Pterygium is a common ocular surface disease induced by a variety of factors. The exact pathogenesis of pterygium remains unclear. Numbers of genes and proteins are discovered in pterygium and they function differentl...Pterygium is a common ocular surface disease induced by a variety of factors. The exact pathogenesis of pterygium remains unclear. Numbers of genes and proteins are discovered in pterygium and they function differently in the occurrence and development of this disease. We searched the Web of Science and PubMed throughout history for literatures about the subject. The keywords we used contain pterygium, gene, protein, angiogenesis, fibrosis, proliferation, inflammation, pathogenesis and therapy. In this review, we summarize the aberrant expression of a range of genes and proteins in pterygium compared with normal conjunctiva or cornea, including growth factors, matrix metalloproteinases and tissue inhibitors of mefalloproteinases, interleukins, tumor suppressor genes, proliferation related proteins, apoptosis related proteins, cell adhesion molecules, extracellular matrix proteins, heat shock proteins and tight junction proteins. We illustrate their possible mechanisms in the pathogenesis of pterygium as well as the related intervention based on them for pterygium therapy.展开更多
FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expression showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investig...FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expression showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investigate the biological function of FUS1 protein, FUS1 cDNA from MRC-5 cells was amplified by RT-PCR and cloned into prokaryotic expression vector pQE-30. The recombinant expression plasmids were transformed into M15 strain and grown at 20℃ or 37℃. SDS–PAGE analysis revealed that the accumulation of the recombinant protein FUS1 (rFUS1) in inclusion body forms reached maxium amount when induced with 0.5 mM IPTG for 5 h at 37℃. The inclusion bodies were solubilized in 2M urea and purified by a 6 ×His tagged affinity column under denaturing condition. The purified rFUS1 was identified by electrospray ionization-mass spectrometry (ESI-MS) and tested for purity by HPLC chromatography. The purified rFUS1 proteins were then used to immunize rabbits to obtain anti-human FUS1 polyclonal antibodies, which were suitable to detect both the recombinant exogenous FUS1 and the endogenous FUS1 from tissues and cells by western blot and immunohistochemistry, Available purified rFUS1 proteins and self-prepared polyclonal antibodies against FUS1 may provide effective tools for further studies on biological function and application of FUS1.展开更多
Objective:To investigate the effect of hepatitis C virus non-structural protein 4B(HCV NS4B) on c-Myc, P53, ras gene expression" and apoptosis in hepatic cells and study the possible role that NS4B played in the c...Objective:To investigate the effect of hepatitis C virus non-structural protein 4B(HCV NS4B) on c-Myc, P53, ras gene expression" and apoptosis in hepatic cells and study the possible role that NS4B played in the carcinogenesis of heparoma. Methods: The recombinant plasmid(PCXN2-NS4B, PCXN2-P53) and the empty, vector were transfected or co-transfected into Chang liver cells with liposome. Screening was performed with G418. Plasmid mRNA was detected by RT-PCR. The pro rein expressions of c-Myc and ras genes were analyzed by immunocytochemistry. The expressions of wild-type P53 (wtp53) gene were detected by in situ hybridization. TUNEL(flow cytometry) was used for assessing the rate of apoptosis. Results:No expression of c-Myc gene was found in PCXN2 group. The expression of c-Myc gene in NS4B group was 21.3% + 1.2%. The ex pression of ras gene in PCXN2 group was lower than that in NS4B group. Compared with PCXN2 group, the expression of P53 mRNA was not promoted or inhibited in NS4B group. But the expression of P53 mRNA in NS4B-P53 group was lower than that in P53 group. In PCXN2, NS4B, P53 and NS4B-P53 group, the rates of apoptosis were 17.02% ± 1.24%, 11.94% ± 2.24%, 25.84% ± 3.49% and 18.34% ± 1.55% respectively. Conclusion :HCV NS4B induces the expression of c-Myc and ras gene. HCV NS4B may play a role in the inhibition of cell death through P53-dependent manner. Results from this study suggested that HCV NS4B might contribute to the viral carcinogenesis.展开更多
