Anbazhagan Arivarasu N.,
Voss Frank R.,
Haqqi Tariq M.
Publication year - 2010
Publication title -
arthritis & rheumatism
Language(s) - English
Resource type - Journals
eISSN - 1529-0131
pISSN - 0004-3591
DOI - 10.1002/art.27329
Subject(s) - microrna , dicer , microbiology and biotechnology , messenger rna , gene expression , three prime untranslated region , untranslated region , biology , reporter gene , transfection , chemistry , small interfering rna , gene , genetics
Objective Aberrant posttranscriptional regulation of matrix metalloproteinases (MMPs) by microRNA has emerged as an important factor in human diseases. The aim of this study was to determine whether the expression of MMP‐13 in human osteoarthritis (OA) chondrocytes is regulated by microRNA. Methods Chondrocytes were stimulated with interleukin‐1β (IL‐1β) in vitro. Total RNA was prepared using TRIzol reagent. Polymerase chain reaction (PCR)–based arrays were used to determine the expression profile of 352 human microRNA. Gene expression was quantified using TaqMan assays, and microRNA targets were identified using bioinformatics. Transfection with reporter construct and microRNA mimic was used to verify suppression of target messenger RNA (mRNA). Gene expression of argonaute and Dicer was determined by reverse transcription–PCR, and expression of protein was determined by immunoblotting. The role of activated MAP kinases (MAPKs) and NF‐κB was evaluated using specific inhibitors. Results In IL‐1β–stimulated OA chondrocytes, 42 microRNA were down‐regulated, 2 microRNA were up‐regulated, and the expression of 308 microRNA remained unchanged. In silico analysis identified a sequence in the 3′‐untranslated region (3′‐UTR) of MMP‐13 mRNA complementary to the seed sequence of microRNA‐27b (miR‐27b). Increased expression of MMP‐13 correlated with down‐regulation of miR‐27b. Overexpression of miR‐27b suppressed the activity of a reporter construct containing the 3′‐UTR of human MMP‐13 mRNA and inhibited the IL‐1β–induced expression of MMP‐13 protein in chondrocytes. NF‐κB and MAPK activation down‐regulated the expression of miR‐27b. Conclusion Our data demonstrated the expression of miR‐27b in both normal and OA chondrocytes. Furthermore, IL‐1β–induced activation of signal transduction pathways associated with the expression of MMP‐13 down‐regulated the expression of miR‐27b. Thus, miR‐27b may play a role in regulating the expression of MMP‐13 in human chondrocytes.