Overview

With the aging of the population and the increase of obese people, the prevalence of OA is rising. OA is a degenerative disease characterized by cartilage destruction and loss, subchondral bone thickening and sclerosis, osteophyte formation with varying degrees of synovitis and joint capsule thickening[1][ Choi WS, Lee G, Song WH, Koh JT, Yang J, et al. The CH25H-CYP7B1-RORalpha axis of cholesterol metabolism regulates osteoarthritis. Nature. 2019 Feb;566(7743):254-58.]. Recent studies have found that the expression of OGN in articular cartilage tissues of OA patients is significantly reduced, and OGN is involved in the regulation of matrix metabolism in various tissues[2][ Nulali J, Zhan M, Zhang K, et al. Osteoglycin: An ECM Factor Regulating Fibrosis and Tumorigenesis. Biomolecules. 2022 Nov 11;12(11):1674.]. However, its regulatory role in matrix metabolism of OA chondrocytes needs further exploration. In this study, we will construct a cell model of chondrocyte matrix metabolism disorder induced by inflammatory factor IL-1β, and use the intervention method of gene overexpression to clarify the regulation of OGN expression in chondrocytes by inflammatory factors(Figure 1). To determine the effect of OGN on IL-1β-regulated chondrocyte matrix metabolism.

Figure 1. The subject design idea of osteocalcin

I.pCMV-3Tag-1A-OGN

1)Construction of OGN overexpressed plasmid

Choi WS, Lee G, Song WH, Koh JT, Yang J, et al. The CH25H-CYP7B1-RORalpha axis of cholesterol metabolism regulates osteoarthritis. Nature. 2019 Feb;566(7743):254-58.

Nulali J, Zhan M, Zhang K, et al. Osteoglycin: An ECM Factor Regulating Fibrosis and Tumorigenesis. Biomolecules. 2022 Nov 11;12(11):1674.

In this work, we first constructed OGN overexpressed plasmid. In brief, DNA fragment of OGN was amplified by using the cDNA of ATDC5 cells and then inserted into the pCMV-3Tag-1A vector. The E. coli strains solution transformed with constructed plasmid were spread onto LB solid medium plate containing corresponding antibiotic and cultured at 37℃ overnight (Figure 2).

Figure 2. Monoclonal colonies of E. coli transformed with pCMV-3Tag-1A-OGN plasmid

We selected several monoclonal colonies from the plate for cultivation, and extracted plasmid DNA from the cultured bacterial solution. The oligo DNA in plasmid was identified by using agarose gel electrophoresis (Figure 3A). Our results show that a band of about 5000 bp is present in sample 2 and 8, suggested that pCMV-3Tag-1A-OGN plasmid were constructed successfully. At the same time, we sent the positive monoclonal antibody to the company for sequencing. Figure 3B showed that the gene sequence was correct and there was no mutation. It proves that pCMV-3Tag-1A-OGN plasmid was successfully constructed.

Figure 3. Electrophoretic gel (left) and Sequencing (right) Verification of pCMV-3Tag-1A-OGN plasmid

2)Identification of ITS-induced ATDC5 cells

In order to explore the function of OGN on the matrix metabolism of chondrocytes. We firstly added ITS to the growth medium to induce the differentiation of ATDC5 cells. Since mature chondrocytes contain a large amount of proteoglycan, which can be stained blue by Alcian Blue, we performed related assays in ITS-induced ATDC5 cells. As shown in Figure 4, our cultured ATDC5 cells have differentiated into chondrocytes.

Figure 4. Microscope image of chondrocytes

3)The increase of OGN expression improved matrix metabolism in IL-1β-treated chondrocytes

Next, we established an in vitro research model of OA by using IL-1β as stimulator on chondrocytes and detected the mRNA and protein levels of OGN in the cells by using qRT-PCR and western blotting. As shown in the Figure 5, the mRNA and protein levels of OGN in IL-1β-stimulated chondrocytes were significantly decreased compared with those of control group, that is consistent with the clinical observation that OGN expression in chondrocytes from osteoarthritis patients is reduced. Moreover, both the mRNA and protein levels of OGN in chondrocytes transfected with previously constructed pCMV-3Tag-1A-OGN plasmid were notably increased compared to the mock group, further indicating the overexpressed plasmid was successfully constructed and worked successfully.

