Objective To establish stable prostate cancer bone metastasis cell line overexpressing microRAN-145 (miR-145)for the study of the mechanism of miR-145 in bone metastasis.Methods pMSCV-miR-145 plasmids and retroviruses...Objective To establish stable prostate cancer bone metastasis cell line overexpressing microRAN-145 (miR-145)for the study of the mechanism of miR-145 in bone metastasis.Methods pMSCV-miR-145 plasmids and retroviruses of pMSCV-vector展开更多
To investigate exogenous PTEN gene transfected human breast cancer cell line MDA-MD-468.Methods Using the lipofectamine 2000 transfection technique,wild type PTEN gene was transducted into an in vitro cultured highly ...To investigate exogenous PTEN gene transfected human breast cancer cell line MDA-MD-468.Methods Using the lipofectamine 2000 transfection technique,wild type PTEN gene was transducted into an in vitro cultured highly metastatic breast cancer cell line MDA-MD-468.After transfection,the cells were selected by G418.The resistant clones were chosen and expanded in DMEM culture medium.RT-PCR,immunohistochemical method and western blot were used to determine the expression of target genes.Results An anti-G418 cell clone was established and expanded in culture.The transfected PTEN gene MDA-MD-468 cells showed expression of PTEN mRNA and PTEN protein.Conclusion Human breast cancer cell line MDA-MB-468 established in this study expresses consistently exogenous PTEN genes.4 refs,6 figs.展开更多
Objective: To investigate the inhibitory effect of apogossypolone (ApoG2) on prostate cancer cell line PC-3 in vivo, and explore its mechanism. Methods: The models of transplantation tumors in Balb/c nu/nu mice were e...Objective: To investigate the inhibitory effect of apogossypolone (ApoG2) on prostate cancer cell line PC-3 in vivo, and explore its mechanism. Methods: The models of transplantation tumors in Balb/c nu/nu mice were established via subcutaneous injection of PC-3 cells and the tumor-transplanted mice were divided into 4 groups: control group and three ApoG2 treatment groups, with 10 mice in each group. Volumes of the tumor were estimated every 2 d and the morphology of tumor tissues was observed. Immunohistochemistry was employed to observe the expression of Bcl-2, PCNA, CD31, caspase-3 and caspase-8 in tumor tissues. Results: ApoG2 (2.5 mg/kg-10 mg/kg) given intraperitoneally once a day can obviously inhibit the growth of subcutaneous prostatic carcinoma implant. The tumor volume decreased obviously when the treatment dosage was bigger than 5.0 mg/kg (P<0.01). Meanwhile, ApoG2 decreased the expression of PCNA and CD31, and enhanced the expression of caspases-3, caspase-8 in tumor tissues. Conclusion: ApoG2 exert an inhibitory effect on prostatic carcinoma possibly by inducing apoptosis and inhibiting tumor angiogenesis.展开更多
Objective:To investigate the effects of miR-25-3p on the occurrence,development and proliferation of tongue squamous cell carcinoma cells.Methods:To establish tongue squamous cell carcinoma cell line Tca8113 that stab...Objective:To investigate the effects of miR-25-3p on the occurrence,development and proliferation of tongue squamous cell carcinoma cells.Methods:To establish tongue squamous cell carcinoma cell line Tca8113 that stably and highly express miR-25-3p using recombinant reiroviral vector-mediated gene transfer method.The proliferation of transfected Tca8113 was detected by thiazolyl blue tetrazolium bromide(MTT)and cell colony formation assays.eyclnD1,p21^(cipt)and p27^(kipt)mRNA expressions in the transfected Tca-8113 were detected by quantitative PCR.cyclinD1,p21^(cipt),p27^(kipt),AKT,p-AKT,FOXOt and p-FOX01 expressions in the transfected Tca8113 were detected by western blot analysis.In addition,miR-25-3p expression in the tongue squamous cell carcinoma cell line and tissue specimen was also detected by quantitative PCR.Results:Quantitative PCR showed that mitt-25-3p expression in the tongue squamous cell carcinoma cell lines and tissue specimen was significantly lower than that in the adjacent tissue.MTT and cell colony formation assays showed that after miR-25-3p overexpression,the proliferation of transfected Tca8113 was obviously attenuated.Western blot analysis and quantitative PCR showed that after miR-25-3p overexpression.p21^(cipt)and p27^(kipt)expressions were upregulated,while cyclinD1,AKT,FOXO1 expressions were downregulated,and AKT and FOXO1 phosphorylation was inactivated in the transfected Tca8113 cells.Conclusions:MiR-25-3p inhibited the proliferation of tongue squamous cell carcinoma cells and regulated cell cycle-related protein expression,playing an important role in the occurrence and development of squamous cell carcinoma of the tongue.展开更多
