Regulation of the IGFBP-5 and MMP-13 genes by the microRNAs miR-140 and miR-27a in human osteoarthritic chondrocytes

Abstract

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.

Conclusion: 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.

Figure 1

IGFBP-5 expression and regulation in human chondrocytes. (A) Total RNA was extracted from normal (n = 6) and OA (n = 6) human chondrocytes and processed for real-time PCR/SybrGreen. (B) Human OA chondrocytes (n = 5) were treated with cytokines and growth factors; total RNA was extracted and processed for real-time PCR/SybrGreen. Levels of the untreated (CTL) cells were given an arbitrary value of 1.

Figure 2

Predicted recognition sequences for miR-140 and miR-27a in the 3'-UTR of the MMP-13 and IGFBP-5 mRNAs. The 3'-UTRs were analyzed by computational programs to predict the presence of functional miR-140 and miR-27a sites. (A) Schematic representation of the mRNAs; ORF (open reading frame), ATG (start codon), TAA and TGA (stop codons), bp (base pair). The numbering starts at the first base following the stop codon. The arrows indicate the location of the potential pairing sites for the miRNAs. (B) Sequences of the potential pairing sites for miR-27a and miR-140. The bars indicate base pair homology.

Figure 3

Effect of pre- and anti-miR-140 and miR-27a on MMP-13 gene expression. OA chondrocytes (n = 8) were transfected with the pre-miR (A) and anti-miR (B) molecules and incubated for 24, 48 and 72 hours. Total RNA was extracted and processed for real-time PCR/TaqMan. Levels from untreated chondrocytes (-) were assigned an arbitrary value of 1.

Figure 4

Effect of pre- and anti-miR-140 and -27a on IGFBP-5 gene expression levels. OA chondrocytes (n = 7-8) were transfected with the pre-miR (A) and anti-miR (B) molecules and incubated for 24, 48 and 72 hours. Total RNA was extracted and processed for real-time PCR/TaqMan. Levels from untreated chondrocytes (-) were assigned an arbitrary value of 1.

Figure 5

Expression and regulation of miR-27a and miR-140 levels in human chondrocytes. (A) Total RNA was extracted from normal (n = 6) and OA (n = 6) human chondrocytes and processed for real-time PCR/TaqMan. (B) OA chondrocytes (n = 5) were treated with cytokines and growth factors and miRNAs were extracted and processed for real-time PCR/TaqMan. Levels of the untreated (CTL) cells were given an arbitrary value of 1.