The effect of matrix attachment regions (MARs) on foreign gene expression in transgenic plants was studied, The beta-glucuronidase (GUS) gene (uidA) was flanked by the MARs isolated from the genome of maize to form pl...The effect of matrix attachment regions (MARs) on foreign gene expression in transgenic plants was studied, The beta-glucuronidase (GUS) gene (uidA) was flanked by the MARs isolated from the genome of maize to form plant expression vector. The vectors with and without MARs were transferred into tobacco ( Nicotiana tabacum L.) through Agrobacterium-mediated transformation procedure. GUS activity assays indicated that MARs could increase expression level of uidA gene. The mean GUS activity could be increased twofold as compared to that of transformants without MARs, and the highest GUS activity of transformant could reach tenfold. The correspondence between GUS activity and mRNA accumulation was positive and indicated that MARs could improve transcription of foreign gene.展开更多
Objective: To elucidate the relation between human tissue factor pathwayinhibitor-2 (TFPI-2) expression and ovarian tumor migration and invasion. Methods: Human TFPI-2expression vector pBos-Cite-neo/TFPI-2 was transfe...Objective: To elucidate the relation between human tissue factor pathwayinhibitor-2 (TFPI-2) expression and ovarian tumor migration and invasion. Methods: Human TFPI-2expression vector pBos-Cite-neo/TFPI-2 was transfected into ovarian tumor cells line A2780- Afterthe transfected cells were selected by G418, transfected and nontransfected cells were screened forTFPI-2 mRNA and protein by reverse transcription-polymerase chain reaction and Western blotanalysis, respectively. The number of transfected or nontransfected cells passing through membraneof Boyden chamber was counted as the basis assessing tumor cells migratory and invasive behaviors.Results: Expression of mRNA and protein of TFPI-2 was detectable in transfected cells. In invasionassay, the number of TFPI-2-expressing cells to traverse a Matrigel-coated membrane was obviouslydecreased compared with that of nonexpressing cells (59.3±6.5 vs 109.7±5.5, P 【 0.01); While inmigration assay, no significant difference through a noncoated membrane was observed amongtransfected and nontransfected cells (114.7±8.6 vs 127.3±7.1, P 】 0.05). Conclusion: Expression ofTFPI-2 may strongly inhibit the invasive ability of ovarian tumor cells in vitro, but has no effecton the migratory ability which provides an experimental basis for genotherapy of human ovariantumor.展开更多
AIM： To examine the association of beta-catenin with the clinicopathologic features and prognosis of esophageal squamous cell carcinoma （ESCC）. METHODS： Beta-catenin mRNA expression level in 40 ESCC patients （28 ...AIM： To examine the association of beta-catenin with the clinicopathologic features and prognosis of esophageal squamous cell carcinoma （ESCC）. METHODS： Beta-catenin mRNA expression level in 40 ESCC patients （28 males and 12 females, age range 38-82 years, median 60 years） was analyzed by real- time PCR. Beta-catenin mRNA expression levels in tumor cells were categorized as weaker （level 1） or equal to or stronger （level 2） than those in endothelial cells. We examined the correlation between the beta-catenin expression and the clinicopathological factors and prognosis of ESCC patients. RESULTS： Level 2 beta-catenin expression was found in 29 patients. ESCC with level 2 expression had a higher rate of lymphnode metastasis （0.0776±0.0369 vs 0.3413±0.1803, P 〈 0.001） and deeper tumor invasion （0.0751±0.0356 vs 0.3667±0.1928, P 〈 0.001）, and a poorer survival rate （P = 0.0024） than ESCC with level I expression. CONCLUSION： Beta-catenin expression in ESCC is of great importance.展开更多
AIM:To investigate the contribution of periostin in nicotine-promoted gastric cancer cell proliferation,survival,invasion,drug resistance,and epithelial-mesenchymal transition(EMT).METHODS:Gastric cancer cells were tr...AIM:To investigate the contribution of periostin in nicotine-promoted gastric cancer cell proliferation,survival,invasion,drug resistance,and epithelial-mesenchymal transition(EMT).METHODS:Gastric cancer cells were treated with nicotine and periostin protein expression was determined by immunoblotting.Periostin mRNA in gastric cancer cells was silenced using small interfering RNA(siRNA) techniques and periostin gene expression was evaluated by quantitative reverse transcription-polymerase