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Role of cysteine‑rich angiogenic inducer 61 in fibroblast‑like synovial cell proliferation and invasion in rheumatoid arthritis.

Jie LG, Huang RY, Sun WF, Wei S, Chu YL, Huang QC, Du HY - Mol Med Rep (2014)

Bottom Line: The study found that the Cyr61 gene was highly expressed in the synovial cells from patients with RA compared with those from patients with OA.Conversely, overexpression of Cyr61 in normal FLS cells led to opposite effects.In conclusion, these results indicate that Cyr61 is capable of promoting RA‑FLS cell proliferation and invasion via the suppression of apoptosis and the regulation of MMP expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Chinese Medicine, Guangzhou General Hospital of Guangzhou Command of PLA, Guangzhou, Guangdong 510010, P.R. China.

ABSTRACT
Cysteine‑rich angiogenic inducer 61 (Cyr61) is a novel molecule that has been shown to be increased in the synovial tissues of patients with rheumatoid arthritis (RA). The present study was conducted in order to investigate the role of Cyr61 in the pathogenesis of RA. A human genome‑wide gene assay was used to screen gene expression in synovial tissues obtained from four patients with RA and three patients with osteoarthritis (OA). To examine the role of Cyr61 in the phenotype of RA‑fibroblast‑like synovial (FLS) cells, Cyr61 expression in RA‑FLS cells was knocked down using small interfering RNA (siRNA). Normal FLS cells transduced with lentiviral vectors encoding Cyr61 cDNA were used to further explore the effects of this molecule on FLS cell apoptosis, proliferation and invasion. The study found that the Cyr61 gene was highly expressed in the synovial cells from patients with RA compared with those from patients with OA. Downregulation of Cyr61 by siRNA led to impaired cell proliferation and invasion. Furthermore, it decreased the levels of matrix metalloproteinase (MMP)‑3 and MMP‑13, and induced apoptosis in RA‑FLS cells. Conversely, overexpression of Cyr61 in normal FLS cells led to opposite effects. In conclusion, these results indicate that Cyr61 is capable of promoting RA‑FLS cell proliferation and invasion via the suppression of apoptosis and the regulation of MMP expression. Therefore, Cyr61 may be a good target molecule for the treatment and prevention of RA.

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Role of Cyr61 in FLS cell proliferation. (A) Reverse transcription-quantitative polymerase chain reaction evaluation for the efficacy of Cyr61 siRNA or cDNA transfection in FLS cells. The mRNA level of Cyr61 in normal FLS cells was used as a control. **P<0.01. (B) Western blotting evaluation for the efficacy of Cyr61 siRNA or cDNA transfection in FLS cells. Cyr61 (40 kDa) was detected using a mouse IgG1 monoclonal antibody specific for Cyr61. GAPDH served as the loading control. (C) Detection of levels of Cyr61 secreted into the culture medium using enzyme-linked imunosorbent assays. *P<0.05 and **P<0.01. (D) MTS assay detection of cell proliferation. Data are presented as the mean ± standard deviation of three independent experiments conducted in triplicate. *P<0.05 and **P<0.01, compared with RA-NC; #P<0.05 and ##P<0.01, compared with NOR-NC; ^P<0.05, compared with NOR-FLA. RA, rheumatoid arthritis; FLS, fibroblast-like synoviocytes; siRNA, small interfering RNA; NOR-FLS, normal FLS cells; NOR-CYR61, normal FLS cells transduced with lentivirus vector encoding Cyr61 cDNA; NOR-NC, normal FLS cells transduced with control lentivirus vector; RA-NC, RA-FLS cells transfected with control siRNA; RA-siRNA, RA-FLS cells transfected with Cyr61-siRNA.
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f2-mmr-11-02-0917: Role of Cyr61 in FLS cell proliferation. (A) Reverse transcription-quantitative polymerase chain reaction evaluation for the efficacy of Cyr61 siRNA or cDNA transfection in FLS cells. The mRNA level of Cyr61 in normal FLS cells was used as a control. **P<0.01. (B) Western blotting evaluation for the efficacy of Cyr61 siRNA or cDNA transfection in FLS cells. Cyr61 (40 kDa) was detected using a mouse IgG1 monoclonal antibody specific for Cyr61. GAPDH served as the loading control. (C) Detection of levels of Cyr61 secreted into the culture medium using enzyme-linked imunosorbent assays. *P<0.05 and **P<0.01. (D) MTS assay detection of cell proliferation. Data are presented as the mean ± standard deviation of three independent experiments conducted in triplicate. *P<0.05 and **P<0.01, compared with RA-NC; #P<0.05 and ##P<0.01, compared with NOR-NC; ^P<0.05, compared with NOR-FLA. RA, rheumatoid arthritis; FLS, fibroblast-like synoviocytes; siRNA, small interfering RNA; NOR-FLS, normal FLS cells; NOR-CYR61, normal FLS cells transduced with lentivirus vector encoding Cyr61 cDNA; NOR-NC, normal FLS cells transduced with control lentivirus vector; RA-NC, RA-FLS cells transfected with control siRNA; RA-siRNA, RA-FLS cells transfected with Cyr61-siRNA.

