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SUMMARY: This study is to investigate the effect of survivin down-regulation by Egr1-survivin shRNA combined with radiotherapy on the apoptosis and radiosensitivity of esophageal squamous cell carcinoma ECA109 and KYSE150 cells. ECA109 and KYSE150 cells were transfected with Egr1-survivin shRNA, and then treated with radiotherapy. After 24 h, the mRNA and protein levels of Egr1-survivin were detected by qPCR and Western-Blot. Cell cycle and apoptosis were detected by flow cytometry. Western blot also detected levels of cleavaged Caspase 3 and Caspase 9. YM155 was used as a positive control to inhibit survivin expression. The levels of survivin mRNA and protein in ECA109 and KYSE150 cells treated with Egr1-survivin shRNA combined with radiotherapy were significantly lower than those of the blank control group, the empty vector control group, and, the YM155 + radiotherapy group (P<0.05). Meanwhile, after survivin down-regulation, the ratio of G2 to S phase of ECA109 and KYSE150 cells increased significantly, leading to significant G2 and S phase arrest. Additionally, apoptosis of ECA109 and KYSE150 cells increased significantly (P <0.01). Further, protein levels of cleavaged Caspase 3 and Caspase 9 significantly increased in Egr1-survivin shRNA combined with radiotherapy group. Egr1-survivin shRNA combined with radiotherapy can down-regulate survivin expression, which further increases the apoptosis, and enhances the radiosensitivity of ECA109 and KYSE150 cells.
Este estudio tuvo como objetivo investigar el efecto de la regulación negativa de survivina por el shRNA de Egr1-survivina combinado con radioterapia sobre la apoptosis y la radiosensibilidad del carcinoma de células escamosas de esófago Células ECA109 y KYSE150. Las células ECA109 y KYSE150 se transfectaron con shRNA de survivina Egr1 y luego se trataron con radioterapia. Después de 24 h, los niveles de ARNm y proteína de Egr1-survivina se detectaron mediante qPCR y Western-Blot. El ciclo celular y la apoptosis se detectaron mediante citometría de flujo. La transferencia Western también detectó niveles de Caspasa 3 y Caspasa 9 escindidas. Se usó YM155 como control positivo para inhibir la expresión de survivina. Los niveles de ARNm y proteína de survivina en células ECA109 y KYSE150 tratadas con shRNA de survivina Egr1 combinado con radioterapia fueron significativamente más bajos que los del grupo control en blanco, el grupo control de vector vacío y el grupo de radioterapia YM155 + (P <0,05). Mientras tanto, después de la regulación negativa de survivina, la proporción entre las fases G2 y S de las células ECA109 y KYSE150 aumentó significativamente, lo que llevó a una detención significativa de las fases G2 y S. Además, la apoptosis de las células ECA109 y KYSE150 aumentó significativamente (P <0,01). Además, los niveles de proteína de Caspasa 3 y Caspasa 9 escindidas aumentaron significativamente en el shRNA de Egr1- survivina combinado con el grupo de radioterapia. El shRNA de survivina de Egr1 combinado con radioterapia puede regular negativamente la expresión de survivina, lo que aumenta aún más la apoptosis y mejora la radiosensibilidad de las células ECA109 y KYSE150.
Subject(s)
Humans , Esophageal Neoplasms/therapy , Survivin , Esophageal Squamous Cell Carcinoma/therapy , Radiation-Sensitizing Agents , Radiation Tolerance , RNA, Messenger , Esophageal Neoplasms/genetics , Esophageal Neoplasms/radiotherapy , Transfection , Down-Regulation , Blotting, Western , Apoptosis , Combined Modality Therapy , RNA, Small Interfering , Cell Line, Tumor/radiation effects , Early Growth Response Protein 1 , Caspase 3 , Caspase 9 , Real-Time Polymerase Chain Reaction , Flow Cytometry , Esophageal Squamous Cell Carcinoma/genetics , Esophageal Squamous Cell Carcinoma/radiotherapyABSTRACT
Objective To investigate the effect of TES gene on the radiosensitivity of nasopharyngeal carcinoma 5-8F cells. Methods Specimens of 5-8F cells (unprocessed group) and 5-8F cells with high TES expression (TES group) were irradiated at 0, 2, 4, 6, and 8 Gy radiation dose points.Cell clone formation experiment was conducted to draw the survival curve.Twenty-four BALB/c nude mice were randomly divided into four groups: nontransfection group, TES group, irradiation group, and TES irradiation group.A nude mouse model of nasopharyngeal carcinoma 5-8F cells was established.The length and diameter of the transplanted tumor were measured every three days, the tumor volume was calculated, and the growth curve of the transplanted tumor was drawn.After the mice were killed one month later, the tumor block was taken and weighed.The apoptosis of the transplanted tumor cells in each group was detected by flow cytometry. Results Compared with that in the unprocessed group, the survival rate of cells in the TES group was significantly lower (P < 0.01).The tumor growth rate and tumor mass of all four groups decreased in turn, while the apoptosis rate increased in turn.The TES irradiation group had the slowest tumor growth rate, greatest decrease in tumor weight, and highest apoptosis rate among the four groups.Multiple comparison revealed statistically significant differences between the groups (P < 0.05). Conclusion The testin gene can effectively improve the radiosensitivity of nasopharyngeal carcinoma 5-8F cells cultured in vitro.
