We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constr...We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by sequentially inserting capsid precursor protein gene(P1) of FMDV and enhanced green fluorescent protein gene(EGFP) into pBABEpuro.The recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packaging cells(GP2-293) by liposomemediated transduction to produce the pseudovirus.The pseudovirus was used to infect BHK-21 cells and resistant cells were screened with puromycin.Green fluorescent proteins were observed by fluorescence microscopy and expression of the capsid precursor protein gene of FMDV was detected by indirect immunofluorescence.The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed successfully.The capsid precursor protein of FMDV and green fluorescent protein were expressed in BHK-21 cells.The mammalian cell expression system for the capsid precursor protein of FMDV has been constructed successfully,which lays the foundation of development of a FMDV subunit vaccine.展开更多
Objective Endometrial carcinoma(EC)is a prevalent gynecological malignancy characterized by increasing incidence and mortality rates.This underscores the critical need for novel therapeutic targets.One such potential ...Objective Endometrial carcinoma(EC)is a prevalent gynecological malignancy characterized by increasing incidence and mortality rates.This underscores the critical need for novel therapeutic targets.One such potential target is cell division cycle 20(CDC20),which has been implicated in oncogenesis.This study investigated the effect of the CDC20 inhibitor Apcin on EC and elucidated the underlying mechanism involved.Methods The effects of Apcin on EC cell proliferation,apoptosis,and the cell cycle were evaluated using CCK8 assays and flow cytometry.RNA sequencing(RNA-seq)was subsequently conducted to explore the underlying molecular mechanism,and Western blotting and coimmunoprecipitation were subsequently performed to validate the results.Animal studies were performed to evaluate the antitumor effects in vivo.Bioinformatics analysis was also conducted to identify CDC20 as a potential therapeutic target in EC.Results Treatment with Apcin inhibited proliferation and induced apoptosis in EC cells,resulting in cell cycle arrest.Pathways associated with apoptosis and the cell cycle were activated following treatment with Apcin.Notably,Apcin treatment led to the upregulation of the cell cycle regulator p21,which was verified to interact with CDC20 and consequently decrease the expression of downstream cyclins in EC cells.In vivo experiments confirmed that Apcin treatment significantly impeded tumor growth.Higher CDC20 expression was observed in EC tissue than in nonmalignant tissue,and increased CDC20 expression in EC patients was associated with shorter overall survival and progress free interval.Conclusion CDC20 is a novel molecular target in EC,and Apcin could be developed as a candidate antitumor drug for EC treatment.展开更多
Objective: To study the correlation between mi R-21 and Treg/Th17 ratio in the microenvironment of rats with hepatocellular carcinoma. Methods: Diethylnitrosamine was used to build the hepatocel ular carcinoma model o...Objective: To study the correlation between mi R-21 and Treg/Th17 ratio in the microenvironment of rats with hepatocellular carcinoma. Methods: Diethylnitrosamine was used to build the hepatocel ular carcinoma model of rats; the content of Treg cells and Th17 cells and the expression of mi R-21 in the peripheral blood of rats with hepatocellular carcinoma were detected. The statistical analysis was performed on the correlation between mi R-21 expression and Treg/Th17 ratio. Results: Hepatocellular carcinoma model of rats was successfully constructed. The proportion of Th17 cells among all CD4+T cells in the peripheral blood of rats with hepatocellular carcinoma was 5.319%, which was higher than the control group; while the proportion of Treg cells was 9.472%, which was higher than the control group. Treg/Th17 ratio in the model group was 1.781, compared with 1.478 in the control group. The expression of mi R-21 was increased in the peripheral blood of rats with hepatocellular carcinoma and it showed a positive correlation with the ratio of Treg/Th17. Conclusions: There is a positive correlation between the expression level of miR-21 and the ratio of Treg/Th17.展开更多
