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Breast cancer is one of the most common malignancies that seriously threaten women's health. In the process of the malignant transformation of breast cancer, metabolic reprogramming and immune evasion represent the two main fascinating characteristics of cancer and facilitate cancer cell proliferation. Breast cancer cells generate energy through increased glucose metabolism. Lipid metabolism contributes to biological signal pathways and forms cell membranes except energy generation. Amino acids act as basic protein units and metabolic regulators in supporting cell growth. For tumor-associated immunity, poor immunogenicity and heightened immunosuppression cause breast cancer cells to evade the host's immune system. For the past few years, the complex mechanisms of metabolic reprogramming and immune evasion are deeply investigated, and the genes involved in these processes are used as clinical therapeutic targets for breast cancer. Here, we review the recent findings related to abnormal metabolism and immune characteristics, regulatory mechanisms, their links, and relevant therapeutic strategies.
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Female , Humans , Breast Neoplasms , Cell Proliferation , Cell Transformation, Neoplastic , Energy Metabolism , Lipid Metabolism , Signal TransductionABSTRACT
Objective To construct the eukaryotic expression vector of PRDX3 labeled with FLAG tag and to study its localization in human tongue cancer cell line SCC15.Methods PRDX3 gene was obtained from the breast library by PCR and cloned into PCDH vector to construct PCDH-FLAG-PRDX3.The plasmid was transiently transfected into 293T cells and the expression was detected by Western blot.Subcellular localization was detected by cellular immunofluorescence.Results The result of double digestion and sequencing showed that PCDH-FLAG-PRDX3 eukaryotic expression vector was constructed.The expression of FLAG-PRDX3 in human 293T cells was positively confirmed by Western blotting.In human tongue cancer cell line SCC15, the result of cellular immunofluorescence showed FLAG-PRDX3 was located in the cytoplasm rather than in the nucleus.Conclusion PRDX3 eukaryotic expression vector labeled with FLAG tag is constructed successfully, which is located in cytoplasm in human SCC15 cells.Construction and identification of PRDX3 could shed light on the function and mechanism of PRDX3 in tongue cancer.
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Objective To construct the eukaryotic expression vector of human telomerase RNA component ( hTR) and study its biological function tentatively .Methods hTR Gene was obtained by PCR from cDNA template , which was reverse transcribed from 293T mRNA and cloned into pCDNA3.0 vector.The recombinant plasmid and empty vector were trans-fected into 293T cells, and hTR expression was identified by qRT-PCR.HepG2 cells that stably transfected with pCDNA3.0-hTR were constructed and identified by qRT-PCR.These cells were used to assess the interaction of hTR with human telomerase revese transcriptase ( hTERT ) and dyskerin .Telomerase activity was also detected in HepG 2 cells transfected with pCDNA3.0-hTR.Results pCDNA3.0-hTR eukaryotic expression vector was successfully constructed by double digestion identification .The inserted fragment was confirmed by sequencing .The expression of hTR in human 293T cells and HepG2 pCDNA3.0-hTR stable cell line was identified.In addition, qRT-PCR and Western blotting results showed that hTR could interact with hTERT and dyskerin , while hTR overexpression could not regulate the telomerase activity in HepG2 cells.Conclusion The eukaryotic expression vector of pCDNA 3.0-hTR is successfully constructed and expressed.This study will contribute to the further study of cancer therapy targeting hTR .
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Objective To purify and prokaryotically express the histone methyltransferase SET7.Methods The coding sequence of full length SET7 was amplified from breast library by PCR and cloned into the pGEX-KG vector.The correct recombinant plasmid was introduced into E.coli.The expressed protein was identified by SDS-PAGE.Results DNA sequencing indicated the SET7 was constructed.GST-SET7 fusion protein was identified by SDS-PAGE.Conclusion The prokaryotically expressed protein of GST-SET7 is obtained,which will falilitate further study of SET7 function.
