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We previously found that the position of matrix attachment regions (MARs) within the vector significantly affects its ability to enhance transgenic expression in the recombinant protein production. This study aims to systematically investigate the position-dependent impacts of MAR on transgene expression. We observed a significant increase in enhanced green fluores-cent protein (eGFP) expression levels in stably transfected CHO-K1 cells with either MAR 1-68 or MAR X-29 when MARs located upstream of the promoter. This increase was especially evident with MAR flanked the expression cassette. Concurrently, a substantial increase was observed in the percentage of eGFP-expressing cells, with 97.8% and 96.0% in MAR-containing constructs versus 73.7% in MAR-absent constructs. Further analysis of erythropoietin (EPO) expression revealed that constructs with flanking MARs induced the highest EPO productivity. Bioinformatics analysis revealed that certain specific transcription factors are important in modulating the transcription process. In conclusion, vectors harboring both MARs around the expression cassette constitute an optimal construct for enhanced recom-binant protein production in CHO-K1 cells. This insight underscores the importance of strate-gic MAR incorporation in vector design for optimized recombinant protein expression.
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Liver cancer is a major malignant tumor, which seriously threatens human health and increases the economic burden on patients. At present, gene therapy has been comprehensively studied as an excellent therapeutic measure in liver cancer treatment. Oncolytic virus (OV) is a kind of virus that can specifically infect and kill tumor cells. After being modified by genetic engineering, the specificity of OV infection to tumor cells is increased, and its influence on normal cells is reduced. To date, OV has shown its effectiveness and safety in experimental and clinical studies on a variety of tumors. Thus, this review primarily introduces the current status of different genetically engineered OVs used in gene therapy for liver cancer, focuses on the application of OVs and different target genes for current liver cancer therapy, and identifies the problems encountered in OVs-based combination therapy and the corresponding solutions, which will provide new insights into the treatment of liver cancer.
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Neoplasias Hepáticas , Neoplasias , Viroterapia Oncolítica , Virus Oncolíticos , Humanos , Virus Oncolíticos/genética , Neoplasias/genética , Neoplasias Hepáticas/terapia , Inmunoterapia , Terapia GenéticaRESUMEN
@#The recombinant protein drugs have the advantages of high activity,high specificity and low toxicity,which have a very broad application prospect in the world.At present,the recombinant therapeutic proteins in vitro are produced from prokaryotic and eukaryotic expression system in order to achieve efficient expression.In view of the advantages such as perfect post-translational modification and protein folding,eukaryotic expression system has been widely used in the production of recombinant protein drugs.There are four commonly used eukaryotic expression systems,including yeast,insect cell,mammalian cell and plant cell expression systems.In this paper,the characteristics,applications and prospects of several common eukaryotic expression systems are reviewed,in view to providing a reference for the selection of suitable expression systems for recombinant protein drugs and promoting the research and development of new drugs.
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Previous work has shown that the EF-1α promoter of episomal vectors maintains high-level transgene expression in stably transfected Chinese hamster ovary (CHO) cells. However, the transgene expression levels need to be further increased. Here, we first incorporated matrix attachment regions (MARs), ubiquitous chromatin opening element (UCOE), stabilizing anti repressor elements 40 (STAR 40) elements into episomal vector at different sites and orientations, and systemically assessed their effects on transgene expression in transfected CHO-K1 cells. Results showed that enhanced green fluorescent protein (eGFP) expression levels increased remarkably when MAR X-29 was inserted upstream of the promoter, followed by the insertion of MAR1 downstream of the poly A, and the orientation had no significant effect. Moreover, MAR X-29 combined with human cytomegalovirus intron (hCMVI) yielded the highest transgene expression levels (4.52-fold). Transgene expression levels were not exclusively dependent on transgene copy numbers and were not related to the mRNA expression level. In addition, vector with MAR X-29+hCMVI can induce herpes simplex virus thymidine kinase (HSV-TK) protein expression, and the HSV-TK protein showed a cell-killing effect and an obvious bystander effect on HCT116 cells. In conclusion, the combination of MAR X-29 and hCMV intron can achieve high efficiency transgene expression mediated by episomal vectors in CHO-K1 cells.