chain reaction.Gastric cancer cells transfected with control or periostin siRNA plasmid were compared in terms of cell proliferation using the methylthiazolyldiphenyl-tetrazolium bromide assay.Cell apoptosis was compared using annexin V-fluoresceine isothiocyanate and propidium iodine double staining.Tumor invasion was determined using the Boyden chamber invasion assay,and the EMT marker Snail expression was evaluated by immunoblotting.RESULTS:Nicotine upregulated periostin in gastric cancer cells through a COX-2 dependent pathway,which was blocked by the COX-2-specific inhibitor NS398.Periostin mRNA expression was decreased by ～87.2% by siRNA in gastric cancer cells,and stable periostinsilenced cells were obtained by G418 screening.Periostin-silenced gastric cancer cells exhibited reduced cell proliferation,elevated sensitivity to chemotherapy with 5-fluorouracil,and decreased cell invasion and Snail expression(P < 0.05).CONCLUSION:Periostin is a nicotine target gene in gastric cancer and plays a role in gastric cancer cell growth,invasion,drug resistance,and EMT facilitated by nicotine.展开更多
MicroRNAs are short regulatory RNAs that negatively modulate gene expression at the post-transcriptional level, and are deeply involved in the pathogenesis of several types of cancers. To investigate whether specific ...MicroRNAs are short regulatory RNAs that negatively modulate gene expression at the post-transcriptional level, and are deeply involved in the pathogenesis of several types of cancers. To investigate whether specific miRNAs and their target genes participate in the molecular pathogenesis of laryngeal carcinoma, oligonucleotide microarrays were used to assess the differential expression profiles of microRNAs and mRNAs in laryngeal carcinoma tissues compared with normal tissues. The oncogeuic miRNA, microRNA-21 （miR-21）, was found to he npregulated in laryngeal carcinoma tissues. Knockdown of miR-21 by specific antisense oligonucleotides inhibited the proliferation potential of HEp-2 cells, whereas overexpression of miR-21 elevated growth activity of the cells, as detected by the colony formation assay. The cell number reduction caused by miR-21 inhibition was due to the loss of control of the G1-S phase transition, instead of a noticeable increase in apoptosis. Subsequently, a new target gene of miR- 21, BTG2, was found to be downregulated in laryngeal carcinoma tissues. BTG2 is known to act as a pan-cell cycle regulator and tumor suppressor. These findings indicate that aberrant expression of miR-21 may contribute to the malignant phenotype of laryngeal carcinoma by maintaining a low level of BTG2. The identification of the oneogenic miR-21 and its target gene, BTG2, in laryngeal carcinoma is potentially valuable for cancer diagnosis and therapy.展开更多
AIM:To investigate the effect of glyceraldehyde-derived advanced glycation end-products(Glycer-AGEs) on hepatocellular carcinoma(HCC)cells.METHODS:Two HCC cell lines(Hep3B and HepG2 cells)and human umbilical vein endo...AIM:To investigate the effect of glyceraldehyde-derived advanced glycation end-products(Glycer-AGEs) on hepatocellular carcinoma(HCC)cells.METHODS:Two HCC cell lines(Hep3B and HepG2 cells)and human umbilical vein endothelial cells(HUVEC)were used.Cell viability was determined using the WST-8 assay.Western blotting,enzyme linked immunosorbent assay,and real-time reverse transcriptionpolymerase chain reactions were used to detect protein and mRNA.Angiogenesis was evaluated by assessing the proliferation,migration,and tube formation of HUVEC.RESULTS:The receptor for AGEs(RAGE)protein was detected in Hep3B and HepG2 cells.HepG2 cells werenot affected by the addition of Glycer-AGEs.GlycerAGEs markedly increased vascular endothelial growth factor(VEGF)mRNA and protein expression,which is one of the most potent angiogenic factors.Compared with the control unglycated bovine serum albumin(BSA) treatment,VEGF mRNA expression levels induced by the Glycer-AGEs treatment were 1.00±0.10 vs 1.92 ±0.09(P<0.01).Similarly,protein expression levels induced by the Glycer-AGEs treatment were 1.63±0.04 ng/mL vs 2.28±0.17 ng/mL for the 24 h treatment and 3.36±0.10 ng/mL vs 4.79±0.31 ng/mL for the 48 h treatment,respectively(P<0.01).Furthermore,compared with the effect of the control unglycated BSA-treated conditioned medium,the Glycer-AGEstreated conditioned medium significantly increased the proliferation,migration,and tube formation of HUVEC,with values of 122.4%±9.0%vs 144.5%±11.3%for cell viability,4.29±1.53 vs 6.78±1.84 for migration indices,and 71.0±7.5 vs 112.4±8.0 for the number of branching points,respectively(P<0.01).CONCLUSION:These results suggest that Glycer-AGEs-RAGE signaling enhances the angiogenic potential of HCC cells by upregulating VEGF expression.展开更多