Figure 5. pCMV-3Tag-1A-OGN plasmid antagonisted IL-1β-induced inhibition on OGN expression in chondrocytes (A) qRT-PCR results; (B) western blot results

Additionally, we investigated whether OGN participates in the pathogenesis of OA. As shown in Figure 6, mRNA levels of matrix metabolism-related genes such as Aggrecan, Col-2 and Sox9 were downregulated, while MMP3 and MMP13 mRNA levels were upregulated in IL-1β-induced chondrocytes. These results indicated the disruption of matrix metabolism in chondrocytes. Notably, OGN overexpression antagonized such IL-1β-induced effects in chondrocytes. Thus, all these results suggests that OGN is a promising therapeutic target for OA.

Figure 6. OGN overexpression antagonisted IL-1β-induced matrix metabolism disorder in chondrocytes

II.RNAi-ReadypSIREN-RetroQ-ShRNA-1;RNAi-ReadypSIREN-RetroQ-ShRNA-2; RNAi-Ready pSIREN-RetroQ-ShRNA-N

1)Construction of OGN gene silencing plasmids

Moreover, we designed and synthesized two interference sequences of OGN gene for the construction of OGN gene silencing plasmids and performed similar experiments to explore whether the OGN is involved in the initiation and progression of OA.

Briefly, the forward and reverse sequences of ShRNA were connected by annealing. The obtained DNA fragments was inserted into the RNAi-Ready pSIREN-RetroQ vector. The E. coli strains solution transformed with constructed plasmid were spread onto LB solid medium plate containing corresponding antibiotic and cultured at 37℃ overnight (Figure 7).

Figure 7. Monoclonal colonies of E. coli transformed with OGN gene silencing plasmids (A) RNAi-Ready pSIREN-RetroQ-ShRNA-1; (B) RNAi-Ready pSIREN-RetroQ-ShRNA-2; (C) RNAi-Ready pSIREN-RetroQ-ShRNA-N

We selected several monoclonal colonies from the plate for cultivation, and extracted plasmid DNA from the cultured bacterial solution. The oligo DNA in plasmid was identified by using agarose gel electrophoresis (Figure 8A). At the same time, we sent the positive monoclonal antibody to the company for sequencing. Figure 8 B. C.D showed that the gene sequence was correct and there was no mutation.

It proves that RNAi-Ready pSIREN-RetroQ-ShRNA-1, RNAi-Ready pSIREN-RetroQ-ShRNA-2, RNAi-Ready pSIREN-RetroQ-ShRNA-N plasmid were successfully constructed.

Figure 8. Electrophoretic gel (A) and Sequencing (B.C.D) Verification of RNAi-Ready pSIREN-RetroQ plasmid

4)OGN gene silencing can induce matrix metabolism disorder in chondrocytes

As shown in Figure 9 and Figure 10, the mRNA and protein levels of OGN were significantly decreased in IL-1βgroup and OGN-Sh-1, 2 group compared with those of Control group or OGN-Sh-N group. In addition, the results of qRT-PCR revealed that the decrease of OGN expression can induce the disruption of matrix metabolism in chondrocytes, indicative of the regulatory role of OGN in OA.

Figure 9. RNAi-Ready pSIREN-RetroQ-ShRNA-1 and 2 plasmids inhibited the expression of OGN in chondrocytes (A) qRT-PCR results; (B) western blot resultsFigure 8. Electrophoretic gel (A) and Sequencing (B.C.D) Verification of RNAi-Ready pSIREN-RetroQ plasmid

Figure 10. The decrease of OGN expression induced matrix metabolism disorder in chondrocytes

References and sources

Choi WS, Lee G, Song WH, Koh JT, Yang J, et al. The CH25H-CYP7B1-RORalpha axis of cholesterol metabolism regulates osteoarthritis. Nature. 2019 Feb;566(7743):254-58.

Nulali J, Zhan M, Zhang K, et al. Osteoglycin: An ECM Factor Regulating Fibrosis and Tumorigenesis. Biomolecules. 2022 Nov 11;12(11):1674.