AIM: To study the inhibitory effect of baculovirus- mediated normal epithelial cell specific-1 (NES1) gene therapy on gastric cancer (GC) in vitro and in vivo. METHODS: We first constructed recombinant baculovirus vec...AIM: To study the inhibitory effect of baculovirus- mediated normal epithelial cell specific-1 (NES1) gene therapy on gastric cancer (GC) in vitro and in vivo. METHODS: We first constructed recombinant baculovirus vectors and then transfected them into gastric cancer cells (SGC-7901). Efficiency of the baculovirus for gene transfer into SGC-7901 cells and cell growth curves were detected by fluorescence microscopy, Western blot and 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro, respectively. The therapeutic effect of this gene therapy on GC was confirmed in xenografted nude mice. Tumor growth was determined by tumor volume, and expression of NES1 in tumor was analyzed by immunohistochemistry. RESULTS: Baculovirus vectors were successfully transfected into SGC-7901 cells. SGC-7901 cells transfected with the NES1 gene inhibited cell growth. In the Bac-NES1 treated group, tumor growth was significantly reduced with a high level of NES1 expression CONCLUSION: Baculovirus-mediated NES1 gene can be used in gene therapy for GC.展开更多
Objective:To explore HtrA1 gene expression aud its regulation in human gastric cancers.Methods:The HtrA1 mRNA levels were examined by QPCR analysis and coufirmed its expression with Northern blot analysis.The HtrA1 pr...Objective:To explore HtrA1 gene expression aud its regulation in human gastric cancers.Methods:The HtrA1 mRNA levels were examined by QPCR analysis and coufirmed its expression with Northern blot analysis.The HtrA1 protein levels in all six gastric epithelial cell lines were investigated by Western blot analysis.Gene copy number was accessed and then sequenced the coding region from each mRNA in all six cell lines.The HtrA1 promoter region DNA methylation status was detected by using bisulfite sequeucing analysis.Effect of decitabine and TSA on HTRA1 expression in gastric cancer cell line was determined by RTPCR.Results:HIC analysis indicated that HtrA1 was highly expressed in normal epithelium,but dramatically down-regulated in gastric carcinoma tissues and variably expressed in tumor-adjacent tissues.HtrA1 gene expression was dramatically decreased in gastric carcinoma cells compared to nontumorigenic counterparts.The HtrA1 gene loss in any of the 4 breast cancer cell lines was not detected.Total 14 CpGs in this region were all methylated in gastric cancer cells,whereas two normal cells.GES-1 and HFI-145,were having several unmethylated cytosines in this region.HtrA1 showed as^Mr 44,000,Expression of HtrA1 protein was not observed in any of the four gastric caucer cell lines.BGC-823.MKN-45.SGC-7901and MKN-28.HtrA1 expression was observed in the HF1-145and GES-1 cell lines.Conclusions:The epigenetic silencing for HtrA1gene expression could provide a possible strategy for re-activating Htrt1 gene expression in gastric cancer cells.thus facilitating further investigation of HtrA1's role in chemotherapy.展开更多
Objective To investigate trichorhinophalangeal syndrome 1 gene(TRPS-1)expression patterns in different subtypes of breast cancer and its correlations with other genes and survival using microarray data sets.Methods Th...Objective To investigate trichorhinophalangeal syndrome 1 gene(TRPS-1)expression patterns in different subtypes of breast cancer and its correlations with other genes and survival using microarray data sets.Methods The transcripts of TRPS-1 and its role in survival in breast cancer were analyzed using published microarray data sets—Netherlands Cancer Institute(NKI)cohort and Wang cohort.Results TRPS-1 expression was lower in basal-like breast cancer.The mRNA levels of TRPS-1negatively correlated with Slug(Pearson correlation coefficient=0.1366,P=0.0189 