AIM: To investigate the expression levels of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), vascular endothelial growth factor receptor-3 (VEGFR-3) and CD44 genes and the relationship between their lev- ...AIM: To investigate the expression levels of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), vascular endothelial growth factor receptor-3 (VEGFR-3) and CD44 genes and the relationship between their lev- els and clinicopathological parameters in gastric cancer.METHODS: Tissue samples were obtained from 33 patients (8 females) with gastric cancer. mRNA levels of LYVE-1, VEGFR-3 and CD44 in normal and tumor tissues were quantitatively measured using real time polymerase chain reaction. The results were correlated with lymph node metastasis, histological type and differentiation of the tumor, T-stage, and presence of vascular, perineural and lymphatic invasions. The distribution of molecules in the tissue was evaluated using immunohistochemistry. RESULTS: LYVE-1, CD44 and VEGFR-3 gene expression levels were significantly higher in gastric cancer than in normal tissue. While there was no correlation between gene expressions and clinicopathologic fea- tures such as histologic type, differentiation and stage, gene expression levels were found to be increased in conjunction with positive lymph node/total lymph node ratio and the presence of perineural invasion. A significant correlation was also found between LYVE-1 and CD44 over-expressions and perineural invasion and lymph node positivity in gastric cancers. When the dis- tribution of LYVE-1 antibody-stained lymphatic vessels in tissue was evaluated, lymphatic vessels were located intra-tumorally in 13% and peri-tumorally in 27% of the patients. Moreover, lymph node metastases were also positive in all patients with LYVE-1-staining. CONCLUSION: LYVE-1, VEGFR-3 and CD44 all play an important role in lymphangiogenesis, invasion and metastasis. LYVE-1 is a perfectly reliable lymphatic vessel marker and useful for immunohistochemistry.展开更多
Dishevelled （Dvl） is a highly conserved protein family that plays an important role in mediating Wnt signal- ing from membrane to cytoplasm. Recently we reported that Dvl also functions in the nucleus by stabilizing...Dishevelled （Dvl） is a highly conserved protein family that plays an important role in mediating Wnt signal- ing from membrane to cytoplasm. Recently we reported that Dvl also functions in the nucleus by stabilizing the β-catenin/TCFs transcriptional complex. Here we describe that Dvl may function as a repressor of NF-kB. Our data show that Dvl directly binds to p65 and their interaction occurs in the nucleus. Dvl expression inhibits p65-mediated or TNF-α-stimulated activation of the NF-KB dependent reporter. This action of Dvl, however, is not dependent on Wnt or its downstream effector β-catenin. Chromatin immunoprecipitation assay shows that recruitment of p65 to the promoters of NF-KB target genes is significantly enhanced when expression of Dvl is knocked down. Consistently, the expression level of a subset of NF-KB target genes is also increased after knock-down of Dvl. Moreover, our data suggest that Dvl may relieve the anti-apoptotic effect of NF-KB, thus play a role in promoting apoptosis. Therefore, this work demonstrates a novel function of Dvl in modulating NF-KB-regulated gene transcription.展开更多
文摘The effect of matrix attachment regions (MARs) on foreign gene expression in transgenic plants was studied, The beta-glucuronidase (GUS) gene (uidA) was flanked by the MARs isolated from the genome of maize to form plant expression vector. The vectors with and without MARs were transferred into tobacco ( Nicotiana tabacum L.) through Agrobacterium-mediated transformation procedure. GUS activity assays indicated that MARs could increase expression level of uidA gene. The mean GUS activity could be increased twofold as compared to that of transformants without MARs, and the highest GUS activity of transformant could reach tenfold. The correspondence between GUS activity and mRNA accumulation was positive and indicated that MARs could improve transcription of foreign gene.