Diabetic kidney disease (DKD)is a microvascular complication of type 2 diabetes.The study of DKD mechanisms is the most important target for the prevention of DKD.Renal senescence is one of the important pathogeneses ...Diabetic kidney disease (DKD)is a microvascular complication of type 2 diabetes.The study of DKD mechanisms is the most important target for the prevention of DKD.Renal senescence is one of the important pathogeneses for DKD,but the mechanism of renal and cellular senescence is unclear.Decreased expression of circulating miR-126 is associated with the development of DKD and may be a promising blood-based biomarker for DKD.This study is to probe the effect and mechanism of miR-126 on the aging of human glomerular mesangial cells (HGMCs)induced by high glucose.HGMCs were cultured with Roswell Park Memorial Institute (RPMI-1640)in vitro.The effect of high glucose on morphology of HGMCs was observed 72h after intervention.The cell cycle was examined by flow cytometry.The telomere length was measured by Southern blotting.The expression levels of p53,p21 and Rb proteins in p53-p21-Rb signaling pathway and p-statl,p-stat3 in JAK/STAT signaling pathway were detected by Western blotting respectively.The expression of miR-126 was examined by qRT-PCR.MiR-126 mimics was transfected into HGMCs.The effects of miR-126 mimics transfection on cell morphology,cell cycle,telomere length,p53,p21,Rb,p-stat1 and p-stat3 were observed. The results showed that high glucose not only arrested the cell cycle in G1phase but also shortened the telomere length.High glucose led to high expression of p53,p21,Rb,p-statl and p-stat3 and premature senescence of HGMCs by activating the telomere-p53-p21-Rb and JAK/STAT signaling pathways.Moreover,the miR-126 was decreased in HGMCs induced by high glucose.It was suggested that the transfection of miR-126 mimics could inhibit the telomere-p53-p21-Rb and JAK/STAT signaling pathway activity in vitro and delay the senescence of HGMCs.The results may serve as a new strategy for the treatment of DKD.展开更多
AIM： To determine the functional significance of TC21 in esophageal squamous cell carcinoma （ESCC）. METHODS： TC21 siRNA transfection was carried out using Hyperfectamine to knock down TC21, and tran- scripts were ...AIM： To determine the functional significance of TC21 in esophageal squamous cell carcinoma （ESCC）. METHODS： TC21 siRNA transfection was carried out using Hyperfectamine to knock down TC21, and tran- scripts were analyzed by reverse transcription-poly- merase chain reaction and protein by Western blotting.We demonstrated the effect of TC21 downregulation of cell signaling in esophageal cancer cells by assess- ing the phosphorylation status of its downstream tar- gets, phosphoinositide 3-kinase （PI3K）, phosphatase and tensin homolog （PTEN）, protein kinase B （pAl〈t）, nuclear factor-KB （NF-~B） and cyclinD1 using specific antibodies. Cell survival analysis after cisplatin treat- ment was carried out by cell viability assay and cell cycle analysis using flow cytometry. RESULTS： TC21 knockdown in human ESCC cell line TEl3 cells, showed only a marginal increase （14.2%） in cell death compared with control cells. The expres- sions of the signaling proteins PI3K and pAkt, transcrip- tion factor NF-KB, and cell cycle protein cyclin D1 were markedly decreased in response to TC21 downregula- tion, whereas the level of pPTEN, an antagonist of PI3K, was increased. In addition, we evaluated the potential of TC21 as a putative target for sensitizing ESCC cells to the chemotherapeutic agent cisplatin. Increased cell death （38.4%） was observed in cells treated with cis- platin after TC21 knockdown compared with cells which were treated with cisplatin alone （20% cell death）. CONCLUSION： Results suggest that TC21 mediates its effects via the PI3K-Akt pathway, NF-KB and cyclin D1, and enhances chemoresistance in esophageal cancer cells.展开更多
