The molecular mechanism study of COMP involved in the articular cartilage damage of Kashin-Beck disease

Abstract

Aims: Kashin-Beck disease (KBD) is a kind of chronic osteochondropathy, thought to be caused by environmental risk factors such as T-2 toxin. However, the exact aetiology of KBD remains unclear. In this study, we explored the functional relevance and biological mechanism of cartilage oligosaccharide matrix protein (COMP) in the articular cartilage damage of KBD.

Methods: The articular cartilage specimens were collected from five KBD patients and five control subjects for cell culture. The messenger RNA (mRNA) and protein expression levels were detected by quantitative reverse transcription PCR (qRT-PCR) and western blot. The survival rate of C28/I2 chondrocyte cell line was detected by MTT assay after T-2 toxin intervention. The cell viability and mRNA expression levels of apoptosis related genes between COMP-overexpression groups and control groups were examined after cell transfection.

Results: The mRNA and protein expression levels of COMP were significantly lower in KBD chondrocytes than control chondrocytes. After the T-2 toxin intervention, the COMP mRNA expression of C28/I2 chondrocyte reduced and the protein level of COMP in three intervention groups was significantly lower than in the control group. MTT assay showed that the survival rate of COMP overexpression KBD chondrocytes were notably higher than in the blank control group. The mRNA expression levels of Survivin, SOX9, Caspase-3, and type II collagen were also significantly different among COMP overexpression, negative control, and blank control groups.

Conclusion: Our study results confirmed the functional relevance of COMP with KBD. COMP may play an important role in the excessive chondrocytes apoptosis of KBD patients.Cite this article: Bone Joint Res 2020;9(9):578-586.

Keywords: Apoptosis; COMP; Chondrocyte; Kashin-Beck disease; T-2 toxin.

Fig. 1

Relative expression ratio of cartilage oligosaccharide matrix protein (COMP) in messenger RNA (mRNA) and protein scales between Kashin-Beck disease (KBD) patients and controls. a) Relative expression ratio of COMP mRNA between KBD patients and normal controls. Compared with the control group, **p < 0.01. b) Relative protein expression ratio of average integrated optical density (IOD) value extracted from western blot band of COMP between KBD patients and controls. IOD compared with the control group, **p < 0.01. c) The western blot band of COMP and reference protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in four KBD patients and four control subjects.

Fig. 2

Cartilage oligosaccharide matrix protein (COMP) messenger RNA (mRNA) and protein relative expression ratio under different concentrations of T-2 toxin intervention. a) COMP mRNA relative expression ratio under different concentrations of T-2 toxin intervention. Note: Compared with the control group, *p < 0.05. Using Dunnett-t test, 2 ng/ml and 5 ng/ml T-2 toxin concentration intervention group were statistically significant compared to 0 ng/ml toxin intervention group. b) COMP protein relative expression of integrated optical density (IOD) values under different T-2 toxin concentrations. Note: Using Dunnett-t test, the values in 1 ng/ml, 2 ng/ml, and 5 ng/ml T-2 toxin concentration intervention groups were found to be statistically significant compared with the 0 ng/ml toxin intervention group, respectively, **p < 0.01. c) The western blot band of COMP and reference protein in four groups with different T-2 toxin treatment concentrations. GADPH, glyceraldehyde-3-phosphate dehydrogenase.

Fig. 3

a) Relative expression ratio of cartilage oligosaccharide matrix protein (COMP) overexpressing lentivirus in articular chondrocytes among the blank control, negative control, and COMP overexpression groups. Compared with the COMP overexpression group, the mean COMP expression levels in the blank control group and negative control group were reduced significantly (7.25, SD 4.69 and 10.12, SD 2.86, respectively; all **p < 0.001, Dunnett-t test). b) Effect of COMP overexpression of lentivirus on the activity of articular chondrocytes in Kashin-Beck disease (KBD) among the blank control, negative control, and COMP overexpression groups. Compared with the COMP overexpression group, the mean survival rate of KBD chondrocytes in the blank control group was reduced (1.31, SD 0.22; *p < 0.05, Dunnett-t test).

Fig. 4

Effects of cartilage oligosaccharide matrix protein (COMP) overexpression on downstream genes in Kashin-Beck disease (KBD) chondrocyte model (the COMP overexpression was only performed for the KBD cells) among the blank control, negative control, and COMP overexpression groups. a) Compared with the COMP overexpression group, the Survivin mean messenger RNA (mRNA) relative expression ratios in the blank control group and negative control group were reduced (8.51, SD 2.88 and 6.77, SD 3.60, respectively; all **p < 0.001, Dunnett-t test). b) Compared with the COMP overexpression group, the sex-determining region Y-box 9 (SOX9) mean mRNA relative expression ratios in the blank control group and negative control group were reduced (5.61, SD 0.87 and 7.71, SD 2.17, respectively; **p < 0.001 , Dunnett-t test). c) Compared with the COMP overexpression group, the Caspase 3 mean mRNA relative expression ratios in the blank control group and negative control group were increased (0.38, SD 0.05 and 0.38, SD 0.04, respectively; all **p < 0.001, Dunnett-t test). d) Compared with the COMP overexpression group, the type II collagen mean mRNA relative expression ratios in the blank control group and negative control group were reduced (4.72, SD 2.17 and 5.66, SD 1.72, respectively; all *p < 0.050, Dunnett-t test). e) Effect on x-linked inhibitor of apoptosis protein (XIAP) (p = 0.948, one-way analysis of variance (ANOVA)). f) Effect on Fas (p = 0.095, ANOVA). g) Effect on Bcl-2-associated x protein (BAX) (p = 0.347, ANOVA). h) Effect on MMP13 (p = 0.263, ANOVA).