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E2F1, a nucleoprotein gene belongs to transcription factor, is closely associated with the development of malignant tumours. Long non-coding RNAs (lncRNAs) are aberrantly expressed in a variety of tumors. In studies of molecular mechanisms associated with lncRNAs and tumours, E2F1 has been identified as a key factor that can play a critical role as an upstream regulator or downstream target of lncRNAs, and even inter-regulate to form a positive feedback loop. This paper reviews the significance of the interaction between E2F1 and lncRNA in malignant tumors in recent years, and aims to provide ideas for the study of tumor mechanisms.
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Objective:To explore the mechanism of miR-205-5p/E2F1 signal axis in regulating the glioma U251, U87 radiotherapy resistance.Methods:X-ray gradual ascending and intermittent induction method was used to irradiate the glioma U251 cells to establish U251/TR, U87/TR radiation-resistant cell lines. Then, the morphology, migration, invasion and proliferation abilities of cells (U251/TR, U87/TR radiation-resistant cells and U251, U87 radiation-sensitive cells) were analyzed. Luciferase gene detection system and point mutation technique were employed to analyze the mechanism of miR-205-5p and E2F1 gene activity on U251 and U87 radiation-resistant cell lines.Results:Compared with the radiation-sensitive U251 cells, the radiation-resistant cells U251/TR, U87/TR showed increased proliferation activity, enhanced migration and invasion abilities and decreased apoptosis under X-ray irradiation. miR-205-5p mimics transfection could down-regulate the expression of E2F1 factor in U251/TR cells, inhibit cell proliferation, invasion and migration and increase the radiosensitivity of U251/TR cells. miR-205-5p mimics transfection combined with with E2F1 down-regulation exerted anti-tumor effect and decreased cell tolerance by suppressing the Wnt/β-catenin signaling pathway activity.Conclusions:The glioma radiation-resistant cell line U251/TR, U87/TR can be established by X-ray gradual ascending and intermittent induction method. The miR-205-5p/E2F1 signal axis exerts tumor-suppressing effect through the classical Wnt/β-catenin signaling pathway, which can be used as an therapeutic target to increase the radiosensitivity of glioma.
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Objective@#To explore the regulatory mechanism of E2F1 transcription factor on M2 macrophages in full-thickness skin defect wounds of mice.@*Methods@#E2F1 gene knockout heterozygotes C57BL/6 mice and wild-type C57BL/6 mice were introduced and self-reproduced. Two weeks after birth, E2F1 gene knockout homozygotes mice and wild-type mice were identified by polymerase chain reaction (PCR). Twelve identified 6-8 weeks old male E2F1 gene knockout homozygotes C57BL/6 mice and wild-type C57BL/6 mice were selected respectively according to the random number table and set as E2F1 gene knockout group and wild-type group. A full-thickness skin defect wound was made on the back of each mouse. On post injury day (PID) 2 and 7, 6 mice in each group were selected according to the random number table and sacrificed, and the wound tissue was excised. The expression of CD68 and CD206 double positive M2 macrophages was observed by immunofluorescence method, and the percentage of CD206 positive cells was calculated. The protein expression of CD206 was detected by Western blotting. The mRNA expression of arginase 1 was detected by real-time fluorescent quantitative reverse transcription PCR (RT-PCR). Wound tissue specimens of the two groups on PID 7 were obtained, and the protein and mRNA expressions of peroxisome proliferator-activated receptor gamma (PPAR-γ) were detected by Western blotting and real-time fluorescent quantitative RT-PCR respectively. The above-mentioned experiments were repeated four times. Three specimens of wound tissue of mice in wild-type group on PID 7 were obtained to detect the relationship between E2F1 and PPAR-γ by co-immunoprecipitation and Western blotting, and this experiment was repeated two times. Data were processed with unpaired t test.@*Results@#The size of PCR products of E2F1 gene knockout homozygotes C57BL/6 mice and wild-type C57BL/6 mice were 227 and 172 bp respectively, which were the same as those of the designed DNA fragments. On PID 2 and 7, the number of CD68 and CD206 double positive M2 macrophages in the wound tissue of mice in E2F1 gene knockout group was more than that of wild-type group, and the percentages of CD206 positive cells in the wound tissue of mice in E2F1 gene knockout group were (0.234±0.032)% and (0.584±0.023)% respectively, which were significantly higher than (0.129±0.017)% and (0.282±0.071)% of wild-type group (t=3.29, 3.54, P<0.05). On PID 2 and 7, the protein expression of CD206 in the wound tissue of mice in E2F1 gene knockout group were 1.00±0.23 and 1.63±0.26 respectively, which were significantly higher than 0.43±0.06 and 0.97±0.08 of wild-type group (t=2.41, 2.45, P<0.05). On PID 2 and 7, the mRNA expressions of arginase 1 in the wound tissue of mice in E2F1 gene knockout group were 0.482±0.105 and 0.195±0.031 respectively, which were significantly higher than 0.163±0.026 and 0.108±0.017 of wild-type group (t=3.04, 2.86, P<0.05). On PID 7, the protein and mRNA expressions of PPAR-γ in the wound tissue of mice in E2F1 gene knockout group were 0.61±0.12 and 0.51±0.13 respectively, which were significantly higher than 0.20±0.04 and 0.20±0.04 of wild-type group (t=3.36, 2.86, P<0.05). On PID 7, detection of the wound tissue of mice in wild-type group showed that PPAR-γ had unidirectional effect on E2F1.@*Conclusions@#E2F1 transcription factor affects the polarization of M2 macrophages by inhibiting the expression of PPAR-γ, thereby inhibiting the healing process of full-thickness skin defect wounds in mice.