The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the go...The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the goat mammary epithelial cell by liposome Tfx^M-20. By screening with G418, the stable transfected goat mammary epithelial cell line was established and the transcription and expression of CecropinB were identified by RT-PCR and agarose diffusion experiment, respectively. Results showed that eukaryotic expression vector pECFP-B was constructed successfully. Stable transfected goat mammary epithelial cell line was established and the CecropinB protein was expressed successfully. It provides the solid foundation for further experimental studies on the function of CecropinB.展开更多
Aim： To investigate the possible role of manganese in the regulation of mitochondrial aconitase （mACON） activity human prostate carcinoma cell line PC-3 cells. Methods： The mACON enzymatic activities of human pros...Aim： To investigate the possible role of manganese in the regulation of mitochondrial aconitase （mACON） activity human prostate carcinoma cell line PC-3 cells. Methods： The mACON enzymatic activities of human prostate carcinoma cell line PC-3 cells were determined using a reduced nicotinamide adenine dinucleotide-coupled assay. Immunoblot and transient gene expression assays were used to study gene expression of the mACON. The putative response element for gene expression was identified using reporter assays with site-directed mutagenesis and electrophoretic mobility-shift assays. Results： In vitro study revealed that manganese chloride （MnCI2） treatment for 16 h inhibited the enzymatic activity of mACON, which induced the inhibition of citrate utility and cell proliferation of PC- 3 cells. Although results from transient gene expression assays showed that MnCI2 treatment upregulated gene translation by approximately 5-fold through the iron response element pathway, immunoblot and reporter assays showed that MnCl2 treatments inhibited protein and gene expression of mACON. This effect was reversed by cotreatment with ferric ammonium citrate. Additional reporter assays with site-directed mutagenesis and electrophoretic mobility-shift assays suggested that a putative metal response element in the promoter of the mACON gene was involved in the regulation of MnCh on the gene expression of mACON. Conclusion： These findings suggest that manganese acts as an antagonist of iron, disrupting the enzymatic activity and gene expression of mACON and citrate metabolism in the prostate.展开更多
Aim:To investigate the possible role of manganese in the regulation of mitochondrial aconitase(mACON)activity human prostate carcinoma cell line PC-3 cells.Methods:The mACON enzymatic activities of human prostate carc...Aim:To investigate the possible role of manganese in the regulation of mitochondrial aconitase(mACON)activity human prostate carcinoma cell line PC-3 cells.Methods:The mACON enzymatic activities of human prostate carcinoma cell line PC-3 cells were determined using a reduced nicotinamide adenine dmucleotide-coupled assay. Immunoblot and transient gene expression assays were used to study gene expression of the mACON.The putative response element for gene expression was identified using reporter assays with site-directed mutagenesis and electro- phoretic mobility-shift assays.Results:In vitro study revealed that manganese chloride(MnCl2)treatment for 16h inhibited the enzymatic activity of mACON,which induced the inhibition of citrate utility and cell proliferation of PC- 3 cells.Although results from transient gene expression assays showed that MnCl_2,treatment upregulated gene translation by approximately 5-fold through the iron response element pathway,immunoblot and reporter assays showed that MnCl_2 treatments inhibited protein and gene expression of mACON.This effect was reversed by co- treatment with fenic ammonium citrate.Additional reporter assays with site-directed mutagenesis and electrophoretic mobility-shift assays suggested that a putative metal response element in the promoter of the mACON gene was involved in the regulation of MnCl_2 on the gene expression of mACON.Conclusion:These findings suggest that manganese acts as an antagonist of iron,disrupting the enzymatic activity and gene expression of mACON and citrate metabolism in the prostate.展开更多