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Vectores Genéticos , Regiones de Fijación a la Matriz , Cricetinae , Animales , Humanos , Cricetulus , Transfección , Células CHO , Intrones/genética , Transgenes/genética , Regiones de Fijación a la Matriz/genética , Vectores Genéticos/genéticaRESUMEN
Esophageal cancer (EC) is the tenth most common cancer worldwide and has high morbidity and mortality. Its main subtypes include esophageal squamous cell carcinoma and esophageal adenocarcinoma, which are usually diagnosed during their advanced stages. The biological defects and inability of preclinical models to summarize completely the etiology of multiple factors, the complexity of the tumor microenvironment, and the genetic heterogeneity of tumors severely limit the clinical treatment of EC. Patient-derived models of EC not only retain the tissue structure, cell morphology, and differentiation characteristics of the original tumor, they also retain tumor heterogeneity. Therefore, compared with other preclinical models, they can better predict the efficacy of candidate drugs, explore novel biomarkers, combine with clinical trials, and effectively improve patient prognosis. This review discusses the methods and animals used to establish patient-derived models and genetically engineered mouse models, especially patient-derived xenograft models. It also discusses their advantages, applications, and limitations as preclinical experimental research tools to provide an important reference for the precise personalized treatment of EC and improve the prognosis of patients.
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Adenocarcinoma , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Ratones , Animales , Humanos , Neoplasias Esofágicas/terapia , Neoplasias Esofágicas/tratamiento farmacológico , Adenocarcinoma/genética , Adenocarcinoma/terapia , Adenocarcinoma/patología , Modelos Animales de Enfermedad , Microambiente TumoralRESUMEN
Abdominal adhesions, a commonly observed complication of abdominal surgery, have a high incidence and adversely affect patients' physical and mental health. The primary causes of abdominal adhesions are intraoperative trauma, acute inflammatory response, bleeding, and foreign body infection. Because most current treatment approaches for abdominal adhesions are limited, improved and novel postoperative anti-adhesion regimens are urgently needed. In this study, we developed calcium polyphenol network (CaPN) microspheres based on the self-assembly of the natural triphenolic compound gallic acid and Ca2+ in solution. The physicochemical properties of CaPNs, including their hemostatic, antibacterial, antioxidant, and anti-inflammatory activities, were investigated in vitro. Bleeding and cecal-abdominal wall adhesion models were established to observe the hemostatic activity of CaPNs and their preventive effect on postoperative abdominal wall adhesion in vivo. The results showed that CaPNs significantly reduced inflammation, oxidative stress, fibrosis, and abdominal adhesion formation and had good hemostatic and antibacterial properties. Our findings suggest a novel strategy for the prevention of postoperative adhesions.
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Calcio , Hemostáticos , Humanos , Polifenoles/farmacología , Polifenoles/uso terapéutico , Adherencias Tisulares/prevención & control , Antibacterianos/farmacologíaRESUMEN
For the advantages of low cost, excellent thermal insulation, and sound absorption properties, the rigid isocyanate-based polyimide foam (RPIF) presents great application prospects as a building insulation material. However, its inflammability and the accompanying toxic fumes create huge safety hazard. In this paper, reactive phosphate-containing polyol (PPCP) is synthesized and employed with expandable graphite (EG) to obtain RPIF with excellent use safety. EG can be considered as an ideal partner for PPCP to weaken the drawbacks in toxic fume release. Limiting oxygen index (LOI), cone calorimeter test (CCT), and toxic gas results show that the combination of PPCP and EG can synergistically enhance flame retardancy and the use safety of RPIF owing to the unique structure of a dense char layer possessing a flame barrier and toxic gas adsorption effects. When EG and PPCP are simultaneously applied to the RPIF system, the higher EG dosage will bring higher positive synergistic effects in the use safety of RPIF. The most preferred ratio of EG and PPCP is 2:1 (RPIF-10-5) in this study; RPIF-10-5 shows the highest LOI, low CCT results and specific optical density of smoke, and low HCN concentration. This design and the findings are of great significance to improving the application of RPIF.
