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1.
Article in Chinese | WPRIM | ID: wpr-1027471

ABSTRACT

Objective:To evaluate the effect of miR-216a-5p on the radiosensitivity of liver cancer cells via targeting Krüppel-like transcription factors 12 (KLF12).Methods:Real-time reverse transcription PCR (RT-qPCR) was used to detect miR-216a-5p, KLF12 mRNA levels in normal human liver cells L-02 cells, and human hepatoma cells Huh7, HepG2, Hep3B and MHCC-97H cell lines. HepG2 cells were divided into 0, 2, 4, 6 and 8 Gy irradiation groups, and miR-216a-5p, KLF12 mRNA levels were compared among different groups. HepG2 cells overexpressing miR-216a-5p and / or KLF12 were constructed by plasmid transfection and divided into the miR-NC group (control group), miR-216a-5p group, miR-216a-5p+NC group, miR-216a-5p+KLF12 group, IR+miR-NC group, IR+miR-216a-5p group, IR+miR-216a-5p+NC group, IR+miR-216a-5p+KLF12 group, respectively. miR-216a-5p, KLF12 mRNA levels were compared among different groups. Clone formation assay was used to detect cell radiosensitivity. CCK-8 assay was employed to detect cell proliferation ability. Flow cytometry was adopted to detect cell apoptosis. Single factor ANOVA was used for inter group comparisons. LSD- t test was used for pairwise comparison. Results:Compared with L-02 cells, KLF12 mRNA levels were up-regulated, whereas miR-216a-5p levels were down-regulated in liver cancer cell lines (all P<0.05). Compared with 0 Gy group, KLF12 mRNA levels were down-regulated, whereas miR-216a-5p levels were up-regulated in 2, 4, 6, and 8 Gy groups (all P<0.05). Overexpression of miR-216a-5p or radiation therapy alone could enhance cell radiosensitivity and apoptosis levels, and reduce cell proliferation ability (all P<0.05). Simultaneous radiation treatment with overexpression of miR-216a-5p exerted more significant effects on cells (all P<0.05). Overexpression of KLF12 could partially reverse the aforementioned effects of overexpression of miR-216a-5p ( P<0.05). Conclusion:MiR-216a-5p reduces cell proliferation ability, enhances cell radiosensitivity and apoptosis via regulating KLF12.

2.
Article in Chinese | WPRIM | ID: wpr-1027485

ABSTRACT

Objective:To screen and verify the key radioresistance genes regulated by m6A methylation in nasopharyngeal carcinoma (NPC) based on the chip data and cell experiments.Methods:The microarray data of NPC radioresistance genes, m6A regulated genes and mRNA expression profiles of NPC genes were downloaded from Gene Expression Omnibus (GEO) database. The differential genes were screened and statistically analyzed by R software. The biological processes, signal pathways and interaction networks of these genes were analyzed by bioinformatics. The m6A regulatory factors were knocked down and the radioresistant strains were constructed. The above m6A differential radioresistant genes of NPC were screened and verified by real-time reverse transcription PCR (qRT-PCR) and Western blot. The m6A modification of screened genes and their direct binding ability with methyltransferase 3 (METTL3) were verified by methylated RNA immunoprecipitation qPCR (MeRIP-qPCR). The siRNA of selected genes was transfected into NPC cells, and after treatment with ionizing radiation, cell proliferation was detected by CCK-8 assay and EdU, apoptosis and cell cycle were detected by flow cytometry, and radiosensitivity was detected by clone formation assay. The trend of differences in the abundance of Fe 2+ and lipid peroxidation between the control and EGLN3 knockdown groups after ionizing radiation treatment was compared by paired t-test. Results:Chip data GSE48501 intersected with GSE200792 and GSE53819 to obtain 6 differential genes, including EGLN3, FOSL2, ADM, JUN, VEGFA and PRDM1. The target genes of EGLN3 and FOSL2 were further screened by TNMplot and KMplot, etc. The mRNA of the target genes directly bound to METTL3 and were subjected to its mediated modification of m6A. The target genes were up-regulated in the parental cells after irradiation in a dose and time gradient manner, which were also significantly up-regulated in radioresistant cells. After EGLN3 and FOSL2 were down regulated, the proliferation activity of NPC cells was more significantly decreased after irradiation, and the radiosensitization ratio was statistically significant compared with that of NPC cells without EGLN3 and FOSL2 down-regulation. After irradiation, EGLN3 down-regulated NPC cells significantly down-regulated glutathione peroxidase 4 (GPX4) expression, increased the abundance of Fe 2+ and lipid peroxidation, which played a role in radiosensitization by inducing ferroptosis. Conclusions:EGLN3 and FOSL2 play a role in radioresistance in NPC through METTL3 mediated m6A methylation. Down-regulation of EGLN3 combined with ionizing radiation can increase the intracellular Fe 2+ abundance and lipid peroxidation and down-reuglate the expression of GPX4 in NPC cells, which can enhance radiosensitization for NPC radiotherapy by the ferroptosis pathway.