文摘Objective: To elucidate the relation between human tissue factor pathwayinhibitor-2 (TFPI-2) expression and ovarian tumor migration and invasion. Methods: Human TFPI-2expression vector pBos-Cite-neo/TFPI-2 was transfected into ovarian tumor cells line A2780- Afterthe transfected cells were selected by G418, transfected and nontransfected cells were screened forTFPI-2 mRNA and protein by reverse transcription-polymerase chain reaction and Western blotanalysis, respectively. The number of transfected or nontransfected cells passing through membraneof Boyden chamber was counted as the basis assessing tumor cells migratory and invasive behaviors.Results: Expression of mRNA and protein of TFPI-2 was detectable in transfected cells. In invasionassay, the number of TFPI-2-expressing cells to traverse a Matrigel-coated membrane was obviouslydecreased compared with that of nonexpressing cells (59.3±6.5 vs 109.7±5.5, P 【 0.01); While inmigration assay, no significant difference through a noncoated membrane was observed amongtransfected and nontransfected cells (114.7±8.6 vs 127.3±7.1, P 】 0.05). Conclusion: Expression ofTFPI-2 may strongly inhibit the invasive ability of ovarian tumor cells in vitro, but has no effecton the migratory ability which provides an experimental basis for genotherapy of human ovariantumor.
文摘AIM： To examine the association of beta-catenin with the clinicopathologic features and prognosis of esophageal squamous cell carcinoma （ESCC）. METHODS： Beta-catenin mRNA expression level in 40 ESCC patients （28 males and 12 females, age range 38-82 years, median 60 years） was analyzed by real- time PCR. Beta-catenin mRNA expression levels in tumor cells were categorized as weaker （level 1） or equal to or stronger （level 2） than those in endothelial cells. We examined the correlation between the beta-catenin expression and the clinicopathological factors and prognosis of ESCC patients. RESULTS： Level 2 beta-catenin expression was found in 29 patients. ESCC with level 2 expression had a higher rate of lymphnode metastasis （0.0776±0.0369 vs 0.3413±0.1803, P 〈 0.001） and deeper tumor invasion （0.0751±0.0356 vs 0.3667±0.1928, P 〈 0.001）, and a poorer survival rate （P = 0.0024） than ESCC with level I expression. CONCLUSION： Beta-catenin expression in ESCC is of great importance.