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Objective To detect the effect of E6AP on gastric cancer cell proliferation and migration.Methods The expression of E6AP in different gastric cancer cell lines and normal gastric mucosa epithelial cell lines was detected by Western blotting.Gastric cancer cells BGC-823 stably expressing E6AP short hairpan RNA(shRNA) were obtained by lentiviral vector of E6AP.The effect of E6AP on BGC-823 cell growth and migration was determined by CCK-8 kit, Tran-swell and wound healing assay.Results Gastric cancer cell line BGC-823 in which E6AP was stably knocked down was established.Knockdown of E6AP inhibited the proliferation and migration of BGC-823 cells.Conclusion E6AP plays a key role in gastric cancer proliferation and migration.
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Objective To construct PES1 shRNA stable expression cell lines in tongue squamous cell carcinoma ( TSCC) cells and to study the effect of knockdown of PES 1 on the growth of TSCC cells .Methods Recombinant lentivirus carrying PES1 shRNA was packaged and obtained in 293T cells.TSCC cells (Tca8113, SCC6 and SCC15) were infected with the lentivirus and selected for stable cells .PES1 expression was identified by Western blot .The effect of inhibition of PES1 on the growth and cell cycle of TSCC cells was detected by growth curve and flow cytometry .Results TSCC cells stably expressing PES1 shRNA were constructed.Knockdown of PES1 inhibited cell proliferation and induced cell cycle ar-rest at G0/G1 phase.Knockdown of PES1 inhibited expression of cyclin D1 in TSCC cells.Conclusion Inhibition of PES1 results in reduced cell proliferation , cell cycle arrest at G 0/G1 phase and reduction of cyclin D 1 expression in TSCC cells . PES1 may be a target for TSCC gene therapy .
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Objective To construct the lentiviral vector of RNA interference(RNAi) for DEK,and to detect its effect on breast cancer cell growth.Methods The DEK siRNA was designed and constructed based on DEK sequence using a lentiviral vector.The lentivirul vector containing DEK siRNA was named PSIH-H1-DEK as confirmed by PCR and sequenceing.PSIH-H1-DEK was then packaged with accessory plasmids into lentivirus in 293T cells and selected for 2 weeks with puromycin ( puro ) before the mixed colonies stably expressing DEK siRNA were obtained and the DEK expression was detected by real time PCR( RT-PCR) and Western blotting.The effect of DEK siRNA on ZR75-1 cell growth was determined by cell counting kit.Results Western blot and RT-PCR showed that PSIH-H1-DEK siRNA could suppress DEK gene expression.Suppression of DEK could markedly inhibit the growth of ZR75-1 cells.Conclusion The lentivirus-mediated DEK siRNA is obtained,which will facilitate further research on DEK function in breast cancer development.
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Objective To construct the lentiviral vector (pSIH-H1) for E6AP small-interfering RNA(siRNA) and to detect its effect on breast cancer ZR 75-1cell growth.Methods E6AP siRNA was designed and constructed based on hu-man papillomavirus E6-associated protein ( E6AP) cDNA sequence.The expression of E6AP was examined by real-time quantitative PCR(qRT-PCR) and Western blotting.The effect of E6AP on ZR75-1 cell growth was determined by cck-8 kit.Results DNA sequencing indicated that E 6AP siRNA expression vector was constructed successfully .qRT-PCR and Western blotting experiments showed that pSIH-H1-E6AP siRNA could suppress the E6AP gene expression.Suppression of E6AP could markedly inhibit the growth of ZR 75-1.Conclusion A lentivirus RNA interference ( RNAi) vector targeting E6AP gene is successfully constructed ,which inhibits the cell growth of ZR 75-1.
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Objective To construct the eukaryotic expression vector of cyclin-dependent kinase ( CDK) 7 labeled with Myc tag, obtain the expressed product , and identify its interaction with FLAG-P53 at the protein level .Methods Human CDK7 coding gene region amplified from the mammary cDNA library by PCR was inserted into the pXJ -40 vector.The recombinant plasmid Myc-CDK7 transfected into human 293T cell lines was investigated and examined by SDS-PAGE and Western blotting.In addition,assay was applied to determine the interaction between Myc-CDK7 and FLAG-p53.Results The coding region of CDK7 was successfully amplified by PCR and cloned into pXJ-40 vector, which was identified by double enzyme digestion and gene sequencing .Myc-CDK7 was successfully expressed in human 293T cell lines according to SDS-PAGE and Western blotting assay indicated that Myc-CDK7 could interact with P53 protein, which verified its known function .Conclusion The eukaryotic expression vector Myc-CDK7 is successfully obtained , which will contribute to further research on CDK 7-mediated cell cycle regulation .