3.
Article in Chinese | WPRIM | ID: wpr-1027510

ABSTRACT

Objective:To evaluate the effect of long non-coding RNA (lncRNA) nuclear enriched abundant transcript 1 (NEAT1) on the radiosensitivity in breast cancer cells by regulating the miR-149-5p/ glutamic pyruvic transaminase 2 (GPT2) axis.Methods:Real-time reverse transcription PCR (RT-qPCR) was used to detect NEAT1, miR-149-5p and glutamic pyruvic transaminase 2 (GPT2) mRNA levels in human breast cells MCF-10A, and human breast cancer cells MCF-7, MDA-MB-231 and MDA-MB-468, respectively. MCF-7 cells were divided into 0, 2, 4, 6 and 8 Gy irradiation groups. MCF-7 cells were divided into NEAT1 knockdown (si-NEAT1) group and control (si-NC) group, NEAT1 knockdown +miR-149-5p knockdown (si-NEAT1+anti-miR-149-5p) group and control (si-NEAT1+anti-miR-NC) group, NEAT1 knockdown + GPT2 overexpression (si-NEAT1+GPT2) group and control (si-NEAT1+NC) group. On the basis of the above grouping, irradiate each group of cells with 4 Gy radiation for 2 h, denoted as IR+si-NEAT1, IR+si-NC, IR+si-NEAT1+anti-miR-149-5p, IR+si-NEAT1+anti-miR-NC, IR+si-NEAT1+GPT2, IR+si-NEAT1+NC groups. Subsequently, MCF-7 cells were irradiated at a dose of 4 Gy and divided into the IR+si-NEAT1, IR+si-NC, IR+si-NEAT1+anti-miR-149-5p, IR+si-NEAT1+anti-miR-NC, IR+si-NEAT1+GPT2 and IR+si-NEAT1+NC groups. RT-qPCR was used to detect NEAT1, miR-149-5p, GPT2 mRNA levels in cells. Colony formation assay was used to detect cell radiosensitivity. CCK-8 assay was adopted to detect cell proliferation ability. The binding sites of NEAT1 and miR-149-5p were predicted by StarBase database. The binding sites of miR-149-5p and GPT2 were predicted by Targetscan database, and validated by dual luciferase assay. Single factor ANOVA was used for inter-group comparisons. LSD- t test was used for pairwise comparison. Results:Compared with MCF-10A cells, NEAT1 and GPT2 mRNA levels in cell lines were up-regulated, whereas miR-149-5p level was down-regulated (all P<0.05). Compared with the 0 Gy dose group, NEAT1 and GPT2 mRNA levels were down-regulated, while miR-149-5p levels were up-regulated in the 2, 4, 6, and 8 Gy dose groups (all P<0.05). Knockdown of NEAT1 expression or radiation alone could enhance cell radiosensitivity, and reduce cell proliferation ability (all P<0.05). Simultaneous radiation treatment with knockdown of NEAT1 expression could strengthen the above effects upon cells (all P<0.05). Knockdown of miR-149-5p expression or overexpression of GPT2 could partially reverse the aforementioned effects of knockdown of NEAT1 expression (all P<0.05). Conclusion:Knockdown of NEAT1 expression enhances breast cancer cell radiosensitivity, and reduces cell proliferation ability by regulating the miR-149-5p/GPT2 signal axis.

4.
Int. j. morphol ; 41(6): 1712-1719, dic. 2023.
Article in English | LILACS | ID: biblio-1528776

ABSTRACT

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/radiotherapy
5.
Article | IMSEAR | ID: sea-231712

ABSTRACT

Breast cancer mortality rate is fifth among all cancer and increasing day by day due to modern lifestyles. Its molecular subtype is classified as per their significant receptor expression, such as estrogen receptor (ER), progesterone receptor (PR) & human epidermal growth receptor 2 (Her2). Triple-negative breast cancer (TNBC) is an aggressive subgroup among breast cancer subtypes and clinically challenging to treat due to loss of all three receptor (ER/PR/Her2) expression. Treatment modalities of TNBC include surgery, chemotherapy, radiotherapy and immunotherapy. Postoperative radiation therapy (RT) improves locoregional control and overall survival in TNBC patients. The powerful ionizing radiation (IR) response to RT is contributed by the inherent radiosensitivity of the tumor, which is influenced by genes associated with the cell cycle, DNA damage repair, apoptosis, etc. This review article narrates the role of biomarkers obtained through data mining and manual curation of published literature to predict radioresistance in patients receiving radiotherapy. Further, the role of natural radiosensitizers in overcoming radioresistance for effectively managing TNBC is also discussed.