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Objective To study the effect of microRNA-320 (miR-320) targeting E2F1 gene on tumor glycometabolism in colorectal cancer.Methods The miR-320 expression level in colorectal cancer cell lines and cancer tissues was detected using quantitative real-time polymerase chain reaction (qRT-PCR).The binding sites of miR-320 and E2F1 were predicted by bioinformatics.Luciferase assay was used to detect the targeting regulation of miR-320 on E2F1.The relationship between E2F1 and miR-320 was verified in mRNA level and protein level.When the miR-320 in SW480 and LOVO ceils was up-regulated and the E2F1 was down-regulated,the changes of glycometabolism in tumor cells were analyzed using glucose/glucose oxidase kit and lactate test kit.Results The qRT-PCR results showed low expressions of miR-320 in colorectal cancer cell lines and cancer tissues (F =42.327,P ＜ 0.001;t =4.345,P =0.023).Luciferase assay showed that miR-320 could negatively regulate the expression of E2F1 (t =4.716,P =0.042).The expression levels of E2F1 protein and mRNA (t =4.780,P =0.041;t =5.506,P =0.031) confirmed that miR-320 could interact with E2F1 in LOVO and SW480 cells.Overexpression of miR-320 could reduce the contents of glucose (t =5.262,P=0.034;t =21.079,P=0.002) and lactic acid (t =9.609,P=0.011;t =18.582,P=0.003) in the cellular supematant in SW480 and LOVO ceils.Down-regulating the expression of E2F1 at the same time could enhance the inhibitory effect of miR-320 on glucose (t =5.128,P =0.036;t =5.089,P =0.037) and lactic acid (t =8.573,P =0.013;t =13.364,P =0.006).Conclusion E2F1 is the target gene of miR-320,and miR-320 can regulate the glycometabolism of colorectal cancer cells by targeting E2F1 gene.
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Objective To explore the effect of transcription factor E2F1 on the apoptosis of small cell cancer line H446. Methods Plamid vector- mediated E2F1 small hairpin RNA (shRNA) was used to silence E2F1 in H446 cell. RT-PCR and western-blot assay were used to detect the expressions of E2F1 and Bcl-2. The apoptosis rate in H446 cell line was detected by flow cytometry assay. Result E2F1 protein was suppressed in shRNA1-modified H446 cell. Sgnificant difference of the apoptosis was shown between E2F1 shRNA1 group and the other two groups. Additionaly, the expression of Bcl-2 protein increased in E2F1 shRNA1-modified cell line. Conclusions E2F1 is highly expressed in H446 small cell lung cancer cell line. E2F1 promotes apoptosis of H446 through upregulating Bcl-2 expression.
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The E2F-1 transcription factor is post-translationally modified and stabilized in response to various forms of DNA damage to regulate the expression of cell-cycle and pro-apoptotic genes. The sustained overexpression of E2F-1 is a characteristic feature of gastric cancer. In this study, we investigated the role of short hairpin RNA (shRNA) targeting E2F-1 gene on human gastric cancer MGC-803 cell growth in vivo, and preliminarily revealed the mechanism. Thus, we constructed recombinant pGCSIL-GFP-shRNA-E2F-1 lentiviral vector to knock down E2F-1 expression in human gastric cancer MGC-803 cells in vivo, and studied the effect of E2F-1 shRNA on growth of MGC-803 tumor and evaluated its treatment efficacy. Our data demonstrated that in a mouse model of established gastric cancer, intratumor injection of lentiviral shRNA targeting E2F-1 definitely decreased the endogenous E2F-1 mRNA and protein expression in MGC-803 tumor, and inhibited tumor growth and promoted tumor cells apoptosis. Moreover, we found that E2F-1 shRNA increased the expression of phosphatase and tensin homolog (PTEN), activated caspase-3 and caspase-9, and suppressed nuclear factor (NF)-kappaB expression in tumor tissue as determined by reverse transcription (RT)-PCR and western blotting. In summary, shRNA targeting of E2F-1 can effectively inhibits human gastric cancer MGC-803 cell growth in vivo and may be a potential therapeutic strategy for gastric cancer.