Mentions: Due to the fact that Cyr61 was found to be overexpressed in synovial tissues, its involvement in the pathophysiological events associated with RA was investigated. RA-FLS cells were transfected with Cyr61-siRNA or control-siRNA, and effects on in vitro proliferation, apoptosis and invasion were determined. In vitro experiments were performed in RA-FLS cells without treatment and normal FLS cells. In addition, normal FLS cells were transduced with lentivirus vectors encoding Cyr61 cDNA or control lentivirus vectors. siRNA-mediated downregulation of Cyr61 resulted in a >80% reduction in Cyr61 mRNA expression in RA-FLS cells compared with normal FLS cells (P<0.01). Conversely, transduction of lentivirus vectors encoding Cyr61 led to a 304.43±24.14 fold increase in Cyr61 mRNA expression in normal FLS cells compared with normal FLS cells transfected with a control lentivirus (P<0.0001; Fig. 2A). The transfection efficacies were confirmed by western blot analysis (Fig. 2B). To further examine whether Cyr61 activity was affected by manipulation of its expression, the levels of Cyr61 secreted into culture media were assessed by ELISA assays. As hypothesized, the culture medium collected from RA-FLS cells contained higher levels of Cyr61 than normal FLS cells (P<0.0001; Fig. 2C). Levels of Cyr61 secretion in culture media were consistent with the status of Cyr61 expression; the level of secreted Cyr61 was elevated in normal FLS cells transfected with Cyr61 cDNA, whilst it was significantly decreased in RA-FLS cells transfected with Cyr61-siRNA (P<0.0001 and P<0.05, respectively; Fig. 2C).


Role of cysteine‑rich angiogenic inducer 61 in fibroblast‑like synovial cell proliferation and invasion in rheumatoid arthritis.

Jie LG, Huang RY, Sun WF, Wei S, Chu YL, Huang QC, Du HY - Mol Med Rep (2014)