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Objective:To evaluate the effect of niraparib, the poly (ADP-ribose) polymerase (PARP) inhibitor, on the radiosensitivity of esophageal squamous cell carcinoma (ESCC) and to preliminarily investigate its mechanism.Methods:Human esophageal squamous cell carcinoma cells ECA-109 and KYSE-150 were divided into the control, niraparib, single irradiation, combined (niraparib+irradiation) groups. Cell proliferation was measured by CCK-8 assay. The changes of cell survival rate were detected by colony formation assay. The changes of cell cycle and apoptosis were analyzed by flow cytometry. The number of γH2AX foci was detected by immunofluorescence, and the expression levels of PARP-1, cleaved-PARP, RAD51, mitogen-activated protein kinase (MAPK) [extracellular signal-regulated kinase 1 and 2 (ERK1/2) ] and p-MAPK (ERK1/2) proteins were determined by Western blot. All data were expressed as Mean±SD. Data between two groups conforming to normal distribution through the normality test were subject to independent sample t-test and multiple groups were analyzed using one-way ANOVA. Results:In human ESCC cells ECA-109 and KYSE-150, the proliferation of ESCC cells was significantly inhibited by niraparib combined with irradiation, and the values of average lethal dose (D 0), quasi-threshould dose(D q), survival fraction after 2 Gy irradiation (SF 2) in the combined group were decreased compared with those in the single irradiation group. The effect of irradiation alone on apoptosis of ECA-109 and KYSE-150 cells was limited. Compared to single irradiation group, irradiation combined with niraparib further increased the apoptosis rate in ESCC cells ( P=0.015, P=0.006). In ECA-109 cells, G 2/M phase arrest was significantly increased in combined group compared with irradiation alone group ( P<0.001). In ECA-109 cells, the number of γH2AX foci in combined group was higher than that in the single irradiation group after 2 h, and showed a significantly slower decay of γH2AX foci ( P<0.001). Moreover, niraparib combined with irradiation enhanced the radiation-induced cleavage of PARP-1 and down-regulated the expression of Rad51 and p-MAPK(ERK1/2). Conclusion:Niraparib can increase the radiosensitivity of esophageal cancer cells by inhibiting cell proliferation, promoting cell apoptosis, inhibiting the repair of DNA damage and regulating the MARK-ERK signaling pathway.
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Brain metastases are the most common intracranial malignancies, and radiotherapy is an effective treatment of controlling the localized lesions. However, conventional radiotherapy techniques have their own shortcomings that limit the effectiveness of radiotherapy. Metastatic brain tumors are highly likely to recur or progress after treatment. Clinical studies have shown that arginine can penetrate the blood-brain barrier and subsequently improve the radiosensitization of metastatic brain tumors. In this article, the mechanisms underlying the effect of arginine on the radiosensitization of metastatic brain tumors by inhibition of tumor glycolytic metabolism, reduction of DNA damage repair and alteration of tumor hemodynamic parameters were reviewed, aiming to provide new ideas for clinical research and treatment of brain metastases.
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As one of the most effective treatments for cancer, radiotherapy(RT)can eliminate the tumor cells and improve the survival rate of cancer patients. However, radiation resistance of tumor cells remarkably reduces the efficacy of RT. Therefore, it is significant to investigate the resistance mechanism and explore corresponding therapy for cancer. Ferroptosis is a novel mode of programmed cell death. Numerous studies have indicated an intimate connection between ferroptosis and tumor radiosensitivity. The mechanism involves lipid reactive oxygen species accumulation, glutathione metabolism and iron metabolism. It has been reported that ferroptosis inducers can act effectively and synergistically with RT, promote radiosensitivity and further improve tumor prognosis. In this article, relevant mechanisms and research progress were reviewed, and the future development direction of this field was discussed, aiming to provide reference for clinical trials and mechanism research of RT for malignant tumors.