MCM10 protein is an essential replication factor involved in the initiation of DNA replication. A mcm10 mutan (mcm10-1) of budding yeast shows a growth arrest at 37 C. In the present work, we have isolated a mcm10-1 s...MCM10 protein is an essential replication factor involved in the initiation of DNA replication. A mcm10 mutan (mcm10-1) of budding yeast shows a growth arrest at 37 C. In the present work, we have isolated a mcm10-1 suppressor strain, which grows at 37 C. Interestingly, this mcm10-1 suppressor undergoes cell Cycle arrest at 14 C. A novel gene, YLR003c, is identified by high-copy complementation of this suppressor. We called it as Cms1 (Complementation of Mcm 10 Suppressor). Furthermore, the experiments of trans- formation show that cells of mcm10-1 suppressor with high-copy plasmid but not low-copy plasmid grow at 14 C, indicating that overexpression of Cms1 can rescue the growth arrest of this mcm10 suppressor at non-permissive temperature. These results suggest that CMS1 protein may functionally interact with MCM10 protein and play a role in the regulation of DNA replication and cell cycle control.展开更多
目的探索乳腺癌/卵巢癌易感基因1相关蛋白1(breast/ovarian cancer susceptibility gene 1 associated protein 1,BAP1)对人源恶性胶质瘤发生、发展的作用与BAP1作为恶性胶质瘤临床诊断标志物的可行性。方法基于基因表达综合数据库(gene...目的探索乳腺癌/卵巢癌易感基因1相关蛋白1(breast/ovarian cancer susceptibility gene 1 associated protein 1,BAP1)对人源恶性胶质瘤发生、发展的作用与BAP1作为恶性胶质瘤临床诊断标志物的可行性。方法基于基因表达综合数据库(gene expression omnibus,GEO)的子数据集GSE4290,GSE90598,分析BAP1在正常组织及胶质瘤组织中的差异性表达情况;受试者工作特征(receiver operating characteristic,ROC)曲线分析BAP1对恶性胶质瘤的早期诊断价值;选取自主收集的非配对28例恶性胶质瘤患者的原发灶组织、5例颅脑外伤患者内减压术切除的非瘤脑组织,采用实时荧光定量PCR(quantitative real-time polymerase chain reaction,qRT-PCR)检测BAP1的表达水平;利用靶向BAP1的特异性小干扰RNAs(small interfering RNAs,siRNAs)瞬时转染U251细胞系,进一步检测其干涉效率;基于流式细胞仪分析BAP1下调的U251细胞系,其细胞周期、凋亡的变化情况。结果生物信息学结果显示,BAP1在恶性胶质瘤组织中的表达水平均低于正常脑组织(GSE4290:1209±18.49 vs 1476±53.90;GSE90598:5.19±0.10 vs 5.65±0.21),差异具有统计学意义(t=5.115,2.267,均P<0.05)。ROC曲线显示,BAP1可高效区分恶性胶质瘤组织与正常脑组织(GSE4290:AUC=0.78;GSE90598:AUC=0.75,均P<0.05)。临床标本结果显示,BAP1在恶性胶质瘤原发灶组织中的表达水平显著低于非瘤脑组织(0.27±0.04 vs 1.06±0.07),差异具有统计学意义(t=10.22,P<0.001)。在U251细胞系中下调BAP1的表达,其细胞周期中S期细胞比例明显增多,由17.59%分别增至27.21%(siBAP1-1)和25.79%(siBAP1-2),差异具有统计学意义(t=6.576,6.642,均P<0.01),而细胞凋亡水平则有所下降,由10.17%分别降至2.70%(siBAP-1)和3.00%(siBAP-2),差异具有统计学意义(t=10.31,9.428,均P<0.01)。结论组蛋白H2A去泛素化酶BAP1能够通过抑制恶性胶质瘤细胞周期快速进展并促进其凋亡,进而发挥肿瘤抑癌基因的功能,可作为潜在的恶性胶质瘤临床诊断标志物。展开更多
AIM:To investigate the promoter region methylation status of retinoblastoma protein-interacting zinc finger gene 1(RIZ1) in the human esophageal squamous cell carcinoma(ESCC) cell lines and tissues and verify the rela...AIM:To investigate the promoter region methylation status of retinoblastoma protein-interacting zinc finger gene 1(RIZ1) in the human esophageal squamous cell carcinoma(ESCC) cell lines and tissues and verify the relationship between methylation of RIZ1 and oncogenesis,tumor progression and metastasis etc of ESCC.METHODS:Methylation-specific polymerase chain reaction(MSP) was used to investigate the promoter region methylation status of RIZ1 in 6 ESCC cell lines.One cell line where RIZ1 promoter region methylation was detected was selected for the next study,where the cell line was treated with 5-aza-CdR.Real-time polymerase chain reaction was used to investigate its influence on the transcription