in NKI data set and Pearson correlation coefficient=0.1571,P=0.0078 in Wang data set),FOXC1(Pearson correlation coefficient=0.1211,P=0.0376 in NKI data set and Pearson correlation coefficient=0.1709,P=0.0037 in Wang data set),and CXCL1(Pearson correlation coefficient=0.1197,P=0.0399 in NKI data set and Pearson correlation coefficient=0.3436,P<0.0001 in Wang data set),but positively correlated with BRCA1(Pearson correlation coefficient=0.1728,P=0.0029 in NKI data set and Pearson correlation coefficient=0.1805,P=0.0022 in Wang data set).Low TRPS-1 expression associated with poor overall survival(hazard ratio 1.79,95%CI of ratio 0.9894 to 3.238,P=0.054)and relapse-free survival(hazard ratio 1.913,95%CI of ratio1.159 to 3.156,P<0.05).The low TRPS-1 mRNA levels predicted poor outcome in breast cancer patients by the 70-gene signature.Conclusion The strong expression of TRPS-1 may serve as a good prognostic marker in breast cancer.展开更多
AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of...AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.展开更多
基金The Doctoral Fund from the Ministry of Education of China, No. BXJ0710
文摘AIM: To study the inhibitory effect of baculovirus- mediated normal epithelial cell specific-1 (NES1) gene therapy on gastric cancer (GC) in vitro and in vivo. METHODS: We first constructed recombinant baculovirus vectors and then transfected them into gastric cancer cells (SGC-7901). Efficiency of the baculovirus for gene transfer into SGC-7901 cells and cell growth curves were detected by fluorescence microscopy, Western blot and 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in vitro, respectively. The therapeutic effect of this gene therapy on GC was confirmed in xenografted nude mice. Tumor growth was determined by tumor volume, and expression of NES1 in tumor was analyzed by immunohistochemistry. RESULTS: Baculovirus vectors were successfully transfected into SGC-7901 cells. SGC-7901 cells transfected with the NES1 gene inhibited cell growth. In the Bac-NES1 treated group, tumor growth was significantly reduced with a high level of NES1 expression CONCLUSION: Baculovirus-mediated NES1 gene can be used in gene therapy for GC.
基金supported by Natural Science and Technology Fund of Hubei Povince(012726334)
文摘Objective:To explore HtrA1 gene expression aud its regulation in human gastric cancers.Methods:The HtrA1 mRNA levels were examined by QPCR analysis and coufirmed its expression with Northern blot analysis.The HtrA1 protein levels in all six gastric epithelial cell lines were investigated by Western blot analysis.Gene copy number was accessed and then sequenced the coding region from each mRNA in all six cell lines.The HtrA1 promoter region DNA methylation status was detected by using bisulfite sequeucing analysis.Effect of decitabine and TSA on HTRA1 expression in gastric cancer cell line was determined by RTPCR.Results:HIC analysis indicated that HtrA1 was highly expressed in normal epithelium,but dramatically down-regulated in gastric carcinoma tissues and variably expressed in tumor-adjacent tissues.HtrA1 gene expression was dramatically decreased in gastric carcinoma cells compared to nontumorigenic counterparts.The HtrA1 gene loss in any of the 4 breast cancer cell lines was not detected.Total 14 CpGs in this region were all methylated in gastric cancer cells,whereas two normal cells.GES-1 and HFI-145,were having several unmethylated cytosines in this region.HtrA1 showed as^Mr 44,000,Expression of HtrA1 protein was not observed in any of the four gastric caucer cell lines.BGC-823.MKN-45.SGC-7901and MKN-28.HtrA1 expression was observed in the HF1-145and GES-1 cell lines.Conclusions:The epigenetic silencing for HtrA1gene expression could provide a possible strategy for re-activating Htrt1 gene expression in gastric cancer cells.thus facilitating further investigation of HtrA1's role in chemotherapy.
基金Supported by grants from Chinese National Science Fund for Young Scholars(81101707)Traditional Chinese Medicine Foundation Project of Zhejiang Province(2011ZA104)Science and Technology Bureau of Jiaxing(2012AY1071-2)
文摘Objective To investigate trichorhinophalangeal syndrome 1 gene(TRPS-1)expression patterns in different subtypes of breast cancer and its correlations with other genes and survival using microarray data sets.Methods The transcripts of TRPS-1 and its role in survival in breast cancer were analyzed using published microarray data sets—Netherlands Cancer Institute(NKI)cohort and Wang cohort.Results TRPS-1 expression was lower in basal-like breast cancer.The mRNA levels of TRPS-1negatively correlated with Slug(Pearson correlation coefficient=0.1366,P=0.0189 in NKI data set and Pearson correlation coefficient=0.1571,P=0.0078 in Wang data set),FOXC1(Pearson correlation coefficient=0.1211,P=0.0376 in NKI data set and Pearson correlation coefficient=0.1709,P=0.0037 in Wang data set),and CXCL1(Pearson correlation coefficient=0.1197,P=0.0399 in NKI data set and Pearson correlation coefficient=0.3436,P<0.0001 in Wang data set),but positively correlated with BRCA1(Pearson correlation coefficient=0.1728,P=0.0029 in NKI data set and Pearson correlation coefficient=0.1805,P=0.0022 in Wang data set).Low TRPS-1 expression associated with poor overall survival(hazard ratio 1.79,95%CI of ratio 0.9894 to 3.238,P=0.054)and relapse-free survival(hazard ratio 1.913,95%CI of ratio1.159 to 3.156,P<0.05).The low TRPS-1 mRNA levels predicted poor outcome in breast cancer patients by the 70-gene signature.Conclusion The strong expression of TRPS-1 may serve as a good prognostic marker in breast cancer.
基金Supported by National Natural Science Foundation of China(No.81273212,81100651)Project of Science and Technology of Shandong Province(No.2014GSF118044)
文摘AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.
文摘目的:探讨上皮膜蛋白1(epithelial membrane protejn-1,EMP1)在人结直肠癌(colorectal carcinoma,CRC)组织中的表达水平及其过表达对CRC SW-480细胞增殖、凋亡和侵袭的影响。方法:免疫组织化学、Western blotting方法检测63例CRC组织及31例癌旁组织中EMP1的表达水平,分析EMP1表达与CRC临床病理参数的关系。慢病毒介导将pLenti6-EMP1质粒转染SW-480细胞,建立EMP1过表达细胞,转染plenti6/V5-DEST空白质粒为对照。Real-time PCR及Western blotting检测转染后SW-480细胞株中EMP1的表达,MTT、流式细胞术及Transwell实验分别检测EMP1过表达对SW-480细胞增殖、凋亡及侵袭能力的影响。结果:EMP1蛋白在人CRC组织的表达显著低于癌旁组织(0.257±0.022 vs 0.863±0.086,P<0.05),并且其表达水平在不同T分期、有无淋巴结转移、临床分期以及组织分级组间表达有显著差异(P<0.05),而与患者年龄、性别、肿瘤部位无关(P>0.05)。成功构建EMP1过表达的LeEMP1细胞。与LeEmpty细胞相比,LeEMP1细胞的增殖能力显著下降[(60.94±4.04)%vs(100.00±0.00)%,P<0.05],凋亡率显著升高[(12.10±1.30)%vs(3.10±0.60)%,P<0.05]、侵袭转移能力显著降低[穿膜细胞数:(87.00±12.00)vs(178.00±21.00)个,P<0.05]。LeEMP1细胞Caspase-9表达显著高于LeEmpty细胞(0.764±0.073 vs 0.231±0.029,P<0.05),VEGFC表达显著降低(0.185±0.022 vs 0.663±0.065,P<0.05)。结论:人CRC组织中EMP1蛋白表达明显减低,过表达EMP1能够抑制CRC SW-480细胞的恶性生物学行为,其可能通过调控Caspase-9和VEGFC的表达来发挥作用。