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Objective:To investigate the effect of heat shock protein 90 (Hsp90) inhibitor PU-H71 combined with X-ray on radioresistant human cervical cancer cells.Methods:The expression levels of Hsp90 gene between cervical cancer tissues and adjacent tissues were analyzed by bioinformatics. Radioresistant cervical cancer cell lines HeLa RR and SiHa RR were obtained by fractional irradiations (2 Gy per fraction, 30 fractions). The cell lines were divided into the control group (treated with dimethyl sulfoxide), irradiation alone group, PU-H71 group (treated with 0.5 μmol/L PU-H71), and PU-H71+irradiation group (irradiation at 24 h after treatment with 0.5 μmol/L PU-H71). Cell survival was detected by clonal formation assay. Immunofluorescence assay was used to detect γH2AX foci at 1, 6, and 24 h after cell treatment. The expression level of Rad51 protein at 1, 2, 6, 12, and 24 h after cell treatment was detected using Western blot. The expression level of phosphorylated DNA-dependent protein kinase catalytic subunit (p-DNA-PKcs) was measured at 2 h after cell treatment. Cell apoptosis at 48 h after cell treatment was assessed by flow cytometry. Results:PU-H71 enhanced the sensitivity of radioresistant cervical cancer cells to X-ray. Compared with the irradiation alone group, the radiation sensitization ratios (SER) of HeLa RR and SiHa RR cells at 10% survival were 1.36 and 1.27, and the apoptosis rates were increased by approximately 72.1% and 63.1% in the PU-H71+irradiation group, respectively. PU-H71 delayed the duration of γH2AX foci induced by X-ray, inhibited the phosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), thus preventing non-homologous end joining (NHEJ) repair and delaying homologous recombination repair.Conclusion:PU-H71 increases the radiosensitivity of radioresistant cervical cancer cells by inhibiting the repair pathway of DNA double-strand break, which is expected to be a radiosensitizer to enhance the efficacy of radiotherapy for cervical cancer.
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Objective:To investigate the effect and mechanism of long non-coding RNA (lncRNA) TTN-AS1 on the radiosensitivity of breast cancer cells. Methods:The expression of TTN-AS1 in breast cancer cells was detected by real-time reverse transcription PCR (qRT-PCR). MDA-MB-231 cells were divided into the 0 Gy group, 4 Gy group, negative control (NC) +4 Gy group, si- TTN-AS1+4 Gy group, si- TTN-AS1+ miR-107 inhibitor+4 Gy group, and si- TTN-AS1+ miR-107 inhibitor+si- HMGA1+4 Gy group. CCK-8 assay and flow cytometry were used to detect the proliferation and apoptosis rates in each group. Results:Compared with breast epithelial cells, TTN-AS1 was significantly highly expressed in breast cancer cell lines ( P<0.001). Compared with the NC+4 Gy group, the cell proliferation ability was significantly decreased ( P<0.05) and cell apoptosis was significantly increased ( P<0.001) in the si- TTN-AS1+4 Gy group. Compared with the 0 Gy group, the expression levels of TTN-AS1 and HMGA1 from 8 h to 24 h after radiotherapy were significantly up-regulated (both P<0.01), whereas the expression of miR-107 was significantly down-regulated from 8 h to 24 h after radiotherapy in the 4 Gy group ( P<0.001). The cell proliferation ability in the si- TTN-AS1+ miR-107 inhibitor+4 Gy group was significantly higher than that in the si- TTN-AS1+4 Gy group ( P<0.001), and cell apoptosis was significantly lower than that in the si- TTN-AS1+4 Gy group ( P<0.001). Compared with the si- TTN-AS1+ miR-107 inhibitor+4 Gy group, cell proliferation ability was significantly decreased ( P<0.001), whereas cell apoptosis was significantly increased in the si- TTN-AS1+ miR-107 inhibitor+si- HMGA1+4 Gy group ( P<0.001). Conclusion:TTN-AS1 can promote the radiosensitivity of breast cancer cells by regulating the miR-107/ HMGA1 signaling axis.