基金Supported by Department of Pathology,Division of Basic Medicine,Central South University Xiangya School of Medicine
文摘AIM:To investigate the contribution of periostin in nicotine-promoted gastric cancer cell proliferation,survival,invasion,drug resistance,and epithelial-mesenchymal transition(EMT).METHODS:Gastric cancer cells were treated with nicotine and periostin protein expression was determined by immunoblotting.Periostin mRNA in gastric cancer cells was silenced using small interfering RNA(siRNA) techniques and periostin gene expression was evaluated by quantitative reverse transcription-polymerase chain reaction.Gastric cancer cells transfected with control or periostin siRNA plasmid were compared in terms of cell proliferation using the methylthiazolyldiphenyl-tetrazolium bromide assay.Cell apoptosis was compared using annexin V-fluoresceine isothiocyanate and propidium iodine double staining.Tumor invasion was determined using the Boyden chamber invasion assay,and the EMT marker Snail expression was evaluated by immunoblotting.RESULTS:Nicotine upregulated periostin in gastric cancer cells through a COX-2 dependent pathway,which was blocked by the COX-2-specific inhibitor NS398.Periostin mRNA expression was decreased by ～87.2% by siRNA in gastric cancer cells,and stable periostinsilenced cells were obtained by G418 screening.Periostin-silenced gastric cancer cells exhibited reduced cell proliferation,elevated sensitivity to chemotherapy with 5-fluorouracil,and decreased cell invasion and Snail expression(P < 0.05).CONCLUSION:Periostin is a nicotine target gene in gastric cancer and plays a role in gastric cancer cell growth,invasion,drug resistance,and EMT facilitated by nicotine.
基金Acknowledgments This work was supported by grants from the National Natural Science Foundation of China （No. 30873017） and the Key Program of the Natural Science Foundation of Tianjing （No. 08JCZDJC23300）. We thank Tianjin First Center Hospital for providing human laryngeal tissue samples. We also thank the College of Public Health of Tianjin Medical University for the technical assistance in fluorescent detection. The ArrayExpress accession numbers of miRNA microarray design and cDNA microarray design are A-MEXP-1506 and A-MEXP-1511. The ArrayExpress accession numbers of miRNA microarray experiment and eDNA microarray experiment are E-MEXP-2039 and E-MEXP-2056.
文摘MicroRNAs are short regulatory RNAs that negatively modulate gene expression at the post-transcriptional level, and are deeply involved in the pathogenesis of several types of cancers. To investigate whether specific miRNAs and their target genes participate in the molecular pathogenesis of laryngeal carcinoma, oligonucleotide microarrays were used to assess the differential expression profiles of microRNAs and mRNAs in laryngeal carcinoma tissues compared with normal tissues. The oncogeuic miRNA, microRNA-21 （miR-21）, was found to he npregulated in laryngeal carcinoma tissues. Knockdown of miR-21 by specific antisense oligonucleotides inhibited the proliferation potential of HEp-2 cells, whereas overexpression of miR-21 elevated growth activity of the cells, as detected by the colony formation assay. The cell number reduction caused by miR-21 inhibition was due to the loss of control of the G1-S phase transition, instead of a noticeable increase in apoptosis. Subsequently, a new target gene of miR- 21, BTG2, was found to be downregulated in laryngeal carcinoma tissues. BTG2 is known to act as a pan-cell cycle regulator and tumor suppressor. These findings indicate that aberrant expression of miR-21 may contribute to the malignant phenotype of laryngeal carcinoma by maintaining a low level of BTG2. The identification of the oneogenic miR-21 and its target gene, BTG2, in laryngeal carcinoma is potentially valuable for cancer diagnosis and therapy.
基金Supported by Grants from the Japan Society for the Promotion of Science,Grant-in-Aid for Scientific Research(B),No.22300264
文摘AIM:To investigate the effect of glyceraldehyde-derived advanced glycation end-products(Glycer-AGEs) on hepatocellular carcinoma(HCC)cells.METHODS:Two HCC cell lines(Hep3B and HepG2 cells)and human umbilical vein endothelial cells(HUVEC)were used.Cell viability was determined using the WST-8 assay.Western blotting,enzyme linked immunosorbent assay,and real-time reverse transcriptionpolymerase chain reactions were used to detect protein and mRNA.Angiogenesis was evaluated by assessing the proliferation,migration,and tube formation of HUVEC.RESULTS:The receptor for AGEs(RAGE)protein was detected in Hep3B and HepG2 cells.HepG2 cells werenot affected by the addition of Glycer-AGEs.GlycerAGEs markedly increased vascular endothelial growth factor(VEGF)mRNA and protein expression,which is one of the most potent angiogenic factors.Compared with the control unglycated bovine serum albumin(BSA) treatment,VEGF mRNA expression levels induced by the Glycer-AGEs treatment were 1.00±0.10 vs 1.92 ±0.09(P<0.01).Similarly,protein expression levels induced by the Glycer-AGEs treatment were 1.63±0.04 ng/mL vs 2.28±0.17 ng/mL for the 24 h treatment and 3.36±0.10 ng/mL vs 4.79±0.31 ng/mL for the 48 h treatment,respectively(P<0.01).Furthermore,compared with the effect of the control unglycated BSA-treated conditioned medium,the Glycer-AGEstreated conditioned medium significantly increased the proliferation,migration,and tube formation of HUVEC,with values of 122.4%±9.0%vs 144.5%±11.3%for cell viability,4.29±1.53 vs 6.78±1.84 for migration indices,and 71.0±7.5 vs 112.4±8.0 for the number of branching points,respectively(P<0.01).CONCLUSION:These results suggest that Glycer-AGEs-RAGE signaling enhances the angiogenic potential of HCC cells by upregulating VEGF expression.