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Animals , Humans , Male , Mice , Apoptosis , Blotting, Western , Caspase 3/metabolism , Caspase 9/metabolism , Cells, Cultured , E2F1 Transcription Factor/antagonists & inhibitors , Genetic Vectors/administration & dosage , Lentivirus/genetics , Mice, Inbred BALB C , Mice, Nude , RNA Interference , RNA, Small Interfering/genetics , Stomach Neoplasms/geneticsABSTRACT
Objective To investigate the effect of morphine on the expression of p53 mRNA and E2F-1 mRNA in human gastric carcinoma cell line MGC-803 .Methods The human gastric cancer cell line MGC-803 was purchased from Cell Biology Research Institute, Chinese Academy of Sciences, and cultured in DMEM liquid culture mediun. The cells were seeded in 6-well plates (1 × 103/ml or 2 × 105/ml, 1 ml/well) and divided into 2 groups (n = 18 wells each):group Ⅰ normal control (group C); group Ⅱ was exposed to 10 μmol/L morphine (group M). The proliferation of the cells was determined by colony formation assay at 7 day of incubation with morphine. The expression of p53 mRNA and E2F-1 mRNA was detected and the ulrastructure of the cells examined with transmission electron microscope after being incubated with morphine for 24 h. Results The proliferation of the cells and E2F-1 mRNA expression were significantly lower and p53 mRNA expression was significantly higher in group M than in group C (P ＜ 0.05). The nuclear evelope was intact and the nucleolus and chromosomes were clearly visible in group C, while in group M fragmentation of nuclear envelope and nucleolus and apoptotic bodies were observed. Conclusion Morphine can inhibit the proliferation of the cells and accelerate the cell apoptosis through up-regulating the expression of p53 gene and down-regulating the expression of E2F-1gene in human gastric carcinoma cell line MGC-803.
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BACKGROUND: E2F1 plays a critical role in the G1-to-S phase transition by inducing various genes that encode S phase-activating proteins and that modulate such diverse cellular functions as DNA synthesis, mitosis and apoptosis. The purpose of this study was to assess the E2F1 expression in relation to the clinicopathologic parameters and other tumor markers in gastrointestinal stromal tumors. METHODS: Immunohistochemical stainings for obtaining the E2F1, p53, and Ki-67 labeling indices were performed on a tissue microarray of 72 gastrointestinal stromal tumor specimens. The clinicopathologic parameters that were analyzed including the risk grade system by Miettinen et al. and the disease-free survival (DFS) rate. RESULTS: 1) An E2F1 expression was correlated with a larger tumor size, a p53 expression and a shorter period of DFS (p=0.014, p=0.007, and p=0.039). 2) A p53 expression was significantly associated with a high risk grade, a larger tumor size, high mitotic counts and a shorter period of DFS (p=0.003, p=0.044, p<0.001, and p<0.0001). 3) A high-risk grade and the epithelioid type were significantly associated with a shorter period of DFS (p=0.0006 and p=0.0008). CONCLUSIONS: E2F1, as well as p53, may be a potentially novel independent prognostic factor for predicting a worse outcome for those patients suffering with Gastrointestinal stromal tumors.
Subject(s)
Humans , Apoptosis , Disease-Free Survival , DNA , E2F1 Transcription Factor , Gastrointestinal Stromal Tumors , Immunohistochemistry , Ki-67 Antigen , Mitosis , Phase Transition , Proteins , Stress, Psychological , Biomarkers, Tumor , Tumor Suppressor Protein p53ABSTRACT
BACKGROUND: Disturbances of the cell cycle regulatory proteins are key events underlying the development and/or progression of human malignancies. The aim of this study was to evaluate the expression of G1/S cell cycle regulatory proteins in ovarian epithelial tumor. METHODS: We simultaneously evaluated the expression of cyclin D1, cyclin E, CDK4, CDK2, p16, Rb, E2F1, p53 and the Ki67 labelling index (LI) by immunohistochemical methods in 148 cases of ovarian epithelial tumor of the benign (n=47), borderline (n=29), and malignant type (n=72). RESULTS: The expression of cyclin E, CDK2, p16, Rb, E2F1, p53 and the Ki67 LI gradually increased from the benign type, through the borderline type, to the malignant tumors. Between the borderline and malignant tumors, the increased expression of cyclin E, E2F1, and p53, and the decreased expression of Rb were significantly associated with malignancy. The reduced Rb expression and the increased E2F1 expression were correlated with the FIGO stage and the histologic grade in the malignant ovarian epithelial tumors. CONCLUSIONS: Cyclin E, E2F1, and p53 overexpressions and the loss of Rb are the important components during carcinogenesis of ovarian epithelial tumors. Our results suggest that in- creased expression of E2F1 should be considered as a new parameter for the prognosis of patients with malignant ovarian epithelial tumors.