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Objective:To investigate the expression of double-stranded RNA-binding protein nuclear factor 45 (NF45) in laryngeal squamous cell carcinoma (LSCC), and the effect of NF45 on the radiation sensitivity of LSCC cells and its mechanism.Methods:NF45 expression in LSCC and adjacent tissues was detected by real-time reverse transcription PCR (qRT-PCR) and immunohistochemical staining. The NF45-ShRNA lentivirus was transfected into Hep-2 cells, and cell transfection efficiency was determined by qRT-PCR and Western blot . Hep-2 cells were randomly divided into the control group, 2 Gy group, sh-NC+2 Gy group and sh-NF45+2 Gy group. Lentivirus infection and 2Gy X-ray irradiation treatment were carried out. Cell proliferation activity was assessed by CCK-8 assay. Apoptosis rate was determined by flow cytometry. Hep-2 cells in each group were treated with mCherry-EGFP-LC3B. The levels of autophagy were detected by immunofluorescence staining. The ratio of autophagy-related protein microtubule-associated protein 1 light chain 3 (LC3)-Ⅱ/LC3-Ⅰ and the expression levels of Beclin-1 and p62 proteins were determined by Western blot.Results:The expression level of NF45 in LSCC tissues was significantly higher than that in adjacent tissues ( P<0.01). The relative expression levels of NF45 mRNA and protein in Hep-2 cells infected with NF45-shRNA were significantly lower than those in the control and sh-NC groups (all P<0.05). Compared with the control group, the cell proliferation activity was decreased, the apoptosis rate was increased, the intracellular autophagy-lysosome were increased, the ratio of LC3-Ⅱ/LC3-Ⅰ was increased, the relative expression levels of Beclin-1 protein were up-regulated, and the relative expression levels of p62 protein were down-regulated in the 2 Gy, sh-NC+2 Gy and sh-NF45+2 Gy groups (all P<0.05). Compared with the 2 Gy group, the cell proliferation activity was decreased, the apoptosis rate was increased, the intracellular autophagy lysosomes were increased, the LC3-Ⅱ/LC3-Ⅰ ratio was increased, the relative expression of Beclin-1 protein was up-regulated, and the relative expression of p62 protein was down-regulated in the sh-NF45+2 Gy group (all P<0.05). Conclusions:The expression of NF45 is up-regulated in LSCC tissues. Targeted down-regulation of NF45 expression can inhibit the proliferation activity of LSCC cells, promote cell apoptosis, and improve the sensitivity of tumor cells to radiation. The mechanism may be related to the regulation of autophagy levels.
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Nasopharyngeal carcinoma is one of the most common malignant head and neck tumors, and radiotherapy is the main treatment. However, radio-resistance is a key cause of local recurrence of nasopharyngeal carcinoma. Therefore, overcoming the radio-resistance of nasopharyngeal carcinoma and enhancing the radiosensitivity have become urgent problems in the treatment of nasopharyngeal carcinoma, which also play a key role in improving the overall survival rate of patients. In this article, recent studies on DNA, non-coding RNA (ncRNA), protein and cell behaviors related to radio-resistance of nasopharyngeal carcinoma were reviewed, aiming to provide valuable ideas for clinical treatment of nasopharyngeal carcinoma.
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Objective:To evaluate the effects of high mobility group protein box 1 (HMGB1) on clinical prognosis of esophagus squamous cell carcinoma (ESCC) patients treated with chemoradiotherapy and the radiosensitivity of xenograft in nude mice.Methods:A total of 90 endoscopic biopsy specimens were obtained from ESCC patients treated with chemoradiotherapy. The expression level of HMGB1 was determined by immunohistochemical staining. High expression level was defined when staining was observed on ≥50% of the tumor cells. All patients were divided into the high expression group ( n=48) and low expression group ( n=42), and their survival information was retrospectively analyzed. Cell transfection was performed with the plasmid carrying human HMGB1-shRNA to knockdown HMGB1 expression in ECA109 cells and xenograft mouse models were established. The tumor volume and mass were calculated after irradiation with a dose of 15 Gy. The cell apoptosis in xenograft tissues were detected. Survival analysis was performed using Kaplan-Meier method. Univariate prognostic analysis was conducted by log-rank test. Intergroup comparison was performed by analysis of variance (ANOVA). Results:The expression level of HMGB1 was significantly associated with gross tumor volume, longest diameter of tumor, T staging and distant metastasis ( χ2=9.663, 5.625, 4.068, 7.146, all P<0.05). In the low expression group, the overall survival (OS) ( χ2=4.826, P=0.028), progression-free survival (PFS) ( χ2=4.390, P=0.036) were longer compared with that in the high expression group. Further analysis of HMGB1-high expression patients showed that the radiation dose and the combination of chemoradiotherapy did not significantly affect the OS or PFS of ESCC patients. We observed that knockdown of HMGB1 slowed the growth rate of xenograft, decreased the tumor volume and increased the apoptosis rate after irradiation. Conclusions:ESCC patients with high expression level of HMGB1 obtain poor prognosis after chemoradiotherapy, which can be enhanced by increasing the sensitivity to radiotherapy and chemotherapy. HMGB1 knockdown can effectively increase the radiosensitivity of xenograft in ESCC nude mice.