基金Supported by TUBTAK-SBAG (Project Number 104S581)the Turkish Academy of Sciences (TUBA)
文摘AIM: To investigate the expression levels of lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1), vascular endothelial growth factor receptor-3 (VEGFR-3) and CD44 genes and the relationship between their lev- els and clinicopathological parameters in gastric cancer.METHODS: Tissue samples were obtained from 33 patients (8 females) with gastric cancer. mRNA levels of LYVE-1, VEGFR-3 and CD44 in normal and tumor tissues were quantitatively measured using real time polymerase chain reaction. The results were correlated with lymph node metastasis, histological type and differentiation of the tumor, T-stage, and presence of vascular, perineural and lymphatic invasions. The distribution of molecules in the tissue was evaluated using immunohistochemistry. RESULTS: LYVE-1, CD44 and VEGFR-3 gene expression levels were significantly higher in gastric cancer than in normal tissue. While there was no correlation between gene expressions and clinicopathologic fea- tures such as histologic type, differentiation and stage, gene expression levels were found to be increased in conjunction with positive lymph node/total lymph node ratio and the presence of perineural invasion. A significant correlation was also found between LYVE-1 and CD44 over-expressions and perineural invasion and lymph node positivity in gastric cancers. When the dis- tribution of LYVE-1 antibody-stained lymphatic vessels in tissue was evaluated, lymphatic vessels were located intra-tumorally in 13% and peri-tumorally in 27% of the patients. Moreover, lymph node metastases were also positive in all patients with LYVE-1-staining. CONCLUSION: LYVE-1, VEGFR-3 and CD44 all play an important role in lymphangiogenesis, invasion and metastasis. LYVE-1 is a perfectly reliable lymphatic vessel marker and useful for immunohistochemistry.
基金This work was supported by the Ministry of Science and Technology of China （2010CB912100 and 2007CB914500）, the National Natural Science Foundation of China （30821065, 30930052 and 90813024）, and the Science and Technology Commission of Shanghai Municipality.
文摘Dishevelled （Dvl） is a highly conserved protein family that plays an important role in mediating Wnt signal- ing from membrane to cytoplasm. Recently we reported that Dvl also functions in the nucleus by stabilizing the β-catenin/TCFs transcriptional complex. Here we describe that Dvl may function as a repressor of NF-kB. Our data show that Dvl directly binds to p65 and their interaction occurs in the nucleus. Dvl expression inhibits p65-mediated or TNF-α-stimulated activation of the NF-KB dependent reporter. This action of Dvl, however, is not dependent on Wnt or its downstream effector β-catenin. Chromatin immunoprecipitation assay shows that recruitment of p65 to the promoters of NF-KB target genes is significantly enhanced when expression of Dvl is knocked down. Consistently, the expression level of a subset of NF-KB target genes is also increased after knock-down of Dvl. Moreover, our data suggest that Dvl may relieve the anti-apoptotic effect of NF-KB, thus play a role in promoting apoptosis. Therefore, this work demonstrates a novel function of Dvl in modulating NF-KB-regulated gene transcription.