目的构建人MCHR2真核表达载体,转染CHO细胞,建立稳定转染的CHO细胞系。方法采用PCR方法,以人胎脑cDNA文库为模板扩增人MCHR2基因的全长cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pcDNA3.1(+),经酶切和测序鉴定后,用脂...目的构建人MCHR2真核表达载体,转染CHO细胞,建立稳定转染的CHO细胞系。方法采用PCR方法,以人胎脑cDNA文库为模板扩增人MCHR2基因的全长cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pcDNA3.1(+),经酶切和测序鉴定后,用脂质体转染法转染CHO细胞,通过G418筛选,建立稳定转染的CHO细胞系,用RT-PCR、W estern b lot及免疫荧光法检测MCHR2的表达。结果成功构建了pcDNA3.1(+)/MCHR2真核表达载体,并建立了稳定转染的CHO细胞系,成功地表达目的基因。结论真核表达载体成功构建和稳定转染CHO细胞系的建立为进一步研究MCHR2的功能奠定良好的实验基础。展开更多
文摘Objective To establish stable prostate cancer bone metastasis cell line overexpressing microRAN-145 (miR-145)for the study of the mechanism of miR-145 in bone metastasis.Methods pMSCV-miR-145 plasmids and retroviruses of pMSCV-vector
文摘To investigate exogenous PTEN gene transfected human breast cancer cell line MDA-MD-468.Methods Using the lipofectamine 2000 transfection technique,wild type PTEN gene was transducted into an in vitro cultured highly metastatic breast cancer cell line MDA-MD-468.After transfection,the cells were selected by G418.The resistant clones were chosen and expanded in DMEM culture medium.RT-PCR,immunohistochemical method and western blot were used to determine the expression of target genes.Results An anti-G418 cell clone was established and expanded in culture.The transfected PTEN gene MDA-MD-468 cells showed expression of PTEN mRNA and PTEN protein.Conclusion Human breast cancer cell line MDA-MB-468 established in this study expresses consistently exogenous PTEN genes.4 refs,6 figs.
文摘Objective: To investigate the inhibitory effect of apogossypolone (ApoG2) on prostate cancer cell line PC-3 in vivo, and explore its mechanism. Methods: The models of transplantation tumors in Balb/c nu/nu mice were established via subcutaneous injection of PC-3 cells and the tumor-transplanted mice were divided into 4 groups: control group and three ApoG2 treatment groups, with 10 mice in each group. Volumes of the tumor were estimated every 2 d and the morphology of tumor tissues was observed. Immunohistochemistry was employed to observe the expression of Bcl-2, PCNA, CD31, caspase-3 and caspase-8 in tumor tissues. Results: ApoG2 (2.5 mg/kg-10 mg/kg) given intraperitoneally once a day can obviously inhibit the growth of subcutaneous prostatic carcinoma implant. The tumor volume decreased obviously when the treatment dosage was bigger than 5.0 mg/kg (P<0.01). Meanwhile, ApoG2 decreased the expression of PCNA and CD31, and enhanced the expression of caspases-3, caspase-8 in tumor tissues. Conclusion: ApoG2 exert an inhibitory effect on prostatic carcinoma possibly by inducing apoptosis and inhibiting tumor angiogenesis.
基金Supported by Key Disciplines Group Construetion Project of Pudong Health Bureau of Shanghai(Grant No.PWZxk2010-12)
文摘Objective:To investigate the effects of miR-25-3p on the occurrence,development and proliferation of tongue squamous cell carcinoma cells.Methods:To establish tongue squamous cell carcinoma cell line Tca8113 that stably and highly express miR-25-3p using recombinant reiroviral vector-mediated gene transfer method.The proliferation of transfected Tca8113 was detected by thiazolyl blue tetrazolium bromide(MTT)and cell colony formation assays.eyclnD1,p21^(cipt)and p27^(kipt)mRNA expressions in the transfected Tca-8113 were detected by quantitative PCR.cyclinD1,p21^(cipt),p27^(kipt),AKT,p-AKT,FOXOt and p-FOX01 expressions in the transfected Tca8113 were detected by western blot analysis.In addition,miR-25-3p expression in the tongue squamous cell carcinoma cell line and tissue specimen was also detected by quantitative PCR.Results:Quantitative PCR showed that mitt-25-3p expression in the tongue squamous cell carcinoma cell lines and tissue specimen was significantly lower than that in the adjacent tissue.MTT and cell colony formation assays showed that after miR-25-3p overexpression,the proliferation of transfected Tca8113 was obviously attenuated.Western blot analysis and quantitative PCR showed that after miR-25-3p overexpression.p21^(cipt)and p27^(kipt)expressions were upregulated,while cyclinD1,AKT,FOXO1 expressions were downregulated,and AKT and FOXO1 phosphorylation was inactivated in the transfected Tca8113 cells.Conclusions:MiR-25-3p inhibited the proliferation of tongue squamous cell carcinoma cells and regulated cell cycle-related protein expression,playing an important role in the occurrence and development of squamous cell carcinoma of the tongue.
