MicroRNA-9 promotion of interleukin-6 expression by inhibiting monocyte chemoattractant protein-induced protein 1 expression in interleukin-1β-stimulated human chondrocytes.
OBJECTIVE
Enhanced expression of interleukin-6 (IL-6) plays an important role in the pathogenesis of osteoarthritis (OA). Monocyte chemoattractant protein-induced protein 1 (MCPIP-1) is a novel posttranscriptional regulator of IL-6 expression and is targeted by microRNA-9 (miR-9). We investigated the expression of MCPIP-1 in OA cartilage and explored whether targeting of MCPIP-1 by miR-9 contributes to enhanced IL-6 expression in OA.


METHODS
Gene and protein expression in IL-1β-stimulated human OA chondrocytes/cartilage was determined by TaqMan assay and immunoblotting, respectively. Messenger RNA (mRNA) for MCPIP-1 and IL-6 expression at the single-cell level was analyzed using RNAscope. MCPIP-1 protein interaction with IL-6 mRNA was investigated using RNA immunoprecipitation. Transient transfections were used for the small interfering RNA (siRNA)-mediated knockdown and overexpression of MCPIP-1, its RNase-defective mutant miR-9, or antagomir. The role of signaling pathways was evaluated using small-molecule inhibitors. Binding of miR-9 with the "seed sequence" in the 3'-untranslated region of MCPIP-1 mRNA was investigated using a luciferase reporter assay.


RESULTS
MCPIP-1 mRNA expression was low, but expression of miR-9 and IL-6 was high, in damaged OA cartilage. In IL-1β-stimulated OA chondrocytes, the expression of miR-9 and MCPIP-1 was mutually exclusive, and increased expression of miR-9 correlated with reduced MCPIP-1 expression and enhanced IL-6 expression. MCPIP-1 protein directly binds with IL-6 mRNA, and overexpression of wild-type MCPIP-1 destabilized the IL-6 mRNA. MCPIP-1 expression was altered by overexpression or inhibition of miR-9. Transfection with miR-9 mimics inhibited the reporter activity, and mutation of the "seed sequence" abolished the repression of reporter activity.


CONCLUSIONS
These findings implicate miR-9-mediated suppression of MCPIP-1 in the pathogenesis of OA via up-regulation of IL-6 expression in IL-1β-stimulated human OA chondrocytes.
Northeast Ohio Medical University, Rootstown.
Mohammad S
MS
Makki
Northeast Ohio Medical University, Rootstown.
Abdul
A
Haseeb
Northeast Ohio Medical University, Rootstown.
Tariq M
TM
Haqqi
D000368:Aged; D002358:Cartilage, Articular / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D019902:Chondrocytes / Q000187:drug effects / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D047468:Immunoprecipitation; D053583:Interleukin-1beta / Q000494:pharmacology; D015850:Interleukin-6 / Q000235:genetics* / Q000378:metabolism; D035683:MicroRNAs / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D008875:Middle Aged; D015207:Osteoarthritis, Hip / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000187:drug effects / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism
C508585:IL1B protein, human; C508600:IL6 protein, human; D053583:Interleukin-1beta; D015850:Interleukin-6; C541874:MIRN92 microRNA, human; D035683:MicroRNAs; D012333:RNA, Messenger; D014157:Transcription Factors; D012260:Ribonucleases; C510646:ZC3H12A protein, human