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Objective:To investigate the effects of down-regulation of FABP5 (fatty acid binding protein 5) on radiation damage of skin cells, and explore underlying mechanism.Methods:A lentiviral vector with down-regulated FABP5 was constructed to infect human immortalized keratinocytes (HaCaT) cells, and the transfection efficiency was examined. The HaCaT cells were divided into blank control group, FABP5 down-regulation group (FABP5), radiation group (IR), and FABP5 down-regulation combined with radiation group (FABP5+ IR). After 6 MV X-ray radiation, cell proliferation viability was measured by CCK-8 assay, cell migration was detected by scratch assay, apoptosis was analyzed by flow cytometry, radiosensitivity was evaluated by cloning formation assay, and the cellular protein expressions of PARP1, γ-H2AX, AKT and p-AKT were detected by Western blot.Results:FABP5 was successfully knocked-down in both RNA level ( t=25.14, P<0.05) and protein level ( t=20.06, P<0.05). The down-regulation of FABP5 decreased the abilities of cells proliferation ( t=3.55, 5.88, 3.18, P<0.05) and migration ( t=15.44, P<0.05), but increased cell resistance to irradiation with a radiosensitization ratio of 0.782. The apoptosis rate of FABP5+ IR group was significantly lower than IR group (22.05±6.71)% vs. (9.82±1.45)%, t=3.08, P<0.05. The protein levels of PARP1 and γ-H2AX in FABP5+ IR group were also lower than those in the IR group 0.04±0.04, 0.11±0.06, 0.26±0.11, 0.22±0.07, 0.21±0.10, 0.52±0.22, 0.57±0.06, 0.43±0.02( t=2.83, 3.07, 4.50, 5.33, P<0.05), while the protein level of p-Akt in FABP5+ IR group was higher than that in IR group ( t=-16.24—3.02, P<0.05). Conclusions:Down-regulation of FABP5 inhibited cell proliferation and migration, increased radioresistance, and reduced radiation-induced apoptosis and DNA damage of skin cells probably through PI3K/AKT signaling pathway.
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Objective:To investigate the radiosensitizing effect and underlying mechanism of STING agonist (c-di-AMP) on cutaneous melanoma cells.Methods:Human cutaneous melanoma cells (A375) were divided into four groups: the control group, 10 μmol/L c-di-AMP group, X-ray irradiation group and X-ray irradiation combined with c-di-AMP group. The radiosensitizing effect of c-di-AMP on A375 cells was detected by CCK-8-based viability assay, lactate dehydrogenase (LDH) release assay, flow cytometry-based apoptosis assay, and colony formation assay. Western blot analysis was used to determine the expressions of cell death-related proteins.Results:In combination with 10 Gy X-ray irradiation, 10 μmol/L c-di-AMP showed significant radiosensitization effect in A375 cells, which was evidenced by decreased cell activity ( t=5.11, P<0.05), increased cytotoxicity ( t=10.15, P<0.05) and cell apoptosis ( t=4.41, P<0.05) and reduced clone viability( t=6.30, 3.55, 5.45, 3.55, P<0.05). The calculated radiosensitization ratio of c-di-AMP to A375 cells was 1.88. Moreover, 10 μmol/L c-di-AMP further increased the expressions of cell death-related proteins induced by radiation in A375 cells. Conclusions:The STING agonist c-di-AMP can be used as a radiosensitizer for cutaneous melanoma, which may provide a novel strategy for radiotherapy.
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Objective:To investigate the effects of over-expression of E2F transcription factor 1 (E2F1) on proliferation, invasion, apoptosis and radiosensitivity of glioma cell U251.Methods:Real-time quantitative PCR (qRT-PCR) were used to detect the differential expression of E2F1 mRNA in glioma cells LN18, SW1088, U251 and normal brain glial cells. The stable over-expression of E2F1 plasmid was constructed and transfected into U251 cells. qRT-PCR and Western blot test were used to detect the expression of E2F1, pituitary tumor transforming gene 1(PTTG1), C-Myc, B-cell lymphoma-2 (Bcl-2), Bcl2-associated X (Bax) mRNA and protein expression in the control group and E2F1 over-expression group.U251 cells were divided into control group(no X-ray irradiation), irradiation group(6 Gy dose of X-ray), and irradiation + E2F1 over-expression group(transfected with E2F1 first, then irradiated by 6 Gy of X-ray). Cell proliferation ability was detected by cell counting Kit-8(CCK-8) cell viability detection reagent, and cell invasion and migration ability were detected by Transwell chamber. Apoptosis and cell cycle were detected by flow cytometry.GraphPad Prism 8.0 was used for data analysis.The statistical methods were one-way ANOVA and independent sample t-test. Results:qRT-PCR showed that there was statistical difference in the mRNA levels of E2F1( F=201.92, P<0.05) in different cell lines.The expression levels of E2F1 mRNA in LN18(4.04±0.29), SW1088(3.19±0.16)and U251(4.66±0.20) cells were higher than those in HEB(1.02±0.07)cells ( q=27.00, 19.40, 32.52, all P<0.05). After successfully constructing U251 cells