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Objective@#To investigate the expression of discoid domain receptor 2 (DDR2) and interferon-induced transmembrane protein 1 (IFITM1) in nasopharyngeal carcinoma (NPC) patients and their clinical significance.@*Methods@#From November 2014 to November 2015, 65 patients with nasopharyngeal carcinoma were selected. All patients underwent nasopharyngeal biopsy. All specimens were embedded in paraffin after biopsy. All specimens were confirmed by pathology. Another 65 rhinitis mucosa specimens with chronic inflammation confirmed by pathology in the same period were selected as control group. The clinical and pathological data of the two groups were collected. The mRNA and protein expression levels of DDR2 and IFITM1 in nasopharyngeal carcinoma tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry. The correlation between them was analyzed by Pearson. All patients were followed up for 3 years, and the 3-year survival period of the patients was analyzed by Kaplan-Meier method.@*Results@#The results of qRT-PCR showed that the relative expression levels of DDR2 and IFITM1 in nasopharyngeal carcinoma tissues were significantly higher than those in control group (P<0.05). Immunohistochemical results showed that the positive rates of DDR2 and IFITM1 in nasopharyngeal carcinoma were significantly higher than those in control group (P<0.05). The results of expressions of DDR2 and IFITM1 and the clinicopathological analysis of the patients showed that the expression of DDR2 was correlated with TNM stage, differentiation degree and infiltration depth, and IFITM1 was correlated with lymph node metastasis, TNM stage, differentiation degree and infiltration depth (P<0.05). Pearson correlation analysis showed that there was correlation between DDR2 and IFITM1 (r=0.608, P<0.01). Kaplan-Meier analysis showed that the progression-free survival rate (41.17%) and total survival rate (52.94%) in DDR2 negative expression group were significantly higher than those in positive expression group (16.67%, 18.75%, P<0.05). The progression-free survival rate (42.86%) and total survival rate (61.90%) in IFITM1 negative expression group were significantly higher than those in positive expression group (18.18%, 20.45%, P<0.05).@*Conclusions@#DDR2 and IFITM1 are highly expressed in nasopharyngeal carcinoma, which may be involved in the occurrence and development of nasopharyngeal carcinoma and significantly affect the prognosis of patients.
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Objective To investigate the expression of discoid domain receptor 2 (DDR2) and interferon-induced transmembrane protein 1 (IFITM1) in nasopharyngeal carcinoma (NPC) patients and their clinical significance.Methods From November 2014 to November 2015,65 patients with nasopharyngeal carcinoma were selected.All patients underwent nasopharyngeal biopsy.All specimens were embedded in paraffin after biopsy.All specimens were confirmed by pathology.Another 65 rhinitis mucosa specimens with chronic inflammation confirmed by pathology in the same period were selected as control group.The clinical and pathological data of the two groups were collected.The mRNA and protein expression levels of DDR2 and IFITM1 in nasopharyngeal carcinoma tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and immunohistochemistry.The correlation between them was analyzed by Pearson.All patients were followed up for 3 years,and the 3-year survival period of the patients was analyzed by Kaplan-Meier method.Results The results of qRT-PCR showed that the relative expression levels of DDR2 and IFITM1 in nasopharyngeal carcinoma tissues were significantly higher than those in control group (P ＜0.05).Immunohistochemical results showed that the positive rates of DDR2 and IFITM1 in nasopharyngeal carcinoma were significantly higher than those in control group (P ＜ 0.05).The results of expressions of DDR2 and IFITM1 and the clinicopathological analysis of the patients showed that the expression of DDR2 was correlated with TNM stage,differentiation degree and infiltration depth,and IFITM1 was correlated with lymph node metastasis,TNM stage,differentiation degree and infiltration depth (P ＜ 0.05).Pearson correlation analysis showed that there was correlation between DDR2 and IFITM1 (r =0.608,P ＜ 0.01).Kaplan-Meier analysis showed that the progression-free survival rate (41.17％) and total survival rate (52.94％) in DDR2 negative expression group were significantly higher than those in positive expression group (16.67％,18.75％,P ＜ 0.05).The progression-free survival rate (42.86％) and total survival rate (61.90％) in IFITM1 negative expression group were significantly higher than those in positive expression group (18.18％,20.45％,P ＜0.05).Conclusions DDR2 and IFITM1 are highly expressed in nasopharyngeal carcinoma,which may be involved in the occurrence and development of nasopharyngeal carcinoma and significantly affect the prognosis of patients.