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Objective To clone E-cadherin and N-cadherin promoters and insert them into a luciferase reporter gene vector, and to characterize the promoter activity of E-cadherin and N-cadherin.Methods E-cadherin and N-cadherin pro-moter were cloned into pGL 4-basic.The resulting plasmids were determined by DNA sequencing .The promoter activity was analyzed in breast cancer cell line ZR 75-1 and hepatocarcinoma cell line HepG 2.Results DNA sequencing showed that the sequences of the cloned promoter regions were correct .Analysis of the reporter gene activity indicated that the E-cad-herin and N-cadherin promoters had the highest transcriptional activity in ZR 75-1 and HepG2 cells.Conclusion The E-cadherin and N-cadherin promoter genes are cloned successfully , contributing much to screening transcription factors that regulate E-cadherin and N-cadherin expression .
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Objective To construct the prokaryotic expression vector of human autophagy-related LC3B gene,obtain the GST-LC3B recombinant plasmid , purify the GST-LC3B fusion protein and identify its activity in vitro.Methods Human LC3B coding region was amplified from the human mammary gland cDNA by PCR and inserted into the prokaryotic expres -sion vector pGEX-KG.The recombinant plasmid pGEX-KG-LC3B was transformed into E.coli Rossate.The expressed product was purified by GST-Sepharose 4B beads and identified by SDS-PAGE and Western blot analysis .The function of the purified protein GST-LC3B was detected by GST pull-down assay.Results About 400 bp of the LC3B coding region was successfully amplified from the mammary gland library by PCR and inserted into pGEX -KG.The result of double diges-tion and sequencing showed that the GST-LC3B recombinant plasmid was successfully obtained .The GST-LC3B fusion pro-tein of about 40 000 (Mr) was successfully purified and identified by SDS-PAGE and Western blotting analysis.GST pull-down assay showed that GST-LC3B could interact with Atg4B, which identified its known function .Conclusion The pro-karyotic expression vector of GST-LC3B is constructed successfully , which will facilitate further research on the function of LC3B in autophagy.
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Objective To construct eukaryotic expression vector of wild type E 4F1 and the mutant deleting amino acid region 32-81, and to detect the interaction between wild type or mutant E 4F1 and p53 and to study the effect of E4F1 on the expression level of p21.Methods Wild type and mutant sequences of E 4F1 were amplified from the mammary library using standard PCR and recombinant PCR .The sequences were cloned into pXJ 40-MYC vector to generate the MYC-E4F1 and MYC-E4F1(Δ32-81) recombinant plasmids that were transfected into 293T cells and identified by Western blotting . FLAG-p53 and MYC-E4F1 or MYC-E4F1(Δ32-81) were co-transfected into 293T cells and immunoprecipitation assay was performed to detect the interaction of wild type or mutant E 4F1 with p53.Wild type and mutant E4F1 expressing vec-tors were co-transfected into osteosarcoma U2OS cells and the expression of p21was detected.Results Recombinant plas-mids of MYC-E4F1 and MYC-E4F1(Δ32-81) were successfully constructed.Both wild type and mutant E4F1 interacted with p53.Deletion of amino acid region 32-81 of E4F1 increased the interaction .The expression level of p21 was in-creased by wild-type E4F1, but not by mutant E4F1.Conclusion The eukaryotic expression vector of wild type E4F1 and its deletion mutant is successfully constructed .Both of them interact with p53.Deletion of amino acid region 32-81 of E4F1 increases the interaction .This study contributes to further studies on the regulation and mechanism of E 4F1 on p53.