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Chinese hamster ovary (CHO) cells are by far the most commonly used mammalian expression system for recombinant expression of therapeutic proteins in the pharmaceutical industry. The development of high-yield stable cell lines requires processes of transfection, selection, screening and adaptation, among which the screening process requires tremendous time and determines the level of forming highly productive monoclonal cell lines. Therefore, how to achieve productive cell lines is a major question prior to industrial manufacturing. Cell line development (CLD) is one of the most critical steps in the production of recombinant therapeutic proteins. Generation of high-yield cell clones is mainly based on the time-consuming, laborious process of selection and screening. With the increase in recombinant therapeutic proteins expressed by CHO cells, CLD has become a major bottleneck in obtaining cell lines for manufacturing. The basic principles for CLD include preliminary screening for high-yield cell pool, single-cell isolation and improvement of productivity, clonality and stability. With the development of modern analysis and testing technologies, various screening methods have been used for CLD to enhance the selection efficiency of high-yield clonal cells. This review provides a comprehensive overview on preliminary screening methods for high-yield cell pool based on drug selective pressure. Moreover, we focus on high throughput methods for isolating high-yield cell clones and increasing the productivity and stability, as well as new screening strategies used for the biopharmaceutical industry.
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Recombinant antibodies are rapidly developing therapeutic agents; approximately 40 novel antibody molecules enter clinical trials each year, most of which are produced from Chinese hamster ovary (CHO) cells. However, one of the major bottlenecks restricting the development of antibody drugs is how to perform high-level expression and production of recombinant antibodies. The high-efficiency expression and quality of recombinant antibodies in CHO cells is determined by multiple factors. This review provides a comprehensive overview of several state-of-the-art approaches, such as optimization of gene sequence of antibody, construction and optimization of high-efficiency expression vector, using antibody expression system, transformation of host cell lines, and glycosylation modification. Finally, the authors discuss the potential of large-scale production of recombinant antibodies and development of culture processes for biopharmaceutical manufacturing in the future.
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BACKGROUND: Colon cancer is one of the most common types of cancer worldwide. Multiple studies have unveiled the key role of microRNAs (miRNAs) in the development of various types of cancer. However, the mechanism of action of miR-125b in the development and progression of colon cancer remains unknown. OBJECTIVES: In this study, we explored the association of miR-125b and signal transducer and activator of transcription 3 (STAT3) and its role in the proliferation and apoptosis of SW480 colon cancer cells. METHODS: The miR-125b expression in NCM460, SW480, HT29, and HCT8 cells was detected using quantitative real-time polymerase chain reaction (qRT-PCR). SW480 cells were transfected with lentiviruses of GFP-miR-125b and GFP-NC to establish a stable miR-125b overexpression colon cancer cell model and a control model. The targeting relationship between miR-125b and STAT3 was analyzed using bioinformatics and verified by the dual-luciferase reporter gene assay. Cell proliferation and apoptosis were assessed using the Cell Counting Kit-8 assay and TUNEL staining. The expression levels of STAT3, Bcl-2, and Bax were analyzed using Western blot analysis. RESULTS: It was found that the relative mRNA expression of miR-125b was decreased in SW480, HT29, and HCT8 cells compared with that in NCM460 cells (P<0.05). The luciferase reporter gene assay confirmed that miR-125b downregulated the STAT3 gene expression (P<0.05). Overexpression of miR-125b inhibited proliferation and promoted apoptosis in SW480 colon cancer cells and was accompanied by upregulated Bax expression and downregulated Bcl-2 expression (P<0.05). Re-expression of STAT3 promoted cell proliferation and inhibited cell apoptosis, whereas Bcl-2 expression increased, and Bax expression decreased (P<0.05). CONCLUSION: The miR-125b regulates the expression of Bax and Bcl-2 by downregulating the expression of STAT3, thereby inhibiting proliferation and inducing apoptosis of SW480 colon cancer cells.