with stable over-expression of E2F1 plasmid, qRT-PCR and Western blot detection results showed that: the mRNA and protein levels of E2F1, PTTG1, C-Myc and Bcl-2 in E2F1 over-expression group were higher than those in control group ( t=77.16, 57.88, 4.63, 51.13, 7.50, 70.85, 8.38, 48.81, all P<0.05). Bax mRNA(0.20±0.01) and protein(0.66±0.01) levels were lower than those in control group((1.00±0.02), (0.94±0.01)), and the differences were statistically significant ( t=1.74, 54.65, both P<0.05). After X-ray irradiation (6 Gy), CCK8 detection results showed: the proliferation ability of the three groups at 24, 48, 72 and 96 h were significantly different ( F=95.41, 187.53, 1 158.49, 7 883.78, all P<0.05). The proliferation capacity of the irradiation group were lower than those of the control group at 24, 48, 72 and 96 h ( q=19.51, 27.20, 66.60, 174.9, all P<0.05). The proliferation capacity of irradiation + E2F1 over-expression group at 24, 48, 72 and 96 h were higher than those of irradiation group ( q=10.63, 10.81, 21.11, 60.90, all P<0.05). Transwell assay results showed that there were significant differences in cell invasion and migration ability among the three groups ( F=315.38, 681.10, both P<0.05). The invasion and migration ability of cells in the irradiation group were lower than those in the control group ( q=35.09, 12.76, both P<0.05), and the invasion and migration ability of cells in the irradiation + E2F1 over-expression group were higher than those in the irradiation group ( q=52.06, 22.81, both P<0.05). Flow cytometry showed that there were significant differences in apoptosis rate and percentage of cells in each cycle among the three groups ( F=667.63, 3 213.30, 3 011.26, 861.98, all P<0.05). The percentage of the apoptosis rate, S phase and G2 phase cells in the irradiation group were higher than those in the control group ( q=51.10, 89.39, 51.82, all P<0.05), while the percentage of G1 phase cells in the irradiation group was lower than that in the control group ( q=141.2, P<0.05). The apoptosis rate and percentage of S phase and G2 phase cells in the irradiation + E2F1 over-expression group were lower than those in the irradiation group ( q=18.87, 41.42, 29.31, all P<0.05), while the number of G1 phase cells in the irradiation + E2F1 over-expression group was lower than that in the irradiation group ( q=70.73, P<0.05). Conclusion:Over-expression of E2F1 can reduce the radiosensitivity of glioma U251 cells by regulating the expression of mRNA and protein of genes related to cell cycle and apoptosis, and E2F1 may be involved in the radioresistance of glioma cells.
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The biological effects of low-dose radiation (LDR) are still a research hotspot in the field of radiobiology. As research deepens on LDR-induced biological effects and the mechanisms, growing evidence shows that LDR produces distinct biological effects from high-dose radiation, which questions the linear non-threshold model. This article reviews LDR-induced bystander effect, hormesis, adaptive response, and hyper-radiosensitivity, as well as the mechanisms, in order to provide a reference for the transformation of basic research on LDR-related biological effects to clinical application.
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Objective:To evaluate the effect of upregulation of HuR gene on the radiosensitivity of esophageal squamous cell carcinoma cell Kyse450. Methods:The HuR gene of Kyse450 cells was upregulated by lentivirus. At the same time, X-ray irradiation at a dose of 6 Gy was selected as the intervention condition. Western blot and qPCR were used to detect the expression levels of protein and RNA after Kyse450 transfection, respectively. CCK8 kit was employed to determine the cell proliferation rate. Clone formation assay was adopted to evaluate the ability of cell clone formation. Wound healing experiment and the Transwell test were performed to detect changes in cell migration. Results:CCK8 assay showed that the proliferation ability of cells was enhanced after upregulation of HuR gene, and this enhancement trend was more obvious after radiation. The plate cloning experiment showed that with the increase of radiation dose, the clone formation rates were decreased in both groups, but the clone formation rate in the overexpression group was higher than that in the control group. Wound healing experiment and Transwell test demonstrated that the wound healing rate and migration ability in the overexpression group were higher than those in the control group, and the difference was more significant after radiotherapy. Western blot showed that the levels of MMP9 and MMP2 at 24 h after radiotherapy in the overexpression group were higher than those in the control group. Conclusion:The upregulation of HuR can enhance the proliferation, cloning, migration capabilities and decrease the radiosensitivity of esophageal squamous cell carcinoma cells.