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Influenza virus contains three integral membrane proteins: haemagglutinin, neuraminidase, and matrix protein (M1 and M2). Among them, M2 protein functions as an ion channel, important for virus uncoating in endosomes of virus-infected cells and essential for virus replication. In an effort to explore potential new functions of M2 in the virus life cycle, we used yeast two-hybrid system to search for M2-associated cellular proteins. One of the positive clones was identified as human Hsp40/Hdj1, a DnaJ/Hsp40 family protein. Here, we report that both BM2 (M2 of influenza B virus) and A/M2 (M2 of influenza A virus) interacted with Hsp40 in vitro and in vivo. The region of M2-Hsp40 interaction has been mapped to the CTD1 domain of Hsp40. Hsp40 has been reported to be a regulator of PKR signaling pathway by interacting with p58(IPK) that is a cellular inhibitor of PKR. PKR is a crucial component of the host defense response against virus infection. We therefore attempted to understand the relationship among M2, Hsp40 and p58(IPK) by further experimentation. The results demonstrated that both A/M2 and BM2 are able to bind to p58(IPK) in vitro and in vivo and enhance PKR autophosphorylation probably via forming a stable complex with Hsp40 and P58(IPK), and consequently induce cell death. These results suggest that influenza virus M2 protein is involved in p58(IPK) mediated PKR regulation during influenza virus infection, therefore affecting infected-cell life cycle and virus replication.
Subject(s)
Humans , HSP40 Heat-Shock Proteins , Genetics , Metabolism , Orthomyxoviridae , Genetics , Metabolism , Phosphorylation , Protein Binding , Genetics , Signal Transduction , Genetics , Two-Hybrid System Techniques , Viral Matrix Proteins , Metabolism , Virus Replication , Genetics , Virus Uncoating , eIF-2 Kinase , MetabolismABSTRACT
Small ubiquitin-like modifiers (SUMO) are a family of proteins that modulate important functional properties,including protein interaction,subcellular localization,protein dimerization,DNA binding and/or transactivation of transcription factors.It has been suggested that SUMO proteins may play an important role in breast carcinogenesis by sumoylation of estrogen signaling proteins such as co-regulators,and breast cancer-related proteins.
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AIM: To construct human Egr-1 promoter luciferase reporter system and study its activity induced by i-onizing radiation. METHODS: Egr-1 promoter was obtained by human genomic PCR and cloned into pGL3-basic vector. After transfection of recombinant plasmid into human tumor cells, the Egr-1 promoter activity induced by ionizing radiation was detected by luciferase reporter assay. RESULTS: The luciferasy reporter system of Egr-1 promoter was successfully constructed. The activity of Egr-1 promoter was substantially increased after different doses of IR and reached to the peak at the time point of 48h after IR. CONCLUSION: The Egr-1 promoter was constructed in this study showed IR inducible activity in tumor cells, laying foundation for the research of radiation, mediated gene therapy.
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Objective To construct estrogen receptor α (ERα)trans-activation system. Methods The full length ERα and its different function regions [ ( transcriptional activation function 1 ( AF1 ), DNA inding domain ( DBD), and transcriptional activation function 2 ( AF2 ) ] were amplified from pcDNA3 -ERα by PCR and cloned into the pGAL vector. The expressions of the recombinant plasmids constructed were detected via immunoblotting. The 293T cells transfected with recombinant plasmids of full length ERα, its different function regions and empty vector were divided into 5 groups; each group was divided into 2 parts which were treated with or without estrogen (E2). The transcriptional activity of each group was detected in 293T cells after the recombinant plasmid was co-transfected with 0. 2 μg of estrogen receptor element luciferase(ERE-LUC) and 0. 1 μg of plasmid expressing β-galactosidase and treated with or without 10 nmol/L E2 for 24 hours. Results The full length ERα and its different function regions were expressed in the 293T cells. Compared with the empty pGAL vector, the transcription activities of full length ERα, AF1, AF2 and DBD recombinant plasmids were raised about 20. 44±1.01, 2. 09±0. 11, 8. 09±0. 30 and 1.05±0. 09 fold, respectively, with the induction of E2 after transfection in the 293T colls. Conclusion The trans-activation system of ERα has been successfully established.
