AIM： To study the changes of human telomerase reverse transcriptase （hTERT） mRNA expression in human hepatocarcinoma cell lines （HepG2） and cholangiocarcinoma cell lines （QBC939） after HBx gene transfection and...AIM： To study the changes of human telomerase reverse transcriptase （hTERT） mRNA expression in human hepatocarcinoma cell lines （HepG2） and cholangiocarcinoma cell lines （QBC939） after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis. METHODS： HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein （EGFP） coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency. Cells were harvested and total RNA was extracted using TRIzol reagent. The expression of hTERT mRNA in HepG2 and QBC939 cell lines was assayed by reverse transcriptionpolymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting. RESULTS： Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939 cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector. Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939 cells only when transfected with HBx gene. CONCLUSION： HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection. 2005 The WJG Press and Elsevier Inc. All rights reserved展开更多
目的鉴定人肺特异性X蛋白(lung specific X protein,LUNX)基因的增强子及其调控活性。方法以人基因组DNA为模板,PCR扩增生物信息学预测的3个增强子片段(E1:+3770～+3959bp;E2:+6454～+6555bp;E3:+14553～+14652bp),通过荧光素酶报告基...目的鉴定人肺特异性X蛋白(lung specific X protein,LUNX)基因的增强子及其调控活性。方法以人基因组DNA为模板,PCR扩增生物信息学预测的3个增强子片段(E1:+3770～+3959bp;E2:+6454～+6555bp;E3:+14553～+14652bp),通过荧光素酶报告基因表达体系检测转录活性。结果 PCR产物经测序证实后,分别定向连接至pGL3-Promoter载体中报告基因启动子上游的Kpn I和Xho I酶切位点和报告基因下游的BamHI和Sal I酶切位点上,经酶切鉴定后,构建了6种荧光素酶报告基因表达体系。以pSV-β-Galactosidase质粒为内对照,瞬时转染HEK293细胞,培养48h后检测细胞裂解液中荧光素酶活性。当3个DNA片段位于报告基因启动子上游时,均不具有增强转录的能力。将它们分别连接于报告基因下游时,E1和E3所调控的荧光素酶活性分别是对照pGL3-Promoter的2.83倍(P<0.05)和1.59倍(P<0.05)。结论 LUNX基因的+3770～+3959bp和+14553～+14652bp序列具有增强转录的能力,为LUNX表达调控机制的深入研究奠定了基础。展开更多
According to the fact that CEA gene expressed only in lung adenocarcinoma but not in normal lung cells, a retroviral expression vector (pCEATK) of the herpes simplex virus thymidine kinase (HSV-TK) gene regulated by C...According to the fact that CEA gene expressed only in lung adenocarcinoma but not in normal lung cells, a retroviral expression vector (pCEATK) of the herpes simplex virus thymidine kinase (HSV-TK) gene regulated by CEA promoter was constructed and introduced into CEA-producing human lung adenocarcinoma cells GL and non-CEA-producing HeLa cells. The expression of pCEATK and Ganciclovir (GCV) sensitivity of the transfected cells were tested in vitro and in vivo . pCEATK expressed only in CEA-producing GL cells but not in non-CEA-producing HeLa cells. The sensitivity to GCV of pCEATK-transfected GL was 992 times higher compared with that of the parental cell line and there was obvious 'bystander effect' in vitro. HeLa cells transfected wtih pCEATK were still resistant to GCV. Injection of GCV resulted in significant regression of pCEATK-transfected GL tumors in nude mice. In addition, all mice with any fraction of GL cells expressing HSV-TK exhibited a significant reduction in tumor growth, including mice with only 10% of GL cells expressing HSV-TK. These results show the possibility of HSV-TK gene-drug therapy using the tumor-specific promoter of CEA gene against CEA-producing lung cancers which was usually refractory to conventional chemotherapy.展开更多