of RIZ1.Experiments using frozenpathological specimens from 47 ESCC patients were performed using the same MSP methodology.RESULTS:Promoter methylation of RIZ1 gene was detected in TE13,CaEs17 and EC109 cell lines and the cell line TE13 was chosen for further study.The expression of RIZ1 mRNA in TE-13 was up-regulated after treatment with 5-aza-CdR.The rate of methylation in carcinomas tissues was significantly higher than those in matched neighboring normal and distal ending normal tissue,and the deviation of data was statistically significant(χ 2 = 24.136,P < 0.01).Analysis of the gender,age familial history,tumour deviation,tumour saturation,lymph gland displacement and clinical staging of 47 samples from ESCC patients showed that the fluctuation of data was not statistically significant.CONCLUSION:Promoter methylation may play an important role in the epigenetic silencing of RIZ1 gene expression in human ESCC.RIZ1 is considered to be a potential tumor suppressor gene and may be a biological parameter for testing early stage human ESCC.展开更多
AIM:To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element(FUSE)-binding protein-interacting repressor(FIR).METHODS:Endogenous c-Myc suppression and apopt...AIM:To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element(FUSE)-binding protein-interacting repressor(FIR).METHODS:Endogenous c-Myc suppression and apoptosis induction by a transient FIR-expressing vector was examined in vivo via a HA-tagged FIR(HA-FIR)expression vector.A fusion gene-deficient,non-transmissible,Sendai virus(SeV)vector encoding FIR cDNA,SeV/dF/FIR,was prepared.SeV/dF/FIR was examined for its gene transduction efficiency,viral dose dependency of antitumor effect and apoptosis induction in HeLa(cervical squamous cell carcinoma)cells and SW480(colon adenocarcinoma)cells.Antitumor efficacy in a mouse xenograft model was also examined.The molecular mechanism of the anti-tumor effect and c-Myc suppression by SeV/dF/FIR was examined using Spliceostatin A(SSA),a SAP155 inhibitor,or SAP155siRNA which induce c-Myc by increasing FIR△exon2 in HeLa cells.RESULTS:FIR was found to repress c-myc transcription and in turn the overexpression of FIR drove apoptosis through c-myc suppression.Thus,FIR expressing vectors are potentially applicable for cancer therapy.FIR is alternatively spliced by SAP155 in cancer cells lacking the transcriptional repression domain within exon 2(FIR△exon2),counteracting FIR for c-Myc protein expression.Furthermore,FIR forms a complex with SAP155 and inhibits mutual well-established functions.Thus,both the valuable effects and side effects of exogenous FIR stimuli should be tested for future clinical application.SeV/dF/FIR,a cytoplasmic RNA virus,was successfully prepared and showed highly efficient gene transduction in in vivo experiments.Furthermore,in nude mouse tumor xenograft models,SeV/dF/FIR displayed high antitumor efficiency against human cancer cells.SeV/dF/FIR suppressed SSA-activated c-Myc.SAP155 siRNA,potentially produces FIR△exon2,and led to c-Myc overexpression with phosphorylation at Ser62.HA-FIR suppressed endogenous c-Myc expression and induced apoptosis in HeLa and SW480 cells.A c-myc transcriptional suppressor FIR expressing SeV/dF/FIR showed high gene transduction efficiency with significant antitumor effects and apoptosis induction in HeLa and SW480 cells.CONCLUSION:SeV/dF/FIR showed strong tumor growth suppression with no significant side effects in an animal xenograft model,thus SeV/dF/FIR is potentially applicable for future clinical cancer treatment.展开更多
基金Supported by the National Natural Science Foundation of China(No.81570814)Natural Science Foundation of Guangdong Province,China(No.2014A030313363)