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Neoplasias del Colon , MicroARNs , Apoptosis/genética , Línea Celular Tumoral , Proliferación Celular/genética , Neoplasias del Colon/genética , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
We report the development of a compact near-infrared (NIR) laser-based trace methane (CH4) detection system. This detection system relied on a 2334 nm distributed feedback (DFB) fiber laser as the light source. A parallel dense light-spot pattern multipass gas cell (MGC) with 41.5 m effective absorption path length was utilized to improve the system sensitivity. A self-calibration approach based on direct absorption spectroscopy (DAS) calibrated wavelength modulation spectroscopy (WMS) technique was employed to solve the problem of extra concentration calibration requirement in traditional WMS technique, and to improve the accuracy and stability of the system. According to the Allan deviation analysis, 1-s measurement precision of 0.61 ppmv for DAS and 0.16 ppmv for WMS was obtained, which could be further reduced to 0.11 ppmv for DAS and 0.03 ppmv for WMS by averaging up to 80 s and 50 s, respectively. A week-long continuous atmospheric CH4 concentration measurement was also carried out to demonstrate the long-term performance of our CH4 detection system. With a fast dynamic response characteristics, high-accuracy and high-sensitivity, the proposed detection system is suitable for CH4 measurement in many fields such as atmospheric chemistry analyzation, industrial safety monitoring, agricultural information acquisition, etc.
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Rayos Láser , MetanoRESUMEN
In this study, the feasibility of classifying soybean frogeye leaf spot (FLS) is investigated. Leaf images and hyperspectral reflectance data of healthy and FLS diseased soybean leaves were acquired. First, image processing was used to classify FLS to create a reference for subsequent analysis of hyperspectral data. Then, dimensionality reduction methods of hyperspectral data were used to obtain the relevant information pertaining to FLS. Three single methods, namely spectral index (SI), principal component analysis (PCA), and competitive adaptive reweighted sampling (CARS), along with a PCA and SI combined method, were included. PCA was used to select the effective principal components (PCs), and evaluate SIs. Characteristic wavelengths (CWs) were selected using CARS. Finally, the full wavelengths, CWs, effective PCs, SIs, and significant SIs were divided into 14 datasets (DS1-DS14) and used as inputs to build the classification models. Models' performances were evaluated based on the classification accuracy for both the overall and individual classes. Our results suggest that the FLS comprised of five classes based on the proportion of total leaf surface covered with FLS. In the PCA and SI combination model, 5 PCs and 20 SIs with higher weight coefficient of each PC were extracted. For hyperspectral data, 20 CWs and 26 effective PCs were also selected. Out of the 14 datasets, the model input variables provided by five datasets (DS2, DS3, DS4, DS10, and DS11) were more superior than those of full wavelengths (DS1) both in support vector machine (SVM) and least squares support vector machine (LS-SVM) classifiers. The models developed using these five datasets achieved overall accuracies ranging from 91.8% to 94.5% in SVM, and 94.5% to 97.3% in LS-SVM. In addition, they improved the classification accuracies by 0.9% to 3.6% (SVM) and 0.9% to 3.7% (LS-SVM).
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Glycine max/ultraestructura , Procesamiento de Imagen Asistido por Computador/métodos , Micosis/microbiología , Enfermedades de las Plantas/microbiología , Hojas de la Planta , Espectroscopía Infrarroja Corta/métodos , Cercospora , Hojas de la Planta/microbiología , Hojas de la Planta/ultraestructura , Glycine max/microbiología , Máquina de Vectores de SoporteRESUMEN
Oncolytic adenovirus-mediated gene therapy shows promise for cancer treatment; however, the systemic delivery of oncolytic adenovirus to tumors remains challenging. Recently, mesenchymal stem cells (MSCs) have emerged as potential vehicles for improving delivery. Yet, because the oncolytic adenovirus replicates in MSCs, balancing MSC viability with viral load is key to achieving optimal therapeutic effect. We thus developed an all-in-one Tet-on system that can regulate replication of oncolytic adenovirus. Then, we loaded the novel oncolytic adenovirus carrying interleukin (IL)-24 and/or Endostatin in human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) for glioma therapy. In vitro assays demonstrated that this novel oncolytic adenovirus could efficiently replicate and kill glioma cells while sparing normal cells. Moreover, doxycycline effectively regulated oncolytic adenovirus replication in the hUCB-MSCs. The doxycycline induction group with dual expression of IL-24 and Endostatin exhibited significantly greater antitumor effects than other groups in a xenograft model of glioma. Thus, this strategy for systemic delivery of oncolytic adenovirus with its oncolytic activity controlled by a Tet-on system is a promising method for achieving antitumor efficacy in glioma, especially for metastatic tumors.