According to the fact that CEA gene expressed only in lung adenocarcinoma and not in normal lung cells, a retroviral vector (pCEAMR) was constructed which carried the CEA promoter coupled to MDR1 ribozyme gene. pCEAMR...According to the fact that CEA gene expressed only in lung adenocarcinoma and not in normal lung cells, a retroviral vector (pCEAMR) was constructed which carried the CEA promoter coupled to MDR1 ribozyme gene. pCEAMR was introduced into drug-resistant lung adenocarcinoma cells GAOK with CEA expression and HeLaK without CEA expression; the expression of pCEAMR and drug resistance in the infected cells were analyzed in vitro and in vivo ; pCEAMR expressed only in CEA-producing GAOK cells and not in non-CEA-producing HeLa cells. The drug resistance to doxorubicin (DOX) decreased 91.5% in the infected GAOK cells and did not change in the infected HeLa cells. In nude mice, DOX could obviously inhibit the growth of the infected GAOK tumors, and had no effect on the growth of the infected HeLa cells. These results indicated that MDR1 ribozyme gene regulated by CEA promoter expressed only in human adenocarcinoma cells and reversed their drug resistance selectively. This gene-drug therapy might serve as an effective treatment method for patients with CEA-producing lung cancers which was usually refractory to conventional chemotherapy.展开更多
OBJECTIVE: To investigate genes involved in cancer metastasis, mRNA differential display was used to compare the levels of gene expression in two cell sublines derived from human giant cell carcinoma of lung (PG) whic...OBJECTIVE: To investigate genes involved in cancer metastasis, mRNA differential display was used to compare the levels of gene expression in two cell sublines derived from human giant cell carcinoma of lung (PG) which had different metastatic potentials. METHODS: Using in vivo tumorigenicity and a spontaneous metastasis assay in nude mice, two sublines (BE1, LH7) from human giant cell carcinoma of lung (PG) with different metastatic potentials were isolated and characterized. mRNA differential display was used to compare the levels of gene expression between them and the obtained results were confirmed by Northern hybridization. RESULTS: One differentially expressed band was nearly identical (99% homology) to Ras-GTPase-Activating protein SH3 domain binding protein (G3BP). G3BP displayed a strong expression in LH7 (non-metastatic in recipient nude mice) and a very weak expression in BE1 (100% metastatic frequency). The same different expression level of G3BP was detected in Northern hybridization with another panel of cell sublines with different metastatic potentials (established in our lab) derived from human prostate carcinoma cell line PC-3M. CONCLUSION: Our results indicate that G3BP was implicated in cancer metastasis because of its differential expressions in the two panels of cell sublines with different metastatic potentials.展开更多
OBJECTIVE: To investigate whether telomerase activity, human telomerase RNA (HTR) and human telomerase reverse transcriptase (HTERT) expression were associated with tumor development in lung cancer and whether telomer...OBJECTIVE: To investigate whether telomerase activity, human telomerase RNA (HTR) and human telomerase reverse transcriptase (HTERT) expression were associated with tumor development in lung cancer and whether telomerase is regulated at gene level or transcriptional level. METHODS: Expression of HTR and HTERT was detected by reverse transcription-polymerase chain reaction (RT-PCR) in 68 human lung cancer and in 68 adjacent-neoplatic lung tissues. And telomerase activity was examined by a quantitative telomeric repeat amplification protocol (TRAP). RESULTS: In 68 lung cancer tissues, telomerase activity, HTR and HTERT were expressed in 79%, 98.5% and 91.2% respectively, whereas all adjacent non-neoplastic lung tissues were telomerase negative. Most normal lung tissues expressed HTR (91.2%) and HTERT was detected in only 7 of 68 non-neoplastic tissues. CONCLUSIONS: The relatively high frequency of telomerase activity in lung cancer whereas the detection of no telomerase activity in normal lung tissues suggested that telomerase may play an important role in tumorigenesis of lung cancer. Compared to HTR, HTERT expression was better associated with telomerase activity with a concordance of 88.9%. Telomerase activity may be regulated at transcription level or translation level.展开更多