Role of Cyr61 in FLS cell proliferation. (A) Reverse transcription-quantitative polymerase chain reaction evaluation for the efficacy of Cyr61 siRNA or cDNA transfection in FLS cells. The mRNA level of Cyr61 in normal FLS cells was used as a control. **P<0.01. (B) Western blotting evaluation for the efficacy of Cyr61 siRNA or cDNA transfection in FLS cells. Cyr61 (40 kDa) was detected using a mouse IgG1 monoclonal antibody specific for Cyr61. GAPDH served as the loading control. (C) Detection of levels of Cyr61 secreted into the culture medium using enzyme-linked imunosorbent assays. *P<0.05 and **P<0.01. (D) MTS assay detection of cell proliferation. Data are presented as the mean ± standard deviation of three independent experiments conducted in triplicate. *P<0.05 and **P<0.01, compared with RA-NC; #P<0.05 and ##P<0.01, compared with NOR-NC; ^P<0.05, compared with NOR-FLA. RA, rheumatoid arthritis; FLS, fibroblast-like synoviocytes; siRNA, small interfering RNA; NOR-FLS, normal FLS cells; NOR-CYR61, normal FLS cells transduced with lentivirus vector encoding Cyr61 cDNA; NOR-NC, normal FLS cells transduced with control lentivirus vector; RA-NC, RA-FLS cells transfected with control siRNA; RA-siRNA, RA-FLS cells transfected with Cyr61-siRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f2-mmr-11-02-0917: Role of Cyr61 in FLS cell proliferation. (A) Reverse transcription-quantitative polymerase chain reaction evaluation for the efficacy of Cyr61 siRNA or cDNA transfection in FLS cells. The mRNA level of Cyr61 in normal FLS cells was used as a control. **P<0.01. (B) Western blotting evaluation for the efficacy of Cyr61 siRNA or cDNA transfection in FLS cells. Cyr61 (40 kDa) was detected using a mouse IgG1 monoclonal antibody specific for Cyr61. GAPDH served as the loading control. (C) Detection of levels of Cyr61 secreted into the culture medium using enzyme-linked imunosorbent assays. *P<0.05 and **P<0.01. (D) MTS assay detection of cell proliferation. Data are presented as the mean ± standard deviation of three independent experiments conducted in triplicate. *P<0.05 and **P<0.01, compared with RA-NC; #P<0.05 and ##P<0.01, compared with NOR-NC; ^P<0.05, compared with NOR-FLA. RA, rheumatoid arthritis; FLS, fibroblast-like synoviocytes; siRNA, small interfering RNA; NOR-FLS, normal FLS cells; NOR-CYR61, normal FLS cells transduced with lentivirus vector encoding Cyr61 cDNA; NOR-NC, normal FLS cells transduced with control lentivirus vector; RA-NC, RA-FLS cells transfected with control siRNA; RA-siRNA, RA-FLS cells transfected with Cyr61-siRNA.
Mentions: Due to the fact that Cyr61 was found to be overexpressed in synovial tissues, its involvement in the pathophysiological events associated with RA was investigated. RA-FLS cells were transfected with Cyr61-siRNA or control-siRNA, and effects on in vitro proliferation, apoptosis and invasion were determined. In vitro experiments were performed in RA-FLS cells without treatment and normal FLS cells. In addition, normal FLS cells were transduced with lentivirus vectors encoding Cyr61 cDNA or control lentivirus vectors. siRNA-mediated downregulation of Cyr61 resulted in a >80% reduction in Cyr61 mRNA expression in RA-FLS cells compared with normal FLS cells (P<0.01). Conversely, transduction of lentivirus vectors encoding Cyr61 led to a 304.43±24.14 fold increase in Cyr61 mRNA expression in normal FLS cells compared with normal FLS cells transfected with a control lentivirus (P<0.0001; Fig. 2A). The transfection efficacies were confirmed by western blot analysis (Fig. 2B). To further examine whether Cyr61 activity was affected by manipulation of its expression, the levels of Cyr61 secreted into culture media were assessed by ELISA assays. As hypothesized, the culture medium collected from RA-FLS cells contained higher levels of Cyr61 than normal FLS cells (P<0.0001; Fig. 2C). Levels of Cyr61 secretion in culture media were consistent with the status of Cyr61 expression; the level of secreted Cyr61 was elevated in normal FLS cells transfected with Cyr61 cDNA, whilst it was significantly decreased in RA-FLS cells transfected with Cyr61-siRNA (P<0.0001 and P<0.05, respectively; Fig. 2C).

Bottom Line: The study found that the Cyr61 gene was highly expressed in the synovial cells from patients with RA compared with those from patients with OA.Conversely, overexpression of Cyr61 in normal FLS cells led to opposite effects.In conclusion, these results indicate that Cyr61 is capable of promoting RA‑FLS cell proliferation and invasion via the suppression of apoptosis and the regulation of MMP expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Chinese Medicine, Guangzhou General Hospital of Guangzhou Command of PLA, Guangzhou, Guangdong 510010, P.R. China.

ABSTRACT
Cysteine‑rich angiogenic inducer 61 (Cyr61) is a novel molecule that has been shown to be increased in the synovial tissues of patients with rheumatoid arthritis (RA). The present study was conducted in order to investigate the role of Cyr61 in the pathogenesis of RA. A human genome‑wide gene assay was used to screen gene expression in synovial tissues obtained from four patients with RA and three patients with osteoarthritis (OA). To examine the role of Cyr61 in the phenotype of RA‑fibroblast‑like synovial (FLS) cells, Cyr61 expression in RA‑FLS cells was knocked down using small interfering RNA (siRNA). Normal FLS cells transduced with lentiviral vectors encoding Cyr61 cDNA were used to further explore the effects of this molecule on FLS cell apoptosis, proliferation and invasion. The study found that the Cyr61 gene was highly expressed in the synovial cells from patients with RA compared with those from patients with OA. Downregulation of Cyr61 by siRNA led to impaired cell proliferation and invasion. Furthermore, it decreased the levels of matrix metalloproteinase (MMP)‑3 and MMP‑13, and induced apoptosis in RA‑FLS cells. Conversely, overexpression of Cyr61 in normal FLS cells led to opposite effects. In conclusion, these results indicate that Cyr61 is capable of promoting RA‑FLS cell proliferation and invasion via the suppression of apoptosis and the regulation of MMP expression. Therefore, Cyr61 may be a good target molecule for the treatment and prevention of RA.

Show MeSH
Related in: MedlinePlus