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Objective:To explore the effect of miR-21 on cell proliferation, apoptosis, invasion and radiosensitivity of cervical cancer HeLa cells and unravel the underlying mechanism.Methods:RT-qPCR assay was used to detect the expression levels of miR-21 in cervical cancer tissues and adjacent non-tumor tissues, normal cervical epithelial cells (H8) and cervical cancer cell lines (HeLa, SiHa, ME180). HeLa cell line with inhibition of miR-21 or knockdown of RECK were constructed. CCK-8, Caspase3/7 live cell apoptosis detection, wound healing test, Transwell invasion, clone formation assay, Western blot and immunofluorescence were performed to detect cell viability, apoptosis, migration, invasion, radiosensitivity and related proteins. The dual luciferase assay verified whether miR-21 targeted RECK.Results:MiR-21 level in the cervical cancer tissues was significantly higher than that in its corresponding adjacent non-tumor tissues ( P<0.05). The expression levels of miR-21 in cervical cancer cell lines HeLa, SiHa and ME180 were significantly up-regulated compared with those in normal cervical epithelial cells H8(all P<0.05). MiR-21 knockdown significantly inhibited HeLa cell viability, promoted cell apoptosis, reduced radiation tolerance, down-regulated the expression of Cyclin D 1,Bcl-2, MMP-2 and MMP-9, and up-regulated the expression P21 and Bax proteins (all P<0.05). miR-21 targeted the 3’-UTR of RECK mRNA and negatively regulated the expression of RECK. Silencing RECK reversed the effects of miR-21 knockdown on HeLa cell apoptosis, migration, invasion and radiosensitivity. Conclusions:Inhibiting the expression of miR-21 significantly decreases cell viability, induces cell apoptosis, weakens cell migration and invasion capabilities, and enhances the radiosensitivity of HeLa cells. The potential mechanism is closely related to the targeted up-regulation of RECK.
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Objective:To evaluate the effect of compound UC2288 on the radiosensitivity of CNE-2R cell line and nude mouse transplanted tumor.Methods:The UC2288 concentration was referenced to previous experimental results (IC 50=12.20 μmol/L). The effect of UC2288 combined with 2, 4, 6, 8 Gy X-ray irradiation on the radiosensitivity of CNE-2R cell line was detected by clone formation experiment. The effect of UC2288 combined with 2, 4, 6, 8 Gy X-ray irradiation on the proliferation of CNE-2R cell line was determined by CCK8 assay. The nude mouse model of transplanted tumor was constructed with CNE-2R cell line. The radiosensitivity of transplanted tumor of UC2288 combined with 2 Gy/fraction X-ray irradiation for three consecutive days was evaluated. Results:The experimental concentration of UC2288 was 8 μmol/L. The clonality of CNE-2R cell line was reduced under UC2288 combined with X-ray 2, 4, 6, and 8 Gy irradiation, andthe radiosensitizationratio was 1.60. The proliferation of CNE-2R cell line was significantly decreased under UC2288 combined with X-ray 2, 4, 6, and 8 Gy irradiation. UC2288 inhibited the growth of transplanted tumor in nude mice, and the inhibitory effect was strengthened with the extension of observation time, and the most obvious effect was observed at 16 d. ( P<0.01). Theradiosensitizationratio was 4.33. The proliferation of CNE-2R cell line was decreased under UC2288 combined with X-ray irradiation. Conclusion:UC2288 can increase the radiosensitivity of nasopharyngeal carcinoma radioresistant cell line CNE-2R.
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Objective:To evaluate the effect of circLPAR3 on the radiosensitivity of esophageal cancer cells and investigate its mechanism.Methods:The cancer tissues and and adjacent tissues of 37 patients with esophageal cancer were collected, and esophageal cancer cell lines Eca-109, EC9706 and KYSE30 and esophageal epithelial cells HET-1A were cultured in vitro. The expression levels of circLPAR3 and miR-1238 in the tissues and cells were measured by RT-qPCR. Eca-109 cells were transfected with circLPAR3 siRNA and miR-1238 mimics or co-transfected with circLPAR3 siRNA and miR-1238 inhibitor. Cell cloning experiment was conducted to evaluate the effects of silencing circLPAR3, overexpressing miR-1238, or silencing both circLPAR3 and miR-1238 on the radiosensitivity of Eca-109 cells. After Eca-109 cells that silenced circLPAR3, overexpressed miR-1238 or silenced both circLPAR3 and miR-1238 were exposed to 4 Gy irradiation, CCK-8 assay (A value), flow cytometry and Western blot were employed to assess the effects of silencing circLPAR3, overexpressing miR-1238, or silencing both circLPAR3 and miR-1238 combined with 4 Gy irradiation on the proliferation and apoptosis of Eca-109 cells and the expression levels of CyclinD1, p21, Bcl-2 and Bax proteins. Dual luciferase reporter gene experiment and RNA pull down experiment were performed to verify the regulatory relationship between circLPAR3 and miR-1238. Results:Compared with adjacent tissues, the expression level of circLPAR3 was up-regulated in the esophageal cancer tissues ( P<0.05), while that of miR-1238 was down-regulated ( P<0.05). Compared with HET-1A cells, the expression levels of circLPAR3 were up-regulated in the esophageal cancer cell lines Eca-109, EC9706 and KYSE30(all P<0.05), whereas those of miR-1238 were down-regulated (all P<0.05). Silencing circLPAR3 or overexpressing miR-1238 reduced the survival fraction of Eca-109 cells (all P<0.05), and the sensitization ratio was 1.21 and 1.75, respectively. Silencing circLPAR3 or overexpressing miR-1238 decreased the A value of Eca-109 cells and the expression levels of CyclinD1 and Bcl-2 proteins (all P<0.05), while increased the apoptosis rate of Eca-109 cells and the expression levels of p21 and Bax proteins (all P<0.05). After silencing circLPAR3 or overexpressing miR-1238 combined with 4 Gy irradiation, the A value of Eca-109 cells and the expression levels of CyclinD1 and Bcl-2 proteins were decreased (all P<0.05), while Eca-109 cell apoptosis rate and the expression levels of p21 and Bax proteins were increased (all P<0.05). circLPAR3 targeted and negatively regulated the expression level of miR-1238 in Eca-109 cells. After silencing miR-1238 and circLPAR3 simultaneously, the survival fraction of Eca-109 cells was higher than that when only silencing circLPAR3, and the sensitization ratio was 0.59. Silencing miR-1238 reversed the effects of silencing circLPAR3 combined with 4 Gy irradiation on the proliferation and apoptosis of Eca-109 cells. Conclusion:circLPAR3 is highly expressed in esophageal cancer tissues and cell lines, and silencing the expression of circLPAR3 can inhibit the proliferation of esophageal cancer Eca-109 cells, promote their apoptosis, and enhance cell radiosensitivity by up-regulating miR-1238.