文摘AIM： To study the changes of human telomerase reverse transcriptase （hTERT） mRNA expression in human hepatocarcinoma cell lines （HepG2） and cholangiocarcinoma cell lines （QBC939） after HBx gene transfection and to illustrate the significance of transcriptional regulation of hTERT gene by HBx gene in the carcinogenesis. METHODS： HepG2 and QBC939 cell lines were cultured and co-transfected with eukaryotic expression vector containing the HBx coding region and cloning vector containing enhanced green fluorescent protein （EGFP） coding sequence using lipid-mediated gene transduction technique. Thirty-six hours after transfection, EGFP expression in cells was used as the indicator of successful transfection. Flow cytometry was performed to determine the transfection efficiency. Cells were harvested and total RNA was extracted using TRIzol reagent. The expression of hTERT mRNA in HepG2 and QBC939 cell lines was assayed by reverse transcriptionpolymerase chain reaction. The expression of HBx protein in both cell lines was detected by immunocytochemical staining and Western blotting. RESULTS： Flow cytometry showed that the transfection efficiency was 46.4% in HepG2 cells and 29.6% in QBC939 cells for both HBx gene expression vector and blank vector. The expression of hTERT mRNA was meaningfully increased in HepG2 and QBC939 cell lines when transfected with HBx gene expression vector compared to those transfected with OPTI-MEM medium and blank vector. Immunocytochemical staining and Western blotting revealed HBx protein expression in HepG2 and QBC939 cells only when transfected with HBx gene. CONCLUSION： HBx gene transfection can upregulate the transcriptional expression of hTERT mRNA. The transactivation of hTERT gene by HBx gene is a newfound mechanism for pathogenesis of hepatocarcinomas and cholangiocarcinomas after HBV infection. 2005 The WJG Press and Elsevier Inc. All rights reserved
文摘目的鉴定人肺特异性X蛋白(lung specific X protein,LUNX)基因的增强子及其调控活性。方法以人基因组DNA为模板,PCR扩增生物信息学预测的3个增强子片段(E1:+3770～+3959bp;E2:+6454～+6555bp;E3:+14553～+14652bp),通过荧光素酶报告基因表达体系检测转录活性。结果 PCR产物经测序证实后,分别定向连接至pGL3-Promoter载体中报告基因启动子上游的Kpn I和Xho I酶切位点和报告基因下游的BamHI和Sal I酶切位点上,经酶切鉴定后,构建了6种荧光素酶报告基因表达体系。以pSV-β-Galactosidase质粒为内对照,瞬时转染HEK293细胞,培养48h后检测细胞裂解液中荧光素酶活性。当3个DNA片段位于报告基因启动子上游时,均不具有增强转录的能力。将它们分别连接于报告基因下游时,E1和E3所调控的荧光素酶活性分别是对照pGL3-Promoter的2.83倍(P<0.05)和1.59倍(P<0.05)。结论 LUNX基因的+3770～+3959bp和+14553～+14652bp序列具有增强转录的能力,为LUNX表达调控机制的深入研究奠定了基础。
文摘According to the fact that CEA gene expressed only in lung adenocarcinoma but not in normal lung cells, a retroviral expression vector (pCEATK) of the herpes simplex virus thymidine kinase (HSV-TK) gene regulated by CEA promoter was constructed and introduced into CEA-producing human lung adenocarcinoma cells GL and non-CEA-producing HeLa cells. The expression of pCEATK and Ganciclovir (GCV) sensitivity of the transfected cells were tested in vitro and in vivo . pCEATK expressed only in CEA-producing GL cells but not in non-CEA-producing HeLa cells. The sensitivity to GCV of pCEATK-transfected GL was 992 times higher compared with that of the parental cell line and there was obvious 'bystander effect' in vitro. HeLa cells transfected wtih pCEATK were still resistant to GCV. Injection of GCV resulted in significant regression of pCEATK-transfected GL tumors in nude mice. In addition, all mice with any fraction of GL cells expressing HSV-TK exhibited a significant reduction in tumor growth, including mice with only 10% of GL cells expressing HSV-TK. These results show the possibility of HSV-TK gene-drug therapy using the tumor-specific promoter of CEA gene against CEA-producing lung cancers which was usually refractory to conventional chemotherapy.