Epigenetic modifications have been observed as a decline in miRNA-21 expression and breast cancer stem cell(CSC)population after 3 cycles of standard chemotherapy.The epigenetic response(miRNAs expression)and CSCs are...Epigenetic modifications have been observed as a decline in miRNA-21 expression and breast cancer stem cell(CSC)population after 3 cycles of standard chemotherapy.The epigenetic response(miRNAs expression)and CSCs are also correlated in patients with Breast Cancer.In patients who tolerated chemotherapy well,miRNA-21(non-coding RNA)expression decreased significantly after three cycles of chemotherapy.The miRNA-21 expression in breast cancer tissue was quantified by quantitative PCR(real-time PCR)using the standard protocol.In addition,breast CSCs(CD44+/CD24-)were also decreased in these patients.The miRNA-21 regulates cell division,proliferation,and autophagy of cancerous cells(as it targets phosphatase and tensin homolog/AKT/transcription factor EB/programmed cell death 4/autophagy-related protein 5 and chemotherapy also produces similar effects),thereby contributing to these benefits.Therefore,when all of the targets on genes have been explored by mimic miRNA,chemotherapy combined with anti-miRNA21 therapy may prove useful in the care of cancer patients.展开更多
Cigarette smoking is intimately related with the development of chronic obstructive pulmonary diseases, and alveolar epithelium is a major target for the exposure of cigarette smoke extract. In order to investigate th...Cigarette smoking is intimately related with the development of chronic obstructive pulmonary diseases, and alveolar epithelium is a major target for the exposure of cigarette smoke extract. In order to investigate the effect of cigarette smoke extract on the proliferation of alveolar epithelial cell type Ⅱ and its relationship with P21^WAF1, the alveolar epithelial type Ⅱ cell line （A549） cells were chosen as surrogate cells to represent alveolar epithelial type Ⅱ cells. MTT assay was used to detect cell viability after interfered with different concentrations of cigarette smoke extract. It was observed cigarette smoke extract inhibited the growth of A549 cells in a dose- and time-dependent manner. The morphological changes, involving the condensation and margination of nuclear chromatin, even karyorrhexis, were observed by both Hoechst staining and electronic microscopy. Flow cytometry analysis demonstrated the increased cell percentages in G1 and subG1 phases after the cells were incubated with cigarette smoke extract. The expression of p21^WAF1 protein and mRNA was also significantly increased as detected by the methods of Western blot or reverse transcription-polymerase chain reaction respectively. In conclusion, cigarette smoke extract inhibits the proliferation of alveolar epithelial cell type Ⅱ and blocks them in G1/S phase. The intracelhilar accumulation of P21^WAF1 may be one of the mechanisms which contribute to cigarette smoke extract-induced inhibition of cell proliferation.展开更多
基金supported by the National Programs for High Technology Research and Development of China (2006AA10A204)the Gansu Key Technologies R&D Program(ZGS-052-A41-0006-03)the Programs for Director Fund of Lanzhou Veterinary Research Institute
文摘We have constructed a retroviral vector mediated mammalian cell expression system of the capsid precursor protein of foot-and-mouth disease virus(FMDV).The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed by sequentially inserting capsid precursor protein gene(P1) of FMDV and enhanced green fluorescent protein gene(EGFP) into pBABEpuro.The recombinant retroviral vector and the pVSV-G plasmid were co-transfected into packaging cells(GP2-293) by liposomemediated transduction to produce the pseudovirus.The pseudovirus was used to infect BHK-21 cells and resistant cells were screened with puromycin.Green fluorescent proteins were observed by fluorescence microscopy and expression of the capsid precursor protein gene of FMDV was detected by indirect immunofluorescence.The recombinant retroviral vector pBABEpuro-P1-2A-EGFP was constructed successfully.The capsid precursor protein of FMDV and green fluorescent protein were expressed in BHK-21 cells.The mammalian cell expression system for the capsid precursor protein of FMDV has been constructed successfully,which lays the foundation of development of a FMDV subunit vaccine.
文摘Objective Endometrial carcinoma(EC)is a prevalent gynecological malignancy characterized by increasing incidence and mortality rates.This underscores the critical need for novel therapeutic targets.One such potential target is cell division cycle 20(CDC20),which has been implicated in oncogenesis.This study investigated the effect of the CDC20 inhibitor Apcin on EC and elucidated the underlying mechanism involved.Methods The effects of Apcin on EC cell proliferation,apoptosis,and the cell cycle were evaluated using CCK8 assays and flow cytometry.RNA sequencing(RNA-seq)was subsequently conducted to explore the underlying molecular mechanism,and Western blotting and coimmunoprecipitation were subsequently performed to validate the results.Animal studies were performed to evaluate the antitumor effects in vivo.Bioinformatics analysis was also conducted to identify CDC20 as a potential therapeutic target in EC.Results Treatment with Apcin inhibited proliferation and induced apoptosis in EC cells,resulting in cell cycle arrest.Pathways associated with apoptosis and the cell cycle were activated following treatment with Apcin.Notably,Apcin treatment led to the upregulation of the cell cycle regulator p21,which was verified to interact with CDC20 and consequently decrease the expression of downstream cyclins in EC cells.In vivo experiments confirmed that Apcin treatment significantly impeded tumor growth.Higher CDC20 expression was observed in EC tissue than in nonmalignant tissue,and increased CDC20 expression in EC patients was associated with shorter overall survival and progress free interval.Conclusion CDC20 is a novel molecular target in EC,and Apcin could be developed as a candidate antitumor drug for EC treatment.
文摘Objective: To study the correlation between mi R-21 and Treg/Th17 ratio in the microenvironment of rats with hepatocellular carcinoma. Methods: Diethylnitrosamine was used to build the hepatocel ular carcinoma model of rats; the content of Treg cells and Th17 cells and the expression of mi R-21 in the peripheral blood of rats with hepatocellular carcinoma were detected. The statistical analysis was performed on the correlation between mi R-21 expression and Treg/Th17 ratio. Results: Hepatocellular carcinoma model of rats was successfully constructed. The proportion of Th17 cells among all CD4+T cells in the peripheral blood of rats with hepatocellular carcinoma was 5.319%, which was higher than the control group; while the proportion of Treg cells was 9.472%, which was higher than the control group. Treg/Th17 ratio in the model group was 1.781, compared with 1.478 in the control group. The expression of mi R-21 was increased in the peripheral blood of rats with hepatocellular carcinoma and it showed a positive correlation with the ratio of Treg/Th17. Conclusions: There is a positive correlation between the expression level of miR-21 and the ratio of Treg/Th17.
基金This project was supported by grants from the Key Science and Technology Development Program of Nanjing City of the People's Republic of China (No. YKK15057 and No.YKK16097)and the National Natural Science Foundation of China (No.81473684).
文摘Diabetic kidney disease (DKD)is a microvascular complication of type 2 diabetes.The study of DKD mechanisms is the most important target for the prevention of DKD.Renal senescence is one of the important pathogeneses for DKD,but the mechanism of renal and cellular senescence is unclear.Decreased expression of circulating miR-126 is associated with the development of DKD and may be a promising blood-based biomarker for DKD.This study is to probe the effect and mechanism of miR-126 on the aging of human glomerular mesangial cells (HGMCs)induced by high glucose.HGMCs were cultured with Roswell Park Memorial Institute (RPMI-1640)in vitro.The effect of high glucose on morphology of HGMCs was observed 72h after intervention.The cell cycle was examined by flow cytometry.The telomere length was measured by Southern blotting.The expression levels of p53,p21 and Rb proteins in p53-p21-Rb signaling pathway and p-statl,p-stat3 in JAK/STAT signaling pathway were detected by Western blotting respectively.The expression of miR-126 was examined by qRT-PCR.MiR-126 mimics was transfected into HGMCs.The effects of miR-126 mimics transfection on cell morphology,cell cycle,telomere length,p53,p21,Rb,p-stat1 and p-stat3 were observed. The results showed that high glucose not only arrested the cell cycle in G1phase but also shortened the telomere length.High glucose led to high expression of p53,p21,Rb,p-statl and p-stat3 and premature senescence of HGMCs by activating the telomere-p53-p21-Rb and JAK/STAT signaling pathways.Moreover,the miR-126 was decreased in HGMCs induced by high glucose.It was suggested that the transfection of miR-126 mimics could inhibit the telomere-p53-p21-Rb and JAK/STAT signaling pathway activity in vitro and delay the senescence of HGMCs.The results may serve as a new strategy for the treatment of DKD.
基金Supported by Department of Science and Technology,Government of India
文摘AIM： To determine the functional significance of TC21 in esophageal squamous cell carcinoma （ESCC）. METHODS： TC21 siRNA transfection was carried out using Hyperfectamine to knock down TC21, and tran- scripts were analyzed by reverse transcription-poly- merase chain reaction and protein by Western blotting.We demonstrated the effect of TC21 downregulation of cell signaling in esophageal cancer cells by assess- ing the phosphorylation status of its downstream tar- gets, phosphoinositide 3-kinase （PI3K）, phosphatase and tensin homolog （PTEN）, protein kinase B （pAl〈t）, nuclear factor-KB （NF-~B） and cyclinD1 using specific antibodies. Cell survival analysis after cisplatin treat- ment was carried out by cell viability assay and cell cycle analysis using flow cytometry. RESULTS： TC21 knockdown in human ESCC cell line TEl3 cells, showed only a marginal increase （14.2%） in cell death compared with control cells. The expres- sions of the signaling proteins PI3K and pAkt, transcrip- tion factor NF-KB, and cell cycle protein cyclin D1 were markedly decreased in response to TC21 downregula- tion, whereas the level of pPTEN, an antagonist of PI3K, was increased. In addition, we evaluated the potential of TC21 as a putative target for sensitizing ESCC cells to the chemotherapeutic agent cisplatin. Increased cell death （38.4%） was observed in cells treated with cis- platin after TC21 knockdown compared with cells which were treated with cisplatin alone （20% cell death）. CONCLUSION： Results suggest that TC21 mediates its effects via the PI3K-Akt pathway, NF-KB and cyclin D1, and enhances chemoresistance in esophageal cancer cells.
基金Supported by SERB:Department of Science and Technology,New Delhi,No. NPDF:SERB 2015/000322
文摘Epigenetic modifications have been observed as a decline in miRNA-21 expression and breast cancer stem cell(CSC)population after 3 cycles of standard chemotherapy.The epigenetic response(miRNAs expression)and CSCs are also correlated in patients with Breast Cancer.In patients who tolerated chemotherapy well,miRNA-21(non-coding RNA)expression decreased significantly after three cycles of chemotherapy.The miRNA-21 expression in breast cancer tissue was quantified by quantitative PCR(real-time PCR)using the standard protocol.In addition,breast CSCs(CD44+/CD24-)were also decreased in these patients.The miRNA-21 regulates cell division,proliferation,and autophagy of cancerous cells(as it targets phosphatase and tensin homolog/AKT/transcription factor EB/programmed cell death 4/autophagy-related protein 5 and chemotherapy also produces similar effects),thereby contributing to these benefits.Therefore,when all of the targets on genes have been explored by mimic miRNA,chemotherapy combined with anti-miRNA21 therapy may prove useful in the care of cancer patients.
文摘Cigarette smoking is intimately related with the development of chronic obstructive pulmonary diseases, and alveolar epithelium is a major target for the exposure of cigarette smoke extract. In order to investigate the effect of cigarette smoke extract on the proliferation of alveolar epithelial cell type Ⅱ and its relationship with P21^WAF1, the alveolar epithelial type Ⅱ cell line （A549） cells were chosen as surrogate cells to represent alveolar epithelial type Ⅱ cells. MTT assay was used to detect cell viability after interfered with different concentrations of cigarette smoke extract. It was observed cigarette smoke extract inhibited the growth of A549 cells in a dose- and time-dependent manner. The morphological changes, involving the condensation and margination of nuclear chromatin, even karyorrhexis, were observed by both Hoechst staining and electronic microscopy. Flow cytometry analysis demonstrated the increased cell percentages in G1 and subG1 phases after the cells were incubated with cigarette smoke extract. The expression of p21^WAF1 protein and mRNA was also significantly increased as detected by the methods of Western blot or reverse transcription-polymerase chain reaction respectively. In conclusion, cigarette smoke extract inhibits the proliferation of alveolar epithelial cell type Ⅱ and blocks them in G1/S phase. The intracelhilar accumulation of P21^WAF1 may be one of the mechanisms which contribute to cigarette smoke extract-induced inhibition of cell proliferation.