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Objective:To investigate the role of SUMO E3 ligase ZNF451 in DNA damage repair and explore the underlying mechanism in non-small cell lung cancer A549 cells and cervical cancer HeLa cells.Methods:A549 cells and HeLa cells were irradiated with γ-ray irradiation or treated with etoposide. Cell proliferation viability was detected by the cell counting kit-8 assay. Protein expression was detected by Western blot assay. DNA damage repair level was detected by DR-GFP plasmid system, and the spatial positioning was detected by immunofluorescence.Results:Etoposide decreased the expression level of ZNF451 in a dose- and time- dependent manner. After treatment with 30, 50, 80 μmol/L etoposide, the cell viability were reduced after the knockdown of ZNF451 in A549 and HeLa cells(A549: t = 27.62, 25.61, 5.32, P<0.01; HeLa: t = 30.77, 21.28, 4.18, P<0.01). Furthermore, ZNF451 was recruited at DNA damage sites. A co-localization and endogenous interaction were found between ZNF451 and γ-H2AX after the treatment of irradiation or etoposide. Moreover, the expression level of γ-H2AX was significantly increased after treatment with 30, 50, 80 μmol/L etoposide(A549: t = 6.12, 10.67, 4.68, P<0.01; HeLa: t = 7.94, 9.81, 15.12, P<0.01)and the repair efficiency of NHEJ was reduced in ZNF451 knockdown cells( t = 18.60, P<0.05). Finally, the immunofluorescence assay showed that ZNF451 was co-localizated with 53BP1 and MDC1 after irradiation or etoposide treatment. Conclusions:Knockdown of ZNF451 inhibits cell proliferation and increases the level of DNA damage in A549 and HeLa cells. ZNF451 was recruited to DNA damage sites after DSBs and participated in NHEJ repair by co-localizing with DNA damage repair factor 53BP1/MDC1.
ABSTRACT
Objective:To investigate the regulating molecules and acting mechanism of TAB182 in HR pathway.Methods:TAB182 in human breast cancer MCF-7 cells was knocked down by shRNA strategy, the TAB182 knockdown MCF-7 as the TAB182 knockdown group, and the MCF-7 cell using the shRNA negative control as the TAB182 negative control group. RNA sequencing and qRT-PCR were performed to screen and verify the differentially expressed genes of HR pathway related to TAB182 depression. Western blot was used to detect protein expression. Immunofluorescence staining of nuclear RAD51 and BrdU was used to check the 3′ ssDNA formation by the end resection. The cell cycle arrest and apoptosis were measured by flow cytometry. Cloning formation assay was used to evaluate the sensitivity TAB182-knockdown cells to radiation.Results:Both quantitative RNA sequencing and qRT-PCR assays showed that TAB182-knockdown significantly decreased the mRNA expression of RPA2( t=17.97, P<0.05). Compared with the TAB182 negative control group, the protein level of RPA2, the number of RAD51 foci, and the 3′ ssDNA-binding nuclear protein marker BrdU in TAB182-knockdown cells were significantly reduced. At 4, 8, and 12 h after actinomycin D treatment, the attenuation of RPA2 mRNA in the TAB182-knockdown cells was accelerated ( t=5.37, 3.79, 3.69, P<0.05). Compared with the TAB182 negative control group, the radiosensitivity and radiation-induced apoptosis in the TAB182-knockdown group were increased ( t=3.48, 11.05, P<0.05), and at 24 h after irradiation, the cell cycle block time was prolonged ( t=8.40, P<0.01). Conclusions:TAB182 plays a role in maintaining RPA2 mRNA stability, thereby promoting HR repair. TAB182 knockdown cells are highly sensitive to ionizing radiation.