Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes.
BACKGROUND
MMP-13 and IGFBP-5 are important factors involved in osteoarthritis (OA). We investigated whether two highly predicted microRNAs (miRNAs), miR-140 and miR-27a, regulate these two genes in human OA chondrocytes.


METHODS
Gene expression was determined by real-time PCR. The effect of each miRNA on IGFBP-5 and MMP-13 expression/production was evaluated by transiently transfecting their precursors (pre-miRNAs) and inhibitors (anti-miRNAs) into human OA chondrocytes. Modulation of IGFBP-5, miR-140 and miR-27a expression was determined upon treatment of OA chondrocytes with cytokines and growth factors.


RESULTS
IGFBP-5 was expressed in human chondrocytes with its level significantly lower (p < 0.04) in OA. Five computational algorithms identified miR-140 and miR-27a as possible regulators of MMP-13 and IGFBP-5 expression. Data showed that both miRNAs were expressed in chondrocytes. There was a significant reduction (77%, p < 0.01) in miR-140 expression in OA compared to the normal chondrocytes, whereas miR-27a expression was only slightly decreased (23%). Transfection with pre-miR-140 significantly decreased (p = 0.0002) and with anti-miR-140 significantly increased (p = 0.05) IGFBP-5 expression at 24 hours, while pre-miR-27a did not affect either MMP-13 or IGFBP-5. Treatment with anti-miR-27a, but not with anti-miR-140, significantly increased the expression of both MMP-13 (p < 0.05) and IGFBP-5 (p < 0.01) after 72 hours of incubation. MMP-13 and IGFBP-5 protein production followed the same pattern as their expression profile. These data suggest that IGFBP-5 is a direct target of miR-140, whereas miR-27a down-regulates, likely indirectly, both MMP-13 and IGFBP-5.


CONCLUSIONS
This study is the first to show the regulation of these miRNAs in human OA chondrocytes. Their effect on two genes involved in OA pathophysiology adds another level of complexity to gene regulation, which could open up novel avenues in OA therapeutic strategies.
Tardif
Ginette
G
Osteoarthritis Research Unit, University of Montreal Hospital Research Centre (CRCHUM), Notre-Dame Hospital, Montreal, Quebec H2L 4M1, Canada. tardifgi@yahoo.com
Hum
David
D
Pelletier
Jean-Pierre
JP
Duval
Nicolas
N
Martel-Pelletier
Johanne
J
D020413:3' Untranslated Regions; D000368:Aged; D019645:Arthroplasty, Replacement, Knee; D001665:Binding Sites; D016022:Case-Control Studies; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism*; D019295:Computational Biology; D016207:Cytokines / Q000378:metabolism; D030541:Databases, Genetic; D005786:Gene Expression Regulation; D006801:Humans; D018975:Insulin-Like Growth Factor Binding Protein 5 / Q000235:genetics* / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D053509:Matrix Metalloproteinase 13 / Q000235:genetics* / Q000378:metabolism; D035683:MicroRNAs / Q000378:metabolism*; D008875:Middle Aged; D020370:Osteoarthritis, Knee / Q000235:genetics* / Q000378:metabolism / Q000601:surgery; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D012720:Severity of Illness Index; D013997:Time Factors; D014162:Transfection
D020413:3' Untranslated Regions; D016207:Cytokines; D018975:Insulin-Like Growth Factor Binding Protein 5; D036341:Intercellular Signaling Peptides and Proteins; C531388:MIRN27 microRNA, human; D035683:MicroRNAs; C544302:Mirn140 microRNA, human; D012333:RNA, Messenger; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13