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In order to investigate if there exists interaction between mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) protein, and how the interaction regulates tumor necrosis factor-? (TNF-?) transcription activity, the human p38 and extracellular-signal regulated protein kinase 2 (ERK2) genes were amplified from human flag-p38 and flag-ERK2 by polymerase chain reaction (PCR) and cloned into pcDNA3-HA. Protein expression of the plasmids was examined by Western blotting. Co-immunoprecipitation was used to identify if there exists interaction between MAPK and STAT3 proteins. If the interaction was approved to be true, report gene system was applied to find how the interaction affect transcriptional expression of TNF-?. After STAT3 pathway was inhibited by RNA interfering, the action on TNF-? activity was determined. The results of DNA sequencing and enzyme digestion showed that the cloned p38 and ERK2 genes were correct, to be 1 080 bp or so. p38 and ERK2 proteins were expressed in 293T cell to be approximately 40 ku. Co-immunoprecipitation data showed that p38 and ERK2 proteins integrated with STAT3 protein in vivo. TNF-? reporter gene activity results found that protein complex of p38-STAT3 and ERK2-STAT3 coordinately increased TNF-? activity. After STAT3 was interfered, the TNF-? activity markedly decreased. These data indicated that there exists interaction between p38 and STAT3 protein, ERK2 and STAT3 protein. The complex of the proteins can coordinately regulate TNF-? expression. After interfereing STAT3 pathway, the coordinated action on TNF-? transcription activity might be obviously reduced.
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Gsalpha gene mutation has been discovered in some human tumors. In our previous studies, three novel deletants of Gsalpha gene, Gsalpha L-1(500 bp), Gsalpha L-2(300 bp), and Gsalpha L-3(200 bp), and wild type Gsalpha-4(1 200 bp) were found in human leukemia cell lines and detected in leukemic cells from patients with acute leukemia. To investigate the construction, function and biological significance of the deletants, the plasmids of Gsalpha L-1, Gsalpha L-2 and wild Gsalpha-4 were transformed into E. coli DH5, amplified by PCR, and cloned in expression vector pET22b(+), and then transformed into E. coli, respectively. As a result, higher levels of expression of three recombinants were obtained in form of inclusion bodies. The results suggested that these Gsalpha isoforms have an open reading frame of gene and can be expressed in vitro. The data lay a foundation to study the relation of Gsalpha gene to leukemogenesis.
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GST/MCP-1 fusion protein was overexpressed in Escherichia coli as inculstion bodies. After isolation of the inclusion bodies, the optimum conditions of denaturation and renaturation were studied. The renatured produts were purified to be electroporetically pure by affinity chromatography on immobilised glutathione. The purified product remains biological activities and can react specifically with MCP-1 antibodies by western blot analysis. The in vitro antitumor effect showed that GST/MCP-1 could activate monocytes and lymphocytes to inhibit the growth of a lung adenocarcinoma cell line A549. In vivo, GST/MCP-1 could inhibit the tumor growth in nude mice. These results suggested that MCP-1 had antitumor effect.
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Messenger RNA was isolated directly from lung cancer cell line A549 using magnetic particles. First strand synthesis from mRNA was driven by M-MLV(Moloney Murine Leukemia Virus) reverse transcriptase and random hexam-eric primer, followed by second strand synthesis using RNase H and DNA polymerase I After treatment with T4 DNA polymerase to flush the ends, the double-stranded cDNA was cloned into the plasmid expression vector digested with EcoRI and followed by removing cohesive end. The number of independent clones of the resulting cDNA library was about 9.0 x 105. The estimated percentage of colonies with inserts was about 85 % . The insert size ranges from 0.5 kb ~ 4 kb. The CPP32 gene coding for death protease was obtained by PCR with the cDNA library from lung cancer cells as a template for the first time. Construction of the cDNA library laid a foundation for screening other genes regulating death of lung cancer cells.