MicroRNA-27b regulates the expression of matrix metalloproteinase 13 in human osteoarthritis chondrocytes.
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-1beta (IL-1beta) 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-kappaB was evaluated using specific inhibitors.


RESULTS
In IL-1beta-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-1beta-induced expression of MMP-13 protein in chondrocytes. NF-kappaB and MAPK activation down-regulated the expression of miR-27b.


CONCLUSIONS
Our data demonstrated the expression of miR-27b in both normal and OA chondrocytes. Furthermore, IL-1beta-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.
School of Medicine, University of South Carolina, Columbia, USA.
Nahid
N
Akhtar
Zafar
Z
Rasheed
Sangeetha
S
Ramamurthy
Arivarasu N
AN
Anbazhagan
Frank R
FR
Voss
Tariq M
TM
Haqqi
D020413:3' Untranslated Regions / Q000235:genetics; D000368:Aged; D060565:Argonaute Proteins; D019902:Chondrocytes / Q000201:enzymology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053487:DEAD-box RNA Helicases / Q000235:genetics; D015852:Eukaryotic Initiation Factor-2 / Q000235:genetics; D039642:Eukaryotic Initiation Factors; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D017930:Genes, Reporter; D006801:Humans; D053583:Interleukin-1beta / Q000494:pharmacology; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D007976:Leupeptins / Q000494:pharmacology; D008156:Luciferases / Q000235:genetics; D053509:Matrix Metalloproteinase 13 / Q000235:genetics* / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics*; D008875:Middle Aged; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D010003:Osteoarthritis / Q000235:genetics* / Q000378:metabolism*; D043244:Ribonuclease III / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D020413:3' Untranslated Regions; C466422:AGO2 protein, human; C517143:AGO3 protein, human; C517144:AGO4 protein, human; D060565:Argonaute Proteins; D015853:Cysteine Proteinase Inhibitors; D015852:Eukaryotic Initiation Factor-2; D039642:Eukaryotic Initiation Factors; D053583:Interleukin-1beta; D007976:Leupeptins; C531388:MIRN27 microRNA, human; D035683:MicroRNAs; D016328:NF-kappa B; D008156:Luciferases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C408087:DICER1 protein, human; D043244:Ribonuclease III; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; D053487:DEAD-box RNA Helicases; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde