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OBJECTIVE@#To investigate the effect of Cyr61 on imatinib (IM) resistance in chronic myeloid leukemia (CML) and its mechanism.@*METHODS@#Cyr61 level in cell culture supernatant was determined by enzyme-linked immunosorbent assay. The expression of Cyr61 and Bcl-xL were measured by real-time PCR and Western blot. Cell apoptosis was analyzed using an Annexin V-APC Kit. Expression of signal pathways related proteins was determined by Western blot.@*RESULTS@#The level of Cyr61 obviously increased in K562G cells (IM resistance to CML cell line K562). Down-regulating the expression of Cyr61 decreased the resistance of K562G cells to IM and promoted IM induced apoptosis. In CML mouse model, down-regulating the expression of Cyr61 could increase the sensitivity of K562G cells to IM. The mechanism studies showed that Cyr61 mediated IM resistance in CML cells was related to the regulation of ERK1/2 pathways and apoptosis related molecule Bcl-xL by Cyr61.@*CONCLUSION@#Cyr61 plays an important role in promoting IM resistance of CML cells. Targeting Cyr61 or its related effectors pathways may be one of the ways to overcome IM resistance of CML cells.
Subject(s)
Animals , Humans , Mice , Apoptosis , Drug Resistance, Neoplasm , Imatinib Mesylate/pharmacology , K562 Cells , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Signal TransductionABSTRACT
OBJECTIVE@#To investigate the role of Cyr61 in angiotensin Ⅱ (AngⅡ)-induced functional changes in HEK293 cells and explore the mechanism.@*METHODS@#Cyr61 knockdown in cultured HEK293T cells was achieved by transfection of the cells with CRISPR/Cas9 KO plasmid. The changes in apoptosis and expression levels of Cyr61 and Bcl-2 in the cells with or without Cyr61 knockdown in response to treatment with 10 mol/L AngⅡ for 48 h were analyzed using flow cytometry, qRT-PCR and Western blotting.@*RESULTS@#The cells with Cyr61 knockdown showed significantly decreased expression of Cyr61 protein as compared with the control cells ( < 0.05). AngⅡ treatment for 48 h significantly increased the expression of Cyr61 and lowers the expression of Bcl-2 at both the protein and mRNA levels in HEK293T cells. In HEK293T cells with Cyr61 knockdown, AngⅡ treatment resulted in significantly increased expression of Bcl-2 in HEK293T cells as compared with that of the control group ( < 0.05). AngⅡ treatment caused significantly increased apoptotic rate in HEK293T cells as compared with the cells with Cyr61 knockdown [(26.94 ± 3.73)% (3.87 ± 0.83)%, < 0.05), and the apoptosis rate was significantly lowered to (15.76 ± 1.31)% in HEK293T cells with Cyr61 knockdown following AngⅡ treatment ( < 0.05).@*CONCLUSIONS@#The up-regulation of Cyr61 expression is related with AngⅡ-induced injury in HEK293T cells, and down-regulating Cyr61 expression can effectively protect HEK293T cells against AngⅡ-induced injury.
Subject(s)
Humans , Angiotensin II , Apoptosis , Cysteine-Rich Protein 61 , HEK293 Cells , Up-RegulationABSTRACT
Objective To investigate the regeneration and differentiation of HOCs in the 2-AAF/PHx rat models. To explore the expression of Cyr61and its mechanism in differentiation of HOCs in vitro. Methods In 2-AAF/PHx rats model,induction and expansion of HOCs were detected by immunochemistry and HE staining. West-ern blot was used for observing the expression of Cyr61. Furthermore,the expression of Cyr61 andβ-catenin were detected by Western blot in differentiation of WB-F344 cells in vitro. Results Cyr61 protein level increased as a re-sult of HOCs in rats livers after 2-AAF/PHx. In addition,the expression of Cyr61 and β-catenin significantly in-creased during WB-F344 cells differentiation in vitro. Conclusions Cyr61 might play an important role as a signal-ing mediator in HOCs response and closely correlate with Cyr61 andβ-catenin in proliferation and differentiation of HOCs.
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Objective To detect the effect and mechanism of Cyr61 on the apoptosis of renal tissue caused by early stage of ischemic acute kidney injury (AKI). Methods 30 SD rats were randomized into 5 groups, including control group, AKI group, AKI+bicarbonate group, AKI+blank virus group, and AKI+over?expression Cyr61 virus group. After animal models were created for 2h, serum and renal tissue were collected from sacrificed animals. Expression level of TNF?α was determined by ELISA. HE staining was used to observe the histologic changes of renal tissues. The levels of NF?κB p65 and TNFR1 were measured by immunohistochemical method. RT?PCR and Western blotting assay were adopted to detect the mRNA and protein expression levels of NF?κB p65, TNFR1 and Caspase3. Results Compared with control group, AKI group, AKI+bicarbonate group, AKI+blank virus group, AKI+over?expression Cyr61 virus group had obvious kidney injury. The levels of TNF?α, the mRNA and protein expression levels of NF?κB p65, TNFR1 and caspase3 were markedly up?regulated. Over?expression of Cyr61 significantly attenuated the degree of pathological injury, numbers of apoptotic renal tubular epithelial cells and increased the degree of Scr. Although compared with other groups, the level of TNF?α in kidney tissue had no difference, there was obvious decreased protein level of NF?κB p65, while the increase of TNFR1 and Caspase3 protein was moderate. Conclusions During the early stage of AKI, over expression of Cyr61 could inhibit apoptosis, which may be related to the suppression of TNFR1 transcriptional expression and interference of TNF?αpathway. Its underlying mechanism therefore deserves further research.
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Objective To evaluate the expressions of Cyr61 and β‐catenin protein in gallbladder carcinoma tissues and investigate their association with the clinicopathologic features of gallbladder carcinoma patients . Methods The expressions of Cyr61 and β‐catenin protein in 50 cases of gallbladder carcinoma and 19 cases of normal tissue were detected by immunohistochemical S‐P method .Results ① The positive expression rate of Cyr61 in gallbladder carcinoma tissues was 66 .0% (33/50) ,which was significantly higher than that in the normal tissues group (26 .3% ) .The expression of Cyr61 was related to tumor differentiation ,TNM stage and lymph node metastasis of gallbladder carcinoma (P=0 .010 ,P=0 .014 ,P=0 .007;P<0 .05) .② The positive expression rate ofβ‐catenin in gallbladder carcinoma tissues was 84 .0% (42/50) ,which was significantly higher than that in the normal tissues group 15 .7% (3/19);the expression of β‐catenin was related to tumor differentiation ,TNM stage and lymph node metastasis of gallbladder carcinoma (P=0 .018 ,P=0 .002 ,P=0 .024;P<0 .05) .③ Correlation test showed that Cyr61 andβ‐catenin were positively correlated in gallbladder carcinoma and adjacent normal tissues (r=0 .378 , P< 0 .05) .Conclusion Cyr61 and β‐catenin are highly expressed in gallbladder carcinoma tissues . Cyr61 andβ‐catenin expressions are closely related to the clinicopathologic features (tumor differentiation ,TNM staging and lymph node metastasis) in gallbladder carcinoma .Cyr61 and β‐catenin may have a synergistic effect in promoting progression and development of gallbladder carcinoma .Combined detection of Cyr61 and β‐catenin in gallbladder carcinoma tissues will contribute to the clinical diagnosis and prognosis .
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Objective:To investigate the effect of Cyr61 on the proliferation of Fibroblast-like Synoviocytes (FLS) in rheumatoid arthritis (RA).Methods:Cyr61 expression in synovial tissues (ST) and FLS was examined using Real-time PCR,Western blot and immunohistochemistry simultaneously.FLS were isolated from synovial tissue of RA patients and cultured in vitro.The proliferation of FLS stimulated with synovial fluid (SF) was determined by 3 H-TdR incorporation.Cyr61 protein level in RA SF was detected by ELISA.Results:Cyr61 was over expressed in ST,FLS of RA patients while hardly examined in normal individuals and osterarthritis (OA) patients.Meanwhile,Cyr61 protein level was elevated in SF,which promoted the proliferation of RA FLS.This proliferation was abrogated by knockdown the Cyr61 gene of FLS or neutralizing monoclonal antibody against human Cyr61.Moreover,inflammatory factor IFN-γ and TNF-α up-regulated the expression of Cyr61.Conclusion:These results indicate that the elevated level of IFN-γ and TNF-α in RA SF can promote the proliferation of the FLS derived from RA patients by increasing expression of Cyr61,suggesting that Cyr61 may play an important role in the development of RA.
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Objective: To investigate the biological functions of the Cyr61 gene in human lung carcinomas. Methods: The Cyr61 expression levels in the cancerous and peri-cancerous tissues of 60 lung cancer patients were measured by real-time polymerase chain reaction (PCR). Results: The expression of Cyr61 gene was down-regulated in 80% (48/60) primary lung cancer patients compared with the matched pericancerous tissues. The result was consistent with the immunohistochemical staining. Statistical analysis revealed that the difference between expression of cancerous tissues and the matched peri-cancerous tissues was significant [(2.742 ± 4.165) vs (4.933 ± 3.349), t = -5.112, P=0.000]. Furthermore, the clinical variables including tumor grade, tumor metastasis, tumor pathological classification, smoking, and family history influenced the level of Cyr61 expression (P<0.05). Conclusion: Cyr61 may play a key role in the progression of lung carcinoma and its protein might serve as an important target for the therapeutic intervention in clinic.
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Objective To study the induction of integrin molecules mediated by CYR61 in human peripheral blood mononuclear cells(PBMC). Methods A recombinant expression plasmid containing full length of human CYR61 labeled with human IgG Fc fragment was constructed and identified by DNA sequencing.COS7 was used as host cell for identification of secretary CYR61 expression,confirmed by Western blot method.The commercial lipofectin was adopted for recombinant plasmid transfection into PBMC.Real-time PCR was ultilized to analyze expression patterns of CYR61 and integrin molecules in transfected PBMC. Results The insert sequence was correct in the recombinant plasmid.Western blot test showed that CYR61 protein secreted into culture supernatant or was in COS7 cytoplasm.The recombinant plasmid was transfected into PBMC stimulated with phytohemagglutinine(PHA) to induce CYR61 expression and secretion.The results demonstrated that exogenous CYR61 transcribed rapidly after being incubated with PHA and reached the peak after 24 h.But the expression dropped down quickly to a very low level after 48 h.Simultaneously,integrin molecules expressed just after CYR61 transcription.In the set of integrin molecules tested in the study,?v,?M,?3 and ?5 expression were higher than the other integrin molecules(P
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CCN1 is a novel extracellular matrix-associated signaling protein which possesses 381 amino-acid residues to compose 4 distinct structural modules with 38 conserved cysteine residues.This protein has a variety of properties in cardiovascular system,affecting the cellular behaviors such as differentiation,proliferation and migration of vascular endothelial cells,smooth muscle cells and cardiac myocytes,that suggested an essential roles of CCN1 in angiogensis,vascular injury,cardiac development and myocardial infarction.