6.
Article in Chinese | WPRIM | ID: wpr-1027423

ABSTRACT

Objective:To investigate the relationship between the expression level of thymocyte selection-associated high mobility group box protein ( TOX) gene and the radiosensitivity of lower-grade glioma (LGG) patients. Methods:Using bioinformatics research methods, 474 LGG patients from The Cancer Genome Atlas (TCGA) database were selected as the test set (TCGA-474 set), and two different genetic data sets ( n=412 and n=171) from the Chinese Glioma Genome Atlas (CGGA) database were selected as the validation set (CGGA-412 set and CGGA-171 set). Patients were stratified based on whether received radiotherapy, and divided into the high and low TOX expression group according to the expression level of TOX gene in LGG. Survival curves of all patients were plotted. The overall survival (OS) and progression-free survival (PFS) of patients in the high and low TOX expression groups were compared and analyzed using log-rank test. Results:Multivariate analysis of OS in the TCGA-474 set showed that high expression of TOX was a protective factor for OS ( HR=0.061, 95% CI: 0.005-0.791, P=0.044). After stratification analysis based on radiotherapy and adjustment for confounding factors, the HR (95% CI) of patients with high TOX expression in the TCGA-474, CGGA-412, and CGGA-171 sets were 0.405 (0.261-0.629), 0.581 (0.418-0.806), and 0.464 (0.269-0.800), respectively, with P values of <0.001, 0.001, and 0.008, respectively. Among patients receiving radiotherapy in the TCGA-474 set, the OS and PFS of patients with high TOX expression were significantly longer than those in the low TOX expression group, and the differences were statistically significant (both P<0.001). The OS benefit of patients with high expression of TOX was significantly prolonged in both the CGGA-412 and CGGA-171 sets compared to those with low TOX expression, and the differences were statistically significant (both P<0.001). Conclusion:The high expression of TOX may be related to the radiosensitivity of LGG, which may be a gene marker of the radiosensitivity of LGG.

7.
Article in Chinese | WPRIM | ID: wpr-1027450

ABSTRACT

Cancer-associated fibroblasts (CAF) represent a crucial and abundant group of stromal cells in the tumor microenvironment (TME) and are effective targets for anti-tumor therapy. CAFs exhibit high heterogeneity and plasticity, which play a pivotal role in tumor initiation, progression, immune evasion, and therapy resistance. Radiotherapy (RT) is a fundamental treatment modality for malignant tumors that can reshape the TME through various mechanisms. Following RT, CAFs undergo a series of phenotypic changes and interact with multiple cells in the TME, promoting radioresistance and immune evasion through multiple pathways, such as enhancing tumor cell proliferation, modulating immune response, inducing angiogenesis, remodeling extracellular matrix, and reprogramming metabolism, etc., thereby affecting therapeutic effect. Targeting CAFs in combination with RT can improve anti-tumor efficacy and prognosis. In this article, research progress in CAFs in tumor RT was reviewed.

8.
Article in Chinese | WPRIM | ID: wpr-1029769

ABSTRACT

Objective:To investigate the effects of matrine on proliferation, apoptosis and radiotherapy sensitivity of uveal melanoma cells.Methods:An animal experiment study. In vitro experiment: MuM2B cells of human choroidal melanoma were randomly divided into control group and matrine 0.25, 0.50, 1.00, 2.00 g/L groups. The cell morphology was observed by transmission electron microscope. Cell proliferation was detected by thiazole blue colorimetry. The mRNA and relative expression levels of CyclinD D (CyclinD), B lymphoblastoma-2 (Bcl-2) and Bcl2-associated X protein (Bax) were detected by real-time polymerase chain reaction and Western blot. In vivo experiment: BALB/C mice were injected with MuM2B cell suspension subcutaneously on the back of forelimb to prepare transplanted tumor model. After successful modeling, they were randomly divided into blank group and matrine treatment group with different concentrations. Mice in blank group were injected with phosphate buffer subcutaneously. Mice in different matrine treatment groups were injected with 15, 25, 50, 100 mg/kg matrine subcutaneously, respectively, for 7 consecutive days. The tumor was weighed and its volume was measured after the last administration. Single factor analysis of variance was used to compare different groups. The t test was used for pairwise comparison between groups. Results:In the control group, the cell structure was normal, the distribution was uniform, and no or rare nuclear pyknosis was seen. With the increase of matrine dosage, the nuclear pyretosis increased gradually and cell morphology changed obviously. Compared with the control group, the cell survival rate in 0.50, 1.00 and 2.00 g/L groups gradually decreased with matrine concentration increasing and treatment time prolongating, the relative expression levels of CyclinD and Bcl-2 mRNA and protein gradually decreased, and the relative expression levels of Bax mRNA and protein gradually increased. Under the same radiation dose X-ray irradiation, the cