Cyclin-dependent kinase 2-associated protein 1（CDK2AP1）, a cell growth inhibitory factor, is abnormally expressed in cancer cells, and might be implicated in the development of lung cancer. However, no studies on th...Cyclin-dependent kinase 2-associated protein 1（CDK2AP1）, a cell growth inhibitory factor, is abnormally expressed in cancer cells, and might be implicated in the development of lung cancer. However, no studies on the function of CDK2AP1 in human lung cancer have been yet reported. In this study, overexpressing lentiviral vectors containing full-length CDK2AP1 cDNA and CDK2AP1 shRNA（short hairpin RNA） were constructed. Our results show that infecting A549 cells with lentivirus containing CDK2AP1 shRNA or full-length CDK2AP1 cDNA results in significantly down- or up-regulated the expression of CDK2AP1, respectively, thereby providing reliable tools for studying the function of CDK2AP1 in pulmonary carcinogenesis. Our data of MTT assay and flowcytometric analysis demonstrate that CDK2AP1 plays an important role in the proliferation/growth and cell cycling of A549 cells in vitro, and further investigation into its underlying mechanism of pulmonary carcinogenesis is needed.展开更多
Objective:Epidermal growth factor receptor variant III(EGFRvIII)is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme(GBM).Temozolomide(TMZ)is a standa...Objective:Epidermal growth factor receptor variant III(EGFRvIII)is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme(GBM).Temozolomide(TMZ)is a standard chemotherapeutic for GBM,but TMZ treatment benefits are compromised by chemoresistance.This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance.Methods:CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM.Western blot,realtime PCR,flow cytometry,and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1(RAD51AP1).Results:Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells.Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment.Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells.Knockdown of E2F1 increased sensitivity to TMZ.Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1,mediates TMZ resistance,and has a potential E2F1 binding site on the promoter.Knockdown of RAD51AP1 enhanced the sensitivity of TMZ;however,overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells.Furthermore,RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase(MGMT)expression.The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated,but not MGMT-unmethylated TMZ-treated GBM patients.Conclusions:Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment.RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair.Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.展开更多
Objective:Adopting mass spectrometry to analyze the protein spectrum of the exosomes in human hepatocellular carcinoma cells and screening the target membrane protein in order to analyze the relationship between its g...Objective:Adopting mass spectrometry to analyze the protein spectrum of the exosomes in human hepatocellular carcinoma cells and screening the target membrane protein in order to analyze the relationship between its gene expression level and prognosis.Methods:The exosomes of hepatocellular carcinoma cells HepG2 and 7721 were isolated by ultracentrifugation,and the extracted exosomes were verified by transmission electron microscope, western blotting,and nanosight.The relative quantitative proteomics analysis was used to analyze the protein spectrum of HepG2 and 7721 cells in exosomes.The bioinformatics was used to collect the signal pathway,and the target protein AP2M1 was verified by qRTPCR.Gene Expression Profiling Interactive Analysis(GEPIA),OncoLnc,and The Cancer Genome Atlas(TCGA)database were used to analyze the expression level of AP2M1 and the survival rate of patients with liver cancer.Results:The extracellular exosomes of HepG2 and 7721 cells were isolated by ultracentrifugation.High-purity exosomes were obtained and verified by transmission electron microscopy,western blotting,and nanosight.There were 836 proteins co-expressed by the exosomes of HepG2 and 7721.By the analysis of Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway,a total of 34 pathways that were related to liver cancer were collected,and the target membrane protein AP2M1 was screened.AP2M1 highly expressed in HepG2 cells,HepG2 and Huh7 cells' exosomes were detected by qRT-PCR(P<0.05).The data of GEPIA showed that comparing to the adjacent tissues,gene AP2M1 was highly expressed in hepatocarcinoma tissues.Kaplan-Meier survival curve analysis was performed by OncoLnc.It was found that the survival rate of patients with liver cancer and high expression of AP2M1 was significantly lower than patients with liver cancer and low expression of AP2M1(P<0.01).Conclusion:The high expression of membrane protein AP2M1 in the exosomes of hepatoma cells and the high expression of its gene in liver cancer tissues predicted the low survival rate.展开更多
基金Supported by the Grants from the Project of Scientific Innovation and Creative for Jilin Provincial Oversea Scholars(No.2010273)Jilin Provincial Science and Technology Services(Nos.20050408, 20070720, 200805120, 20090732)+2 种基金Jilin Provincial Development and Reformation Committee(No.2009Y042J12314)Jilin Province Talent Development Foundation (No.JRJB2007-2)the Natural Science Foundation of China(Nos.30670301, 30870354)
文摘Cyclin-dependent kinase 2-associated protein 1（CDK2AP1）, a cell growth inhibitory factor, is abnormally expressed in cancer cells, and might be implicated in the development of lung cancer. However, no studies on the function of CDK2AP1 in human lung cancer have been yet reported. In this study, overexpressing lentiviral vectors containing full-length CDK2AP1 cDNA and CDK2AP1 shRNA（short hairpin RNA） were constructed. Our results show that infecting A549 cells with lentivirus containing CDK2AP1 shRNA or full-length CDK2AP1 cDNA results in significantly down- or up-regulated the expression of CDK2AP1, respectively, thereby providing reliable tools for studying the function of CDK2AP1 in pulmonary carcinogenesis. Our data of MTT assay and flowcytometric analysis demonstrate that CDK2AP1 plays an important role in the proliferation/growth and cell cycling of A549 cells in vitro, and further investigation into its underlying mechanism of pulmonary carcinogenesis is needed.
基金supported by the Science and Technology Project of Tianjin Municipal Health Commission(Grant Nos.TJWJ2022MS003 and TJWJ2021ZD008)the Tianjin Science and Technology Plan Project(Grant Nos.21JCYBJC01520 and 20JCYBJC01070)。
文摘Objective:Epidermal growth factor receptor variant III(EGFRvIII)is a constitutively-activated mutation of EGFR that contributes to the malignant progression of glioblastoma multiforme(GBM).Temozolomide(TMZ)is a standard chemotherapeutic for GBM,but TMZ treatment benefits are compromised by chemoresistance.This study aimed to elucidate the crucial mechanisms leading to EGFRvIII and TMZ resistance.Methods:CRISPR-Cas13a single-cell RNA-seq was performed to thoroughly mine EGFRvIII function in GBM.Western blot,realtime PCR,flow cytometry,and immunofluorescence were used to determine the chemoresistance role of E2F1 and RAD51-associated protein 1(RAD51AP1).Results:Bioinformatic analysis identified E2F1 as the key transcription factor in EGFRvIII-positive living cells.Bulk RNA-seq analysis revealed that E2F1 is a crucial transcription factor under TMZ treatment.Western blot suggested enhanced expression of E2F1 in EGFRvIII-positive and TMZ-treated glioma cells.Knockdown of E2F1 increased sensitivity to TMZ.Venn diagram profiling showed that RAD51AP1 is positively correlated with E2F1,mediates TMZ resistance,and has a potential E2F1 binding site on the promoter.Knockdown of RAD51AP1 enhanced the sensitivity of TMZ;however,overexpression of RAD51AP1 was not sufficient to cause chemotherapy resistance in glioma cells.Furthermore,RAD51AP1 did not impact TMZ sensitivity in GBM cells with high O6-methylguanine-DNA methyltransferase(MGMT)expression.The level of RAD51AP1 expression correlated with the survival rate in MGMT-methylated,but not MGMT-unmethylated TMZ-treated GBM patients.Conclusions:Our results suggest that E2F1 is a key transcription factor in EGFRvIII-positive glioma cells and quickly responds to TMZ treatment.RAD51AP1 was shown to be upregulated by E2F1 for DNA double strand break repair.Targeting RAD51AP1 could facilitate achieving an ideal therapeutic effect in MGMT-methylated GBM cells.
基金supported by grants from the National Natural Science Foundation of China(No.81760612)Guangxi Key Research and Development Project(No.AB16380351)
文摘Objective:Adopting mass spectrometry to analyze the protein spectrum of the exosomes in human hepatocellular carcinoma cells and screening the target membrane protein in order to analyze the relationship between its gene expression level and prognosis.Methods:The exosomes of hepatocellular carcinoma cells HepG2 and 7721 were isolated by ultracentrifugation,and the extracted exosomes were verified by transmission electron microscope, western blotting,and nanosight.The relative quantitative proteomics analysis was used to analyze the protein spectrum of HepG2 and 7721 cells in exosomes.The bioinformatics was used to collect the signal pathway,and the target protein AP2M1 was verified by qRTPCR.Gene Expression Profiling Interactive Analysis(GEPIA),OncoLnc,and The Cancer Genome Atlas(TCGA)database were used to analyze the expression level of AP2M1 and the survival rate of patients with liver cancer.Results:The extracellular exosomes of HepG2 and 7721 cells were isolated by ultracentrifugation.High-purity exosomes were obtained and verified by transmission electron microscopy,western blotting,and nanosight.There were 836 proteins co-expressed by the exosomes of HepG2 and 7721.By the analysis of Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway,a total of 34 pathways that were related to liver cancer were collected,and the target membrane protein AP2M1 was screened.AP2M1 highly expressed in HepG2 cells,HepG2 and Huh7 cells' exosomes were detected by qRT-PCR(P<0.05).The data of GEPIA showed that comparing to the adjacent tissues,gene AP2M1 was highly expressed in hepatocarcinoma tissues.Kaplan-Meier survival curve analysis was performed by OncoLnc.It was found that the survival rate of patients with liver cancer and high expression of AP2M1 was significantly lower than patients with liver cancer and low expression of AP2M1(P<0.01).Conclusion:The high expression of membrane protein AP2M1 in the exosomes of hepatoma cells and the high expression of its gene in liver cancer tissues predicted the low survival rate.