基金Supported by Key Teachers Foundation of Education Office of Heilongjiang Province, 2005 (1055G005)
文摘The paper aimed at constructing the eukaryotic expression vector of CecropinB and transfecting it into the goat mammary epithelial cell line. The recombinant plasmid inserted with CecropinB was transfected into the goat mammary epithelial cell by liposome Tfx^M-20. By screening with G418, the stable transfected goat mammary epithelial cell line was established and the transcription and expression of CecropinB were identified by RT-PCR and agarose diffusion experiment, respectively. Results showed that eukaryotic expression vector pECFP-B was constructed successfully. Stable transfected goat mammary epithelial cell line was established and the CecropinB protein was expressed successfully. It provides the solid foundation for further experimental studies on the function of CecropinB.
文摘Aim： To investigate the possible role of manganese in the regulation of mitochondrial aconitase （mACON） activity human prostate carcinoma cell line PC-3 cells. Methods： The mACON enzymatic activities of human prostate carcinoma cell line PC-3 cells were determined using a reduced nicotinamide adenine dinucleotide-coupled assay. Immunoblot and transient gene expression assays were used to study gene expression of the mACON. The putative response element for gene expression was identified using reporter assays with site-directed mutagenesis and electrophoretic mobility-shift assays. Results： In vitro study revealed that manganese chloride （MnCI2） treatment for 16 h inhibited the enzymatic activity of mACON, which induced the inhibition of citrate utility and cell proliferation of PC- 3 cells. Although results from transient gene expression assays showed that MnCI2 treatment upregulated gene translation by approximately 5-fold through the iron response element pathway, immunoblot and reporter assays showed that MnCl2 treatments inhibited protein and gene expression of mACON. This effect was reversed by cotreatment with ferric ammonium citrate. Additional reporter assays with site-directed mutagenesis and electrophoretic mobility-shift assays suggested that a putative metal response element in the promoter of the mACON gene was involved in the regulation of MnCh on the gene expression of mACON. Conclusion： These findings suggest that manganese acts as an antagonist of iron, disrupting the enzymatic activity and gene expression of mACON and citrate metabolism in the prostate.
文摘Aim:To investigate the possible role of manganese in the regulation of mitochondrial aconitase(mACON)activity human prostate carcinoma cell line PC-3 cells.Methods:The mACON enzymatic activities of human prostate carcinoma cell line PC-3 cells were determined using a reduced nicotinamide adenine dmucleotide-coupled assay. Immunoblot and transient gene expression assays were used to study gene expression of the mACON.The putative response element for gene expression was identified using reporter assays with site-directed mutagenesis and electro- phoretic mobility-shift assays.Results:In vitro study revealed that manganese chloride(MnCl2)treatment for 16h inhibited the enzymatic activity of mACON,which induced the inhibition of citrate utility and cell proliferation of PC- 3 cells.Although results from transient gene expression assays showed that MnCl_2,treatment upregulated gene translation by approximately 5-fold through the iron response element pathway,immunoblot and reporter assays showed that MnCl_2 treatments inhibited protein and gene expression of mACON.This effect was reversed by co- treatment with fenic ammonium citrate.Additional reporter assays with site-directed mutagenesis and electrophoretic mobility-shift assays suggested that a putative metal response element in the promoter of the mACON gene was involved in the regulation of MnCl_2 on the gene expression of mACON.Conclusion:These findings suggest that manganese acts as an antagonist of iron,disrupting the enzymatic activity and gene expression of mACON and citrate metabolism in the prostate.
文摘目的构建人MCHR2真核表达载体,转染CHO细胞,建立稳定转染的CHO细胞系。方法采用PCR方法,以人胎脑cDNA文库为模板扩增人MCHR2基因的全长cDNA编码区序列,利用DNA重组技术将其定向插入到真核表达载体pcDNA3.1(+),经酶切和测序鉴定后,用脂质体转染法转染CHO细胞,通过G418筛选,建立稳定转染的CHO细胞系,用RT-PCR、W estern b lot及免疫荧光法检测MCHR2的表达。结果成功构建了pcDNA3.1(+)/MCHR2真核表达载体,并建立了稳定转染的CHO细胞系,成功地表达目的基因。结论真核表达载体成功构建和稳定转染CHO细胞系的建立为进一步研究MCHR2的功能奠定良好的实验基础。