文摘According to the fact that CEA gene expressed only in lung adenocarcinoma and not in normal lung cells, a retroviral vector (pCEAMR) was constructed which carried the CEA promoter coupled to MDR1 ribozyme gene. pCEAMR was introduced into drug-resistant lung adenocarcinoma cells GAOK with CEA expression and HeLaK without CEA expression; the expression of pCEAMR and drug resistance in the infected cells were analyzed in vitro and in vivo ; pCEAMR expressed only in CEA-producing GAOK cells and not in non-CEA-producing HeLa cells. The drug resistance to doxorubicin (DOX) decreased 91.5% in the infected GAOK cells and did not change in the infected HeLa cells. In nude mice, DOX could obviously inhibit the growth of the infected GAOK tumors, and had no effect on the growth of the infected HeLa cells. These results indicated that MDR1 ribozyme gene regulated by CEA promoter expressed only in human adenocarcinoma cells and reversed their drug resistance selectively. This gene-drug therapy might serve as an effective treatment method for patients with CEA-producing lung cancers which was usually refractory to conventional chemotherapy.
基金86 3HighTechnologyProject (No # 10 2 10 0 1 0 9)andSpecialFoundationforPh DTrainingProgramofEducationMinistry (No 19990 0
文摘OBJECTIVE: To investigate genes involved in cancer metastasis, mRNA differential display was used to compare the levels of gene expression in two cell sublines derived from human giant cell carcinoma of lung (PG) which had different metastatic potentials. METHODS: Using in vivo tumorigenicity and a spontaneous metastasis assay in nude mice, two sublines (BE1, LH7) from human giant cell carcinoma of lung (PG) with different metastatic potentials were isolated and characterized. mRNA differential display was used to compare the levels of gene expression between them and the obtained results were confirmed by Northern hybridization. RESULTS: One differentially expressed band was nearly identical (99% homology) to Ras-GTPase-Activating protein SH3 domain binding protein (G3BP). G3BP displayed a strong expression in LH7 (non-metastatic in recipient nude mice) and a very weak expression in BE1 (100% metastatic frequency). The same different expression level of G3BP was detected in Northern hybridization with another panel of cell sublines with different metastatic potentials (established in our lab) derived from human prostate carcinoma cell line PC-3M. CONCLUSION: Our results indicate that G3BP was implicated in cancer metastasis because of its differential expressions in the two panels of cell sublines with different metastatic potentials.
基金theNationalNaturalSciencesFoundationofChina (No 395 70 392 )
文摘OBJECTIVE: To investigate whether telomerase activity, human telomerase RNA (HTR) and human telomerase reverse transcriptase (HTERT) expression were associated with tumor development in lung cancer and whether telomerase is regulated at gene level or transcriptional level. METHODS: Expression of HTR and HTERT was detected by reverse transcription-polymerase chain reaction (RT-PCR) in 68 human lung cancer and in 68 adjacent-neoplatic lung tissues. And telomerase activity was examined by a quantitative telomeric repeat amplification protocol (TRAP). RESULTS: In 68 lung cancer tissues, telomerase activity, HTR and HTERT were expressed in 79%, 98.5% and 91.2% respectively, whereas all adjacent non-neoplastic lung tissues were telomerase negative. Most normal lung tissues expressed HTR (91.2%) and HTERT was detected in only 7 of 68 non-neoplastic tissues. CONCLUSIONS: The relatively high frequency of telomerase activity in lung cancer whereas the detection of no telomerase activity in normal lung tissues suggested that telomerase may play an important role in tumorigenesis of lung cancer. Compared to HTR, HTERT expression was better associated with telomerase activity with a concordance of 88.9%. Telomerase activity may be regulated at transcription level or translation level.
