Overview

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

Cycle 1: BBa_K4840002 (pCMV-3Tag-1A-OGN)

Design

We selected the glycoprotein-Osteoglycin ( OGN ), which is involved in the regulation of matrix metabolism in various tissues. At the same time, the restriction sites EcoRI and NotI were added at both ends of the primer. At the same time, in order to be successfully transfected into cells, we selected the plasmid vector pCMV-3Tag-1A, digested OGN and plasmid vector pCMV-3Tag-1A, and connected OGN and plasmid vector pCMV-3Tag-1A by T4 ligase. Finally, we constructed the overexpression plasmid of OGN , named pCMV-3Tag-1A-OGN ( Figure 2 ).

Figure 2. The map of pCMV-3Tag-1A-OGN plasmid

Build

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 3).

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

We randomly 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 4A). 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 4B showed that the gene sequence was correct and there was no mutation. It proves that pCMV-3Tag-1A-OGN plasmid was successfully constructed.

Figure 4. Electrophoretic gel (left) and Sequencing (right) Verification of pCMV-3Tag-1A-OGN plasmid

Test

Identification of ITS-induced ATDC5 cell

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 5, our cultured ATDC5 cells have differentiated into chondrocytes.

Figure 5. Identification of chondrocytes by microscope

2.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 6, 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.

Figure 6. 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 7, 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 results suggests that OGN is a promising therapeutic target for OA.

Figure 7. OGN overexpression antagonisted IL-1β-induced matrix metabolism disorder in chondrocytes

Learn

All results suggests that OGN is a promising therapeutic target for OA. In the future, we can optimize the vector of OGN. First, OGN plasmid cannot be directly used to overexpress OGN in chondrocytes in vivo. It is necessary to find a suitable delivery vector to carry it into cells. Second, we find a suitable chondrocyte targeting carrier to make OGN highly expressed in chondrocytes.In addition, it can further study the pathogenesis of OA and explore new treatment strategies for OA by using new drug targets of OGN, which has important clinical significance and social value.

Cycle2: BBa_K4840008 ~ BBa_K4840010

(RNAi-ReadypSIREN-RetroQ--ShRNA-1;RNAi-ReadypSIREN-RetroQ-ShRNA-2; RNAi-Ready pSIREN-RetroQ-ShRNA-N)

Design

We selected the glycoprotein-Osteoglycin (OGN)，and further constructed knockout plasmid of OGN. At the same time, in order to be successfully transfected into cells, we selected the plasmid vector RNAi-ReadypSIREN-RetroQ. We were designed two interference sequences (ShRNA-1, ShRNA-2 ) and a negative control sequence (ShRNA-N ) by Ambion siRNA Target Finder. At the same time, the restriction endonuclease BamHI and EcoRI sites were designed at both ends of the sequence. The RNAi-ReadypSIREN-RetroQ and ShRNA-1, ShRNA-2 , ShRNA-N were ligated using T4 ligase, respectively named RNAi-ReadypSIREN-RetroQ--ShRNA-1; RNAi-ReadypSIREN-RetroQ-ShRNA-2; RNAi-Ready pSIREN-RetroQ-ShRNA-N ( Figure 8 ABC).

Figure 8 . The map of RNAi-ReadypSIREN-RetroQ--ShRNA-1; RNAi-ReadypSIREN-RetroQ-ShRNA-2; RNAi-Ready pSIREN-RetroQ-ShRNA-N

Build

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 9).

Figure 9. 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 randomly 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 10A). At the same time, we sent the positive monoclonal antibody to the company for sequencing. Figure 10 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 10. Electrophoretic gel (A) and Sequencing (B.C.D) Verification of RNAi-Ready pSIREN-RetroQ plasmid

Test

1.OGN gene silencing can induce matrix metabolism disorder in chondrocytes

As shown in Figure 11 and Figure 12, 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 11. RNAi-Ready pSIREN-RetroQ-ShRNA-1 and 2 plasmids inhibited the expression of OGN in chondrocytes (A) qRT-PCR results; (B) western blot results

Figure 12. The decrease of OGN expression induced matrix metabolism disorder in chondrocytes

Learn

OGN-Sh-1, and OGN-Sh-2 gene silencing have a significant effect on OGN. In the future, we can design more gene silencing sequences to further study the effect of different silencing sequences on OGN expression and try to find the optimal silencing sequence. In order to clarify whether OGN is involved in the regulation of IL-1β on chondrocyte ECM metabolism ; verify the potential application of OGN as a functional target for relieving OA and provide data support. It provides new ideas for curing arthritis with high morbidity and high disability rate, and brings huge medical care burden and economic burden to the whole society.