Silencing of microRNA-101 prevents IL-1β-induced extracellular matrix degradation in chondrocytes

Abstract

Introduction: Extracellular matrix (ECM) degradation leads to malfunction of the cartilage in osteoarthritis (OA). Inflammatory cytokine interleukin-1 beta (IL-1β) functions in ECM degradation and prevents ECM synthesis by down-regulating the key transcription factor, Sox9, and consequently inhibiting ECM gene expression. Evidence reveals that microRNAs (miRNA) have been associated with OA, but little is known of their function in chondrocyte ECM degradation. This study aimed to identify possible miRNAs that mediate IL-1β-induced down-regulation of Sox9 as well as its known down-stream genes, collagen type II and aggrecan.

Methods: The miRNAs were predicted based on three classical databases. The expression levels of the predicted miRNAs were assessed in IL-1β stimulated chondrocytes by real-time PCR. A luciferase reporter was used to test the binding of the miRNAs to the 3' untranslated regions (3'UTR) of Sox9. The predicted miRNAs were transfected into chondrocytes to validate their relationship with Sox9. Functional analysis of the miRNAs on chondrocytes ECM degradation was performed at both the mRNA and protein levels after miRNA transfection and IL-1β treatment.

Results: Six miRNAs were predicted to target Sox9, and their expression in IL-1β-stimulated chondrocytes was revealed by real-time PCR. The luciferase reporter assay indicated that only miR-101 could bind to the 3'UTR of Sox9. The expression of Sox9 was likewise negatively regulated by miR-101 in rat chondrocytes. Functional analysis showed that miR-101 could aggravate chondrocyte ECM degradation, whereas miR-101 inhibition could reverse IL-1β-induced ECM degradation.

Conclusion: miR-101 participates in IL-1β-induced chondrocyte ECM degradation. Down-regulating miR-101 expression can prevent the IL-1β-induced ECM degradation in chondrocytes. miR-101 probably functions by directly targeting Sox9 mRNA.

Figure 1

microRNA prediction and its expression in IL-1β-treated chondrocytes. (A) Six microRNAs were selected to target Sox9 using three classic prediction databases. Primary rat chondrocytes were treated with IL-1β (5 ng/ml) for 2, 4 and 6 h. Sox9 expression was analyzed by real-time PCR and western blot. (B and C) Sox9 expression was markedly decreased at the mRNA and protein levels with the increasing duration of IL-1β stimulation. The upper panels (C) are representative images of the western blot analysis. The lower panels are densitometric analyses performed with images of three independent experiments, respectively; n = 3,*P < 0.05 versus 0 h. (D) microRNAs expression in IL-1β-induced chondrocytes was analyzed by real-time PCR; n = 3,*P < 0.05 versus 0 h.

Figure 2

Validation of the targeting effect of microRNAs on Sox9. (A) The diagram illustrates the construction of Sox9 3' UTR luciferase reporter. (B) The conserved sequences of miR-101 and the 3'UTRs of Sox9 in different species are compared. The underlined sequences indicate a sequence complementary in miR-101 to a specific binding site within the 3'UTR of Sox9. The sequences in the frame were used in our study. (C) Luciferase activity of the Sox9 3'UTR reporter was analyzed in HeLa cells. miRNAs were co-transfected with the wild-type Sox9 3' UTR or mutant vector. Scrambled 22 nt mimic (miR-Scr) was used as a negative control; n = 3,*P < 0.05 versus miR-Scr group. MT refers to mutant Sox9 3'UTR reporter. Primary rat chondrocytes were transfected with the miRNA mimic and miRNA inhibitor. miR-Scr was used as a negative control. Sox9 expression was analyzed by real-time PCR and western blot 24 h post miRNA transfection (D and E). Lower panels (E) show the densitometric analysis of Sox9 expression (upper panels), which is normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The analysis was performed using images from three independent experiments; n = 3,*P < 0.05 versus miR-Scr group.

Figure 3

Effects of miR-101 on IL-1β-induced chondrocyte extracellular matrix (ECM) degradation. Primary rat chondrocytes were transfected with miR-Scr, miR-101 mimic, and miR-101 inhibitor, and then treated with or without IL-1β 12 h post-miRNA transfection. (A and B) The expression of collagen type II (A) and aggrecan (B) was evaluated by real-time PCR after transfection of miR-101 mimic or inhibitor with or without IL-1β treatment; n = 3, *P < 0.05. (C) The collagen type II expression of transfected chondrocytes was assessed by immunofluorescence staining method. Original magnification: × 60. Bars = 50 μm. (D) The fluorescence intensity of the images (C) was analyzed using Image-Pro Plus 6.0 software. Data are expressed as the average of at least five images; n = 3, *P < 0.05. (E) The sGAG content in the cell suspension was assessed by dimethylmethylene blue (DMMB) assay after transfection of miR-101 mimic or inhibitor with or without IL-1β treatment. Data were normalized by total protein content of the cell lysate in each group; n = 3, *P < 0.05.

Figure 4

miR-101 function during IL-1β-induced chondrocyte extracellular matrix (ECM) degradation probably occurs through regulation of Sox9. Primary rat chondrocytes were transfected with miR-Scr, miR-101 mimic, and miR-101 inhibitor, and then treated with or without IL-1β 12 h post-miRNA transfection in the same manner as mentioned above. (A and B) Sox9 expression was analyzed by real-time PCR (A) and western blot (B) 24 h post miRNA mimic, inhibitor, or miR-Scr transfection. The right panel (B) is the densitometric analysis of Sox9 expression (left panel), normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The analysis was performed using images of three independent experiments, respectively; n = 3, *P < 0.05. (C and D) Primary rat chondrocytes were co-transfected with miR-Scr or miR-101 mimic together with Sox9 full-length vector (Sox9 expression vector containing both UTR and CDS regions) or Sox9 CDS vector (Sox9 expression vector containing only CDS region, no UTR). Sox9 expression was analyzed by real-time PCR (C) and western blot (D) 24 h post transfection; n = 3,*P < 0.05. (E and F) Primary rat chondrocytes were first transfected with miR-Scr or miR-101 inhibitor, and then siRNA against Sox9 (siSox9) or scrambled siRNA (siScr) were transfected. Sox9 expression was analyzed by western blot (E) 24 h post transfection. The lower panel (E) is the densitometric analysis of Sox9 expression (upper panel), normalized by GAPDH. The analysis was performed using images of three independent experiments, respectively; n = 3,*P < 0.05. (F) The sGAG content in the cell suspension was assessed by dimethylmethylene blue (DMMB) assay. Data were normalized by total protein content in the cell lysate in each group; n = 3,*P < 0.05.