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Discoidin domain receptor 2 (DDR2), a tyrosine kinase receptor belonging to DDRs family, is associated with progression of various cancers. Studies have indicated that DDR2 expression is upregulated in various cancer tissues, which could promote tumors proliferation, invasion, and metastases. Furthermore, patients with high DDR2 expression showed poor 5-year overall survival rate. In addition, DDR2 point mutation can enhance the sensitivity of tumor cells to small molecule tyrosine kinase inhibitor dasatinib, which suggests that DDR2 mutation may be a promising target for cancer therapy.
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PURPOSE: The discoidin domain-containing receptor tyrosine kinase 2 (DDR2) is known to contain mutations in a small subset of patients with squamous cell carcinomas (SCC) of the lung. Studying the DDR2 mutations in patients with SCC of the lung would advance our understanding and guide the development of therapeutic strategies against lung cancer. MATERIALS AND METHODS: We selected 100 samples through a preliminary genetic screen, including specimens from biopsies and surgical resection, and confirmed SCC by histologic examination. DDR2 mutations on exons 6, 15, 16, and 18 were analyzed by Sanger sequencing of formalin-fixed, paraffin-embedded tissue samples. The functional effects of novel DDR2 mutants were confirmed by in vitro assays. RESULTS: We identified novel somatic mutations of DDR2 in two of the 100 SCC samples studied. One mutation was c.1745T>A (p.V582E) and the other was c.1784T>C (p.L595P), and both were on exon 15. Both patients were smokers and EGFR/KRAS/ALK-triple negative. The expression of the mutant DDR2 induced activation of DDR2 by the collagen ligand and caused enhanced cell growth and tumor progression. Moreover, dasatinib, a DDR2 inhibitor, showed potential efficacy against DDR2 L595P mutant–bearing cells. CONCLUSION: Our results suggest that a mutation in DDR2 occurs naturally with a frequency of about 2% in Korean lung SCC patients. In addition, we showed that each of the novel DDR2 mutations were located in a kinase domain and induced an increase in cell proliferation rate.
Subject(s)
Humans , Biopsy , Carcinoma, Squamous Cell , Cell Proliferation , Collagen , Dasatinib , Epithelial Cells , Exons , In Vitro Techniques , Lung Neoplasms , Lung , Phosphotransferases , Prevalence , Protein-Tyrosine Kinases , TYK2 Kinase , TyrosineABSTRACT
Discoidin domain receptor 2 (DDR2) is a receptor tyrosine kinase (RTK) that can be activated by fibrillar collagens (Ⅰ-Ⅲ,X).Recently researchers show that the DDR2 is expressed differently in many cancers,which suggests DDR2 is probably correlated with tumorigenesis and cancer development,and it may become a promising cancer therapeutic target.
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Objective To explore the effects of silencing DDR2 expression by siRNA on CCl4-induced liver fibrosis and its mechanism in rats. Methods Liver fibrosis model was induced by intraperitoneal injection of CCl4 twice a week for 6 consecutive weeks. Some rats were administered siRNA targeting DDR2 (0. 3 mg/kg), saline or control siRNA every three days from the beginning of CCl4 injection via tail vein injection, while other rats were treated in the same pattern after 2-week CCl4 injection. Quantitative real-time polymerase chain reaction (QRT-PCR) and Western blot were used to detect the mRNA and protein expressions of DDR2, MMP-2 and COL Ⅰ . Meanwhile, the pathological changes of liver tissues and the levels of liver function were also observed. Results QRT-PCR showed that the DDR2, MMP-2 and COL Ⅰ mRNA in the chemically synthetic cholesterol-modified siRNADDR2 group were significantly decreased as compared with those in the control group (P＜0.01) ,and the protein expressions of DDR2, MMP-2 and COL Ⅰ were also significantly decreased (P＜0. 01,4 wand 6w). In addition, in comparison with those in the control group, the pathological changes of liver tissues in the siRNA-DDR2 treated group were markedly attenuated, and the levels of ALT(1356.17 ±83.80 nkat/L vs 2532. 70±145.11 nkat/L,4w,1367. 60±321.76 nkat/L vs 2604.37±255.02 nkat/L,6w,P＜0. 01 ) and AST (2460. 80 ± 207. 58 nkat/L vs 3983. 70 ± 253. 08 nkat/L, 4w, P＜ 0. 01,2383.27±290.16 nkat/L vs 3227.70±353. 34 nkat/L,6w,P＜0. 05)were also significantly lowered,while the level of TBIL (7. 97 ± 1.60 μmol/L vs 3.80± 0.60 μmol/L, 4w, 10.40±1.61 μmol/L vs 6.10±0.79 μmol/L,6w,P＜0. 01)was markedly increased. Conclusion Systemic administration of cholesterol-modified siRNA targeting DDR2 could significantly suppress the expression of DDR2, decrease the contents of the extracellular matrix,and thus has a potential antifibrotic effect.
