MicroRNA-9 promotion of interleukin-6 expression by inhibiting monocyte chemoattractant protein-induced protein 1 expression in interleukin-1β-stimulated human chondrocytes

Abstract

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.

Conclusion: 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.

Figure 1. MCPIP1 expression is decreased in OA cartilage and induced upon IL-1β treatment in chondrocytes

(A) RNA was isolated from damaged or smooth cartilage and MCPIP1 mRNA levels were determined by TaqMan assay. Each dot represents the value from a single patient sample. (B) Chondrocytes were serum starved for 12 hr and then stimulated with 2ng/ml IL-1β for the indicated time pointsand MCPIP1 and IL-6 mRNA level was measured by TaqMan assay with expression of β-Actin as endogenous control. (C) Chondrocytes were treated with increasing doses of IL-R antagonist in presence or absence of IL-1β. MCPIP1 expression was measured after 16 hrs incubation as above. (D) Cell lysates were prepared and IL-6 or MCPIP1 protein levels were determined by Western blotting using antibodies specific for IL-6 and MCPIP1. Blots were probed with anti β-Actin antibody to ensure equal loading. (E) Supernatants from the control or stimulated cells were analyzed by a sandwich ELISA to determine the secreted IL-6 concentration. (F) For In-situ hybridization chondrocytes were induced for the indicated time points and RNA hybridization was performed on formalin fixed cells using IL-6 or MCPIP1 specific probes. Fluorescence was visualized using Olympus confocal microscope. Data are represented as mean ±SD of three experiments performed in duplicates using at least three patients samples. *p< 0.05, **p<0.005.

Figure 2. MCPIP1 knockdown or overexpression alters IL-6 expression in OA chondrocytes

(A) Chondrocytes were transfected with non-targeted siRNA or siRNA against MCPIP1. Twenty-four hr post-transfection chondrocytes were induced by IL-1β for 8 hr and then the expression of MCPIP1, IL-6, MMP13, COL2A1, ACAN, MMP3 or COL10A1 was measured by TaqMan Assay. (B) Wild type or mutant MCPIP1 encoding cDNA constructs were transfected by electroporation into primary chondrocytes and 24 hr later were stimulated for 12 hr and then the expression of indicated genes was determined. Overexpression of Wild type and the mutant form is shown by Western blot using anti-MCPIP1 antibody. (C) Endogenous MMP13 expression was measured by Western blot upon knockdown or overexpression of MCPIP1 as in B. (D) Chondrocytes were transfected as in A and B and induced by IL-1β and gene expression of IL-10 or IL-1RA was measured by TaqMan assay. (E) Primary chondrocytes were transfected separately by non-targeted siRNA, siRNA against MCPIP1, vector control or wild type MCPIP1 cDNA construct and treated as above and then challenged with 4μM ActD for the indicated time points. IL-6 mRNA expression is represented as percentage of remaining IL-6 mRNA. Data are mean ±SD of three experiments performed in duplicates using at least three patient samples. *p< 0.05, **p<0.005.

Figure 3. MCPIP1 interact with IL-6 mRNA in OA chondrocytes

Primary chondrocytes were stimulated for 8 hr with IL-1β in vitro. Cell lysates were subjected to RIP (RNA immune-precipitation assay) using IgG control antibody or anti-MCPIP1 antibody. Immunoprecipitated RNA was reverse transcribed and was used to measure the expression of IL-6 or MCPIP1 mRNA by TaqMan assay. Data are represented as mean ±SD of three experiments performed in duplicates using at least three patients samples. **p<0.005.

Figure 4. Expression of miR-9 was upregulated in damaged cartilage and induced in chondrocytes upon IL-1β or IL-6 stimulation

(A) miR-9 expression was directly assessed in damage or smooth cartilage. Each dot represents the value from a single patient sample. (B) Chondrocytes were serum starved for 12 hr and then stimulated with 2ng/ml IL-1β for the indicated time points in the presence or absence of IL-1RA (C) or (D) IL-6 for 24 hr and then RNA was extracted and miR-9 expression was measured by TaqMan Assay. Data are represented as Mean ±SD of three experiments performed in duplicates using at least three patients samples. **p<0.005.

Figure 5. miR-9 directly targets the seed sequence in the 3’UTR of MCPIP1 mRNA

(A) Overexpression or inhibition of miR-9 expression was measured by TaqMan assay. Expression of RNU6B was used as endogenous control. (B) Primary OA chondrocytes were transfected with 100 nM each of miR-9 mimic or miR-9 inhibitor by electroporation. Cells were induced for 36 hr and expression of miR-9, MCPIP1 and IL-6 was quantified by TaqMan assays. Protein level was measured by Western blotting using anti-MCPIP1 or anti-IL-6 antibody. Expression of β-Actin was used as an internal control. (C) Culture supernatant from (B) was used to determine the IL-6 protein level by sandwich ELISA. (D) miR-9 seed sequence are indicated in gray box which is complement to the position 148-155 of MCPIP1 3’ UTR. Arrows indicate nucleotide changes (in bold) present in the MCPIP1 mutant 3’UTR. MCPIP1 3’UTR luciferase reporter construct (WT-left panel or mutant-right panel) was transfected in OA chondrocytes alone or co-transfected with miR-9 mimic and 24 hr post-transfection Luciferase activity was measured using the dual reporter assay. Renilla luciferase was co-transfected for normalization purpose. Data are represented as Mean ±SD of three experiments performed in duplicates using at least three patients samples. *p<0.05, **p<0.005.