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Neoplasias Encefálicas/terapia , Trasplante de Células Madre de Sangre del Cordón Umbilical , Endostatinas/biosíntesis , Terapia Genética , Glioma/terapia , Interleucinas/biosíntesis , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/virología , Viroterapia Oncolítica , Virus Oncolíticos/genética , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/virología , Muerte Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Endostatinas/genética , Femenino , Vectores Genéticos , Glioma/genética , Glioma/metabolismo , Glioma/virología , Humanos , Interleucinas/genética , Ratones Endogámicos BALB C , Ratones Desnudos , Virus Oncolíticos/crecimiento & desarrollo , Carga Tumoral , Replicación Viral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The aim of this study was to construct an expression vector mediated by the dual promoter that can simultaneously drive the recombinant protein production in eukaryotic and prokaryotic cells. The prokaryotic T7 promoter and ribosome binding site (RBS) was cloned downstream of CMV promoter in the eukaryotic expression vector pIRES-neo, and T7 termination sequence was inserted upstream of neomycin phosphotransferase gene to generate the dual promoter vector. The enhanced green fluorescent protein (eGFP) gene was used as reporter gene. Then, the resultant vector was transfected into Chinese hamster ovary (CHO) cells and transformed into Escherichia coli (E. coli) BL21, and the eGFP expression levels were analyzed by fluorescence microscopy, flow cytometry and Western blot, respectively. Fluorescence microscopy revealed that the eGFP was expressed in both CHO cells and E. coli BL21. Flow cytometry showed that the eGFP expression level had no significant difference between the dual promoter vector and control vector in transfected CHO cells. Western blot analysis indicated the eGFP expressed in transformed E. coli. In conclusion, a prokaryotic-eukaryotic double expression vector was successfully constructed, which has potential applications in rapid cloning and expression of recombinant proteins in both prokaryotic and eukaryotic expression systems.
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Ingeniería Genética/métodos , Vectores Genéticos/genética , Regiones Promotoras Genéticas , Animales , Células CHO , Cricetinae , Cricetulus , Escherichia coli , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismoRESUMEN
BACKGROUND: Breast cancer is among the most common malignant cancers worldwide, and breast adenocarcinoma in glandular tissue cells has excessive metastasis and invasion capability. However, little is known on the molecular process by which this disease develops and progresses. OBJECTIVE: In this study, we explored the effects of sex-determining region Y-box 4 (SOX4) protein on proliferation, migration, apoptosis and tumourigenesis of breast adenocarcinoma and its possible mechanisms. METHODS: The SOX4 overexpression or knockdown Michigan Cancer Foundation-7 (MCF-7) cell lines were established. Among the SOX4 overexpression or MCF-7 knockdown cell lines, proliferation, migration ability and apoptosis rate were detected. The expression levels of apoptosis-related proteins (Bax and Cleaved caspase-3) were analysed using Western blot. The effect of SOX4 on tumourigenesis was analysed using the clone formation assay in vitro and tumour xenograft experiment in nude mice. RESULTS: Compared with the overexpression of control cells, proliferation and migration ability of SOX4 overexpression cells significantly increased, the apoptosis rate significantly decreased in addition to the expression levels of Bax and Cleaved caspase-3 (P < 0.05). Compared with the knockdown of control cells, proliferation and migration ability of SOX4 knockdown cells significantly decreased, and the apoptosis rate and expression levels of Bax and Cleaved caspase-3 significantly increased (P < 0.05). Clone formation and tumour growth abilities of SOX4 overexpression cells were significantly higher than those of the control cells (P < 0.05), whereas SOX4 knockdown cells had the opposite effect. CONCLUSION: SOX4 plays an oncogenic role in breast adenocarcinoma tumourigenesis by promoting cell proliferation, migration and inhibiting apoptosis. It can be used as a potential molecular target for breast cancer gene therapy.
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Adenocarcinoma/etiología , Apoptosis , Neoplasias de la Mama/etiología , Factores de Transcripción SOXC/fisiología , Adenocarcinoma/patología , Animales , Neoplasias de la Mama/patología , Carcinogénesis , Movimiento Celular , Proliferación Celular , Femenino , Humanos , Células MCF-7 , Ratones , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Colon cancer is one of the most common malignant tumors, and B cell Translocation Gene (BTG)1 is involved in the occurrence and development of colon cancer, however, the underlying molecular mechanism remains unclear. OBJECTIVE: In this study, we investigated the expression of BTG1 protein in colon cancer, and its association with clinicopathology and prognosis. METHODS: The tumor specimens from 59 patients with colon cancer who had undergone radical colectomy were selected as the observation group. Para-carcinoma tissues from the same patients were selected as the control group. The expressions of BTG1 mRNA and protein in the specimen of two groups were analyzed by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Western blot. According to the immunohistochemical results, the patients were divided into BTG1-negative and BTG1-positive groups. The postoperative cumulative survival rate in the two groups was analyzed. The association of the expression of BTG1 protein with the clinicopathological features and postoperative survival was investigated. RESULTS: Compared with the control group, the expression levels of BTG1 mRNA and BTG1 protein were significantly decreased in the observation group (P < 0.05). Immunohistochemical analysis revealed that there were 12 positive tumor samples and 47 negative samples. The expression of BTG1 was negatively associated with the degree of differentiation and lymphatic metastasis. The cumulative survival rate of BTG1-positive patients was significantly increased compared with that of BTG1- negative patients (P < 0.05). Stepwise Cox regression analysis showed that lymphatic metastasis, tumor size and BTG1 expression level were independent prognostic factors for overall survival in patients with colon cancer. CONCLUSION: BTG1 protein in colon cancer tissues were expressed at low levels, which was associated with the clinicopathological features, postoperative recurrence and survival of patients.
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Neoplasias del Colon/patología , Proteínas de Neoplasias/fisiología , Anciano , Neoplasias del Colon/mortalidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/análisis , Proteínas de Neoplasias/genética , Recurrencia Local de Neoplasia , Pronóstico , Modelos de Riesgos Proporcionales , ARN Mensajero/análisisRESUMEN
OBJECTIVES: Previously, we have found that the matrix attachment region (MAR) may confer a 'distance effect' on transgene expression. This work aims to systematically explore the increased transgene expression in transfected Chinese hamster ovary (CHO) cells due to the characteristics of MAR and its mechanism. RESULTS: Compared with the control vector, 500 and 1000 bp DNA distances between MAR and the cytomegalovirus promoter can increase transgene expression by 1.77- and 1.56-fold, respectively. Meanwhile, transgene expression was not affected when 2000 and 2500 bp spacer DNAs were inserted, but a declining trend was observed when a 1500 bp spacer DNA was inserted. The vector containing a 500 bp DNA distance significantly increased the expression of the enhanced green fluorescent protein, and this increase was not related to transgene copy numbers. CONCLUSIONS: A short DNA distance-containing MAR confers high transgene expression level in transfected CHO cells, but a distance threshold does not exist in the vector system.