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Objective To detect the changes in the expression of discoidin domain receptor 2(DDR2)and matrix metalloproteinase (MMP)-13 in different stages of cartilage and synovium damage of osteoarthritis rats.The relation between DDR2 and the degree of cartilage damage was explored.Methods Modified papain knee joint injection approach was adopted to establish animal model of OA.The expression and distribution of protein of DDR2 and MMP-13 were checked in articular cartilage and synovium at different stages of OA.Results The expressions of DDR2 in articular cartilage and synovium of experimental groups were different from those of the normal group (P＜0.01).They were higher in cartilage than those in the corresponding synovium.The expressions of MMP-13 demonstrated the same characteristics with those of DDR2,r=0.93(P＜0.01).Conclusions The important role of DDR2-MMP-13 in cartilage damage has been proven in the pathogenic process of OA.The upregulated expressions of DDR2 in articular cartilage and synovium have a detrimental effect on cartilage degeneration.
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Objective To explore the effects of inhibiting DDR2 expression by siRNA on hepatic stellate cells and evaluate the role of DDR2 gene in hepatic fibrogenesis. Methods (1) Three pairs of chemically synthesized siRNAs targeting DDR2 were respectively transfected into HSC-T6 cells for evaluation of silence efficacy, and the most effective siRNA was used. (2) HSC-T6 cells were divided into three groups, group A served as normal controls, group B served as negative control and group C was RNA interference DDR2 (siRNA-DDR2) expression of HSC. The most effective RNA interference sequences targeting DDR2 gene was chosen to transfect HSC-T6 cells by plasmid transfection. The tendency of DDR2, α-smooth muscle actin(α-SMA) and collagen-Ⅰ mRNA expression were estimated using RT-PCR, and the protein expression of DDR2 was evaluated by Western blot. Meanwhile, MTT assay was employed to analyze the proliferation of HSC. Results (1) DDR2 siRNA, which began at nt 868, inhibited DDR2 gone expression stronger than the other two siRNAs. (2) After transfection of siRNA-DDR2, the mRNA expression of DDR2 (P<0.01) and α-SMA (P<0.01) significantly decreased compared with the normal group, and the protein expression of DDR2 also significantly decreased (P<0.01). In addition, the proliferation of HSC was also markedly suppressed as compared with the normal group (P<0.01). However, compared with the negative control group, none of them was markedly suppressed. Conclusion SiRNA targeting DDR2 significantly suppresses the activation, proliferation of HSC, and thus attenuates hepatic fibrogonesis in vitro.
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Objective Discoidin domain receptor 2 is a kind of receptor tyrosine kinases, which was found to be over-expressed in synovial fibroblasts in rheumatoid arthritis (RA). The present study was to construct and express wild type (FLDDR2), Fc chimera (FcDDR2) and truncated form of mouse discoidin domain receptor 2 (ttDDR2) for further study. Methods Full-length, truncated form of mouse DDR2 were amplified by RT-PCR. A chimera form of mouse DDR2 was constructed by replacing part of the extracellular domain with Fc fragment of human immunoglobulin G1 (IgG1). Eukaryotic expression vectors of the different forms of mouse DDR2 were constructed by subcloning the PCR products into pcDNA3.1(+) or pMKIT-Neo. Then COS-7 cells were transient transfected with the eukaryotic expression vectors of full-length (FLDDR2), truncated (ttDDR2) and chimeric form of mouse DDR2 (FcDDR2). Successful transfection and expression was confirmed by Western blot(WB) and Immunoprecipitation (IP)/WB. With or without stimulation with soluble type I collagen for 3h, phosphorylation level of transfected cells were detected by IP/WB. Eukaryotic expression vectors of full-length, truncated form and chimeric forms of mouse DDR2 were successfully constructed and confirmed by sequencing. After transient transfection, the expression of these three forms in the respectively transfected cells was observed by Western blot. Results The result of IP/WB suggested that the chimeric form of mouse DDR2(FcDDR2) could be properly expressed in the COS-7 cells. Under the condition of collagen, decreased tyrosine phosphorylation of FLDDR2 was detected in the COS-7 cells that were cotransfected with ttDDR2 and FLDDR2, comparing with that in COS-7 cells transected with only FLDDR2. There was no obvious difference in phosphorylation level between FcDDR2 without collagen stimulation and FLDDR2 with the collagen stimulation. Conclusion Three different forms of mouse DDR2 were successfully constructed. FcDDR2 could be an activator for its auto-phosphorylation. And ttDDR2 could be a partially negative competitor.