文摘Pterygium is a common ocular surface disease induced by a variety of factors. The exact pathogenesis of pterygium remains unclear. Numbers of genes and proteins are discovered in pterygium and they function differently in the occurrence and development of this disease. We searched the Web of Science and PubMed throughout history for literatures about the subject. The keywords we used contain pterygium, gene, protein, angiogenesis, fibrosis, proliferation, inflammation, pathogenesis and therapy. In this review, we summarize the aberrant expression of a range of genes and proteins in pterygium compared with normal conjunctiva or cornea, including growth factors, matrix metalloproteinases and tissue inhibitors of mefalloproteinases, interleukins, tumor suppressor genes, proliferation related proteins, apoptosis related proteins, cell adhesion molecules, extracellular matrix proteins, heat shock proteins and tight junction proteins. We illustrate their possible mechanisms in the pathogenesis of pterygium as well as the related intervention based on them for pterygium therapy.
文摘FUS1 is a novel candidate tumor suppressor gene identified in human chromosome 3p21.3. Its expression showed significantly reduction or even loss in lung cancer and other types of cancers. In order to further investigate the biological function of FUS1 protein, FUS1 cDNA from MRC-5 cells was amplified by RT-PCR and cloned into prokaryotic expression vector pQE-30. The recombinant expression plasmids were transformed into M15 strain and grown at 20℃ or 37℃. SDS–PAGE analysis revealed that the accumulation of the recombinant protein FUS1 (rFUS1) in inclusion body forms reached maxium amount when induced with 0.5 mM IPTG for 5 h at 37℃. The inclusion bodies were solubilized in 2M urea and purified by a 6 ×His tagged affinity column under denaturing condition. The purified rFUS1 was identified by electrospray ionization-mass spectrometry (ESI-MS) and tested for purity by HPLC chromatography. The purified rFUS1 proteins were then used to immunize rabbits to obtain anti-human FUS1 polyclonal antibodies, which were suitable to detect both the recombinant exogenous FUS1 and the endogenous FUS1 from tissues and cells by western blot and immunohistochemistry, Available purified rFUS1 proteins and self-prepared polyclonal antibodies against FUS1 may provide effective tools for further studies on biological function and application of FUS1.
基金Scientific Research Fund of Sichuan Provincial Education Department(20003531)
文摘Objective:To investigate the effect of hepatitis C virus non-structural protein 4B(HCV NS4B) on c-Myc, P53, ras gene expression" and apoptosis in hepatic cells and study the possible role that NS4B played in the carcinogenesis of heparoma. Methods: The recombinant plasmid(PCXN2-NS4B, PCXN2-P53) and the empty, vector were transfected or co-transfected into Chang liver cells with liposome. Screening was performed with G418. Plasmid mRNA was detected by RT-PCR. The pro rein expressions of c-Myc and ras genes were analyzed by immunocytochemistry. The expressions of wild-type P53 (wtp53) gene were detected by in situ hybridization. TUNEL(flow cytometry) was used for assessing the rate of apoptosis. Results:No expression of c-Myc gene was found in PCXN2 group. The expression of c-Myc gene in NS4B group was 21.3% + 1.2%. The ex pression of ras gene in PCXN2 group was lower than that in NS4B group. Compared with PCXN2 group, the expression of P53 mRNA was not promoted or inhibited in NS4B group. But the expression of P53 mRNA in NS4B-P53 group was lower than that in P53 group. In PCXN2, NS4B, P53 and NS4B-P53 group, the rates of apoptosis were 17.02% ± 1.24%, 11.94% ± 2.24%, 25.84% ± 3.49% and 18.34% ± 1.55% respectively. Conclusion :HCV NS4B induces the expression of c-Myc and ras gene. HCV NS4B may play a role in the inhibition of cell death through P53-dependent manner. Results from this study suggested that HCV NS4B might contribute to the viral carcinogenesis.