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OBJECTIVES: Previously, we sought to compile a list of genes expressed during early folliculogenesis by using cDNA microarray to investigate follicular gene expression and changes during primordialprimary follicle transition and development of secondary follicles (Yoon et al., 2005). Among those genes, a group of genes related to the cell size growth was characterized during the ovarian development in the present study. METHODS: We determined ovarian expression pattern of six genes related to the cell size growth (cyr61, emp1, fhl1, socs2, wig1 and wisp1) and extended into CCN family (connective tissue growth factor/cysteine-rich 61/nephroblastoma-overexpressed), ctgf, nov, wisp2, wisp3, including cyr61 and wisp1 genes. Expression of mRNA and protein according to the ovarian developmental stage was evaluated by in situ hybridization, and/or semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR), and immunohistochemistry, respectively. RESULTS: Among 6 genes related to the cell size growth, cyr61 and wisp1 mRNA was detected only in oocytes in the postnatal day5 mouse ovaries. cyr61 mRNA expression was limited to the nucleolus of oocytes, while wisp1 was expressed in the cytoplasm and nucleolus of oocytes, except nucleus. cyr61 mRNA expression, however, was found in granulosa cells from secondary follicles. The rest 4 genes in the cell size growth group were detected in oocytes, granulosa and theca cells. Cyr61 and Wisp1 proteins were expressed in the oocyte cytoplasm from primordial follicle stage. Especially, Cyr61 protein was detected in pre-granulosa cells, Wisp1 protein was not. By using RT-PCR, we evaluated and decided that Cyr61 protein is produced by their own mRNA in pre-granulosa cells that was not detected by in situ hybridization. cyr61 and wisp1 genes are happen to be the CCN family members. The other members of CCN family were also studied, but their expression was detected in oocytes, granulose and theca cells. CONCLUSIONS: We firstly characterized the ovarian expression of genes related to the cell size growth and CCN family according to the early folliculogenesis. Cyr61 protein expression in the pre-granulosa cells is profound in meaning. Further functional analysis for cyr61 in early folliculogenesis is under investigation.
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Animals , Female , Humans , Mice , Cell Enlargement , Cell Size , Cysteine-Rich Protein 61 , Cytoplasm , Gene Expression , Genes, vif , Granulosa Cells , Immunohistochemistry , In Situ Hybridization , Oligonucleotide Array Sequence Analysis , Oocytes , Ovary , Reverse Transcriptase Polymerase Chain Reaction , RNA, Messenger , Theca CellsABSTRACT
OBJECTIVE:To study the effect and thepossible mechanism of low molecular weight heparin(LMWH)on immunocytes' adhesion to fibroblast-like synovocytes(FLS)isolated from patients with rheumatoid arthritis(RA)and to study LMWH'S possible anti-inflammatory effect on RA.METHODS:LMWH's interference on the adhesion of peripheral blood monouclear cells(PBMC)isolated from healthy volunteers to FLS of RA patients was determined by quantitative counting using flow cytometry.The expression of CYR61 in the in vitro cultured FLS of RA patients was detected using real-time PCR technology.RESULTS:When FLS culture system was added with PBMC,PBMC were obviously found to adhere to FLS,but the number of adhered PBMC decreased after LMWH treatment,which manifested as increase of deciduous PBMC,increased more with the increase of LMWH dose.There was a high expression of CYR61 in synovium tissue in RA patients.CONCLUSION:LMWH inhibited the adheresion of PBMC to FLS from RA patients in a dose-dependent manner,which might be attributed to its competitive combination with heparin sulfate sites on CYR61.
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Objective To investigate the effect of over-expressed Cyr61 on the expression of extracellular matrix of human renal tubular epithelial cells(HKC), and explore the role of Cyr61 in the pathogenesis and development of autosomal dominant polycystic kidney disease(ADPKD). Methods Cyr61 gene cDNA was amplified by RT-PCR. A recombinant plasmid pcDNA3.1+ Cyr61 was constructed by cloning Cyr61 gene into pcDNA3.1. HKC cells were transfected with wild-type Cyr61 plasmid vector (pcDNA3.1+Cyr61). Then the fusion of Cyr61 gene and expression of its protein were detected by RT-PCR and Western blot. The mRNA expression of Cyr61, collagen Ⅰ, collagen Ⅳ, and laminin were determined in four groups (the untransfected cells, pcDNA3.1 transfected cells, pcDNA3.1+Cyr61 -transfected cells and cyst-lining epithelial cells) by fluorescence quantum PCR. Results The amount of Cyr61 protein in Cyr61-transfected cells and cyst-lining epithelial cells were much greater than the control. The mRNA expression of Cyr61, collagen Ⅰ, collagen Ⅳ and laminin in Cyr61-transfected cells were all amplified significantly, and the level of collagen Ⅳwas much higher than collagen I(P