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Clonación Molecular/métodos , Proteínas Recombinantes/metabolismo , Transgenes , Animales , Células CHO , Cricetinae , Cricetulus , Expresión Génica , Regiones de Fijación a la Matriz , Regiones Promotoras Genéticas , TransfecciónRESUMEN
BACKGROUND: Esophageal carcinoma is one of the common malignant tumors in digestive tract. BECLIN-1 is a key gene that regulates autophagy, and its abnormal expression may be related with many human tumors. However, the mechanism of BECLIN-1 in esophageal carcinoma remains unknown. OBJECTIVE: In this study, we explored the effect of BECLIN-1 overexpression on tumor growth in mice with esophageal carcinoma and its mechanism. METHODS: Recombined lentiviral vector containing BECLIN-1 was used to transfect human esophageal carcinoma Eca109 cells and establish stable cell line. qRT-PCR was used to detect BECLIN-1 mRNA level in the transfected Eca109 cells, CCK-8 assay was used to detect cell proliferation. Beclin-1, P62 and LC3-II protein expression levels in Eca109 cells were detected using Western blot analysis. Subcutaneous xenograft nude mice model of human esophageal carcinoma was established, and the tumor growths in Beclin-1 group, control group and empty vector group were monitored. Beclin-1 protein expression in vivo was detected by immunohistochemistry. RESULTS: Beclin-1 mRNA and protein were overexpressed in Eca109 cells. Compared with empty vector group, the growth rate of cells transfected with BECLIN-1 decreased significantly. Compared with the control group and empty vector group, the expression level of P62 protein in beclin-1 group was significantly decreased, while the expression level of LC3-II protein was significantly increased. The tumor growth rate in nude mice of Beclin-1 group was significantly lower than that of the control group and empty vector group, and Beclin-1 protein was mainly expressed in Beclin-1 group in vivo. CONCLUSION: BECLIN-1 can induce autophagy in esophageal carcinoma Eca109 cells, and it can significantly inhibit the growth of esophageal carcinoma.
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Autofagia/fisiología , Beclina-1/fisiología , Neoplasias Esofágicas/patología , Animales , Beclina-1/análisis , Beclina-1/genética , Proliferación Celular , Neoplasias Esofágicas/terapia , Terapia Genética , Humanos , Lentivirus/genética , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , ARN Mensajero/análisis , Proteínas de Unión al ARN/análisis , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The episomal vector cannot integrate into the host cell chromosome, which has no potential risk in gene therapy. However, the low level of transgene expression driven by episomal vectors needs to be solved. In this study, we investigated the effects of enhancers, promoters and promoter variants on transgene expression levels driven by episomal vectors in HEK293, Chang liver and primary cells. Results showed that all eight cis-acting elements used could increase transfection efficiency and transient eGFP expression in transfected HEK293 and Chang liver cells. In stably transfected mammalian cells, the elongation factor-1 alpha (EF-1α) promoter and mutant-404 showed high and stable transgene expression. The mechanisms might be related to the type and quantity of transcription factor regulatory elements. Moreover, quantitative reverse transcription polymerase chain reaction analysis showed that mRNA expression levels were not directly proportional to protein expression levels. Furthermore, the EF-1α promoter conferred high transgene expression levels in primary cells, and the plasmid was also present in the episomal state. Taken together, these results provided valuable information for improving transgene expression with episomal vectors in mammalian cells.
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Elementos de Facilitación Genéticos , Hígado/metabolismo , Plásmidos/genética , Regiones Promotoras Genéticas , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Hígado/citología , Plásmidos/metabolismo , Cultivo Primario de Células , Transfección , TransgenesRESUMEN
In this study, we systemically investigated the positions and orientations of matrix attachment regions (MARs) in expression vectors to fully explore the mechanism for improving transgene expression. We constructed 14 vectors that incorporated human ß-globin MARs into pIRES-eGFP backbone vectors. The MARs flanked the eGFP expression cassette or promoter in a forward/reverse orientation. After stable transfection into CHO-K1 cells with these vectors, eGFP expression levels were increased significantly relative to that of the control vector (MAR-devoid) when two MARs flanking the expression cassette were incorporated, followed by those at the 5' site (upstream of the promoter). Simultaneously, the percentage of the eGFP-expressing cells was elevated to some extent. The vector with both MARs in forward orientation flanking the expression cassette yielded the highest transgene expression levels (2.5-fold). The orientation (forward or reverse) of the MARs did not present a significant difference when added in the same site. In addition, transgene expression levels were not exclusively dependent on transgene copy numbers. Bioinformatic analysis indicated that some specific transcription factors may contribute to the transcriptional process. In conclusion, two MARs in a forward orientation and flanking the expression cassette comprised the optimal construct for improving the stable transgene expression in the CHO-K1 cells. The effects may be related to specific transcription factors, such as PRDM1 and REL.