文摘MCM10 protein is an essential replication factor involved in the initiation of DNA replication. A mcm10 mutan (mcm10-1) of budding yeast shows a growth arrest at 37 C. In the present work, we have isolated a mcm10-1 suppressor strain, which grows at 37 C. Interestingly, this mcm10-1 suppressor undergoes cell Cycle arrest at 14 C. A novel gene, YLR003c, is identified by high-copy complementation of this suppressor. We called it as Cms1 (Complementation of Mcm 10 Suppressor). Furthermore, the experiments of trans- formation show that cells of mcm10-1 suppressor with high-copy plasmid but not low-copy plasmid grow at 14 C, indicating that overexpression of Cms1 can rescue the growth arrest of this mcm10 suppressor at non-permissive temperature. These results suggest that CMS1 protein may functionally interact with MCM10 protein and play a role in the regulation of DNA replication and cell cycle control.
文摘目的探索乳腺癌/卵巢癌易感基因1相关蛋白1(breast/ovarian cancer susceptibility gene 1 associated protein 1,BAP1)对人源恶性胶质瘤发生、发展的作用与BAP1作为恶性胶质瘤临床诊断标志物的可行性。方法基于基因表达综合数据库(gene expression omnibus,GEO)的子数据集GSE4290,GSE90598,分析BAP1在正常组织及胶质瘤组织中的差异性表达情况;受试者工作特征(receiver operating characteristic,ROC)曲线分析BAP1对恶性胶质瘤的早期诊断价值;选取自主收集的非配对28例恶性胶质瘤患者的原发灶组织、5例颅脑外伤患者内减压术切除的非瘤脑组织,采用实时荧光定量PCR(quantitative real-time polymerase chain reaction,qRT-PCR)检测BAP1的表达水平;利用靶向BAP1的特异性小干扰RNAs(small interfering RNAs,siRNAs)瞬时转染U251细胞系,进一步检测其干涉效率;基于流式细胞仪分析BAP1下调的U251细胞系,其细胞周期、凋亡的变化情况。结果生物信息学结果显示,BAP1在恶性胶质瘤组织中的表达水平均低于正常脑组织(GSE4290:1209±18.49 vs 1476±53.90;GSE90598:5.19±0.10 vs 5.65±0.21),差异具有统计学意义(t=5.115,2.267,均P<0.05)。ROC曲线显示,BAP1可高效区分恶性胶质瘤组织与正常脑组织(GSE4290:AUC=0.78;GSE90598:AUC=0.75,均P<0.05)。临床标本结果显示,BAP1在恶性胶质瘤原发灶组织中的表达水平显著低于非瘤脑组织(0.27±0.04 vs 1.06±0.07),差异具有统计学意义(t=10.22,P<0.001)。在U251细胞系中下调BAP1的表达,其细胞周期中S期细胞比例明显增多,由17.59%分别增至27.21%(siBAP1-1)和25.79%(siBAP1-2),差异具有统计学意义(t=6.576,6.642,均P<0.01),而细胞凋亡水平则有所下降,由10.17%分别降至2.70%(siBAP-1)和3.00%(siBAP-2),差异具有统计学意义(t=10.31,9.428,均P<0.01)。结论组蛋白H2A去泛素化酶BAP1能够通过抑制恶性胶质瘤细胞周期快速进展并促进其凋亡,进而发挥肿瘤抑癌基因的功能,可作为潜在的恶性胶质瘤临床诊断标志物。
基金Supported by Grant-in-aid from Specialized Research Fund for the Doctoral Program of Higher Education,No. 20091202110009Grant-in-aid from Natural Science Foundation of Tianjin,No. 10JCYBJC11300
文摘AIM:To investigate the promoter region methylation status of retinoblastoma protein-interacting zinc finger gene 1(RIZ1) in the human esophageal squamous cell carcinoma(ESCC) cell lines and tissues and verify the relationship between methylation of RIZ1 and oncogenesis,tumor progression and metastasis etc of ESCC.METHODS:Methylation-specific polymerase chain reaction(MSP) was used to investigate the promoter region methylation status of RIZ1 in 6 ESCC cell lines.One cell line where RIZ1 promoter region methylation was detected was selected for the next study,where the cell line was treated with 5-aza-CdR.Real-time polymerase chain reaction was used to investigate its influence on the transcription of RIZ1.Experiments using frozenpathological specimens from 47 ESCC patients were performed using the same MSP methodology.RESULTS:Promoter methylation of RIZ1 gene was detected in TE13,CaEs17 and EC109 cell lines and the cell line TE13 was chosen for further study.The expression of RIZ1 mRNA in TE-13 was up-regulated after treatment with 5-aza-CdR.The rate of methylation in carcinomas tissues was significantly higher than those in matched neighboring normal and distal ending normal tissue,and the deviation of data was statistically significant(χ 2 = 24.136,P < 0.01).Analysis of the gender,age familial history,tumour deviation,tumour saturation,lymph gland displacement and clinical staging of 47 samples from ESCC patients showed that the fluctuation of data was not statistically significant.CONCLUSION:Promoter methylation may play an important role in the epigenetic silencing of RIZ1 gene expression in human ESCC.RIZ1 is considered to be a potential tumor suppressor gene and may be a biological parameter for testing early stage human ESCC.
基金Supported by In part by the 21st Century COE(Center Of Ex-cellence)Programs to Dr.Takenori Ochiaiby a Grant-in-Aid 18591453 to K.M from the Ministry of Education,Science,Sports and Culture of Japan
文摘AIM:To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element(FUSE)-binding protein-interacting repressor(FIR).METHODS:Endogenous c-Myc suppression and apoptosis induction by a transient FIR-expressing vector was examined in vivo via a HA-tagged FIR(HA-FIR)expression vector.A fusion gene-deficient,non-transmissible,Sendai virus(SeV)vector encoding FIR cDNA,SeV/dF/FIR,was prepared.SeV/dF/FIR was examined for its gene transduction efficiency,viral dose dependency of antitumor effect and apoptosis induction in HeLa(cervical squamous cell carcinoma)cells and SW480(colon adenocarcinoma)cells.Antitumor efficacy in a mouse xenograft model was also examined.The molecular mechanism of the anti-tumor effect and c-Myc suppression by SeV/dF/FIR was examined using Spliceostatin A(SSA),a SAP155 inhibitor,or SAP155siRNA which induce c-Myc by increasing FIR△exon2 in HeLa cells.RESULTS:FIR was found to repress c-myc transcription and in turn the overexpression of FIR drove apoptosis through c-myc suppression.Thus,FIR expressing vectors are potentially applicable for cancer therapy.FIR is alternatively spliced by SAP155 in cancer cells lacking the transcriptional repression domain within exon 2(FIR△exon2),counteracting FIR for c-Myc protein expression.Furthermore,FIR forms a complex with SAP155 and inhibits mutual well-established functions.Thus,both the valuable effects and side effects of exogenous FIR stimuli should be tested for future clinical application.SeV/dF/FIR,a cytoplasmic RNA virus,was successfully prepared and showed highly efficient gene transduction in in vivo experiments.Furthermore,in nude mouse tumor xenograft models,SeV/dF/FIR displayed high antitumor efficiency against human cancer cells.SeV/dF/FIR suppressed SSA-activated c-Myc.SAP155 siRNA,potentially produces FIR△exon2,and led to c-Myc overexpression with phosphorylation at Ser62.HA-FIR suppressed endogenous c-Myc expression and induced apoptosis in HeLa and SW480 cells.A c-myc transcriptional suppressor FIR expressing SeV/dF/FIR showed high gene transduction efficiency with significant antitumor effects and apoptosis induction in HeLa and SW480 cells.CONCLUSION:SeV/dF/FIR showed strong tumor growth suppression with no significant side effects in an animal xenograft model,thus SeV/dF/FIR is potentially applicable for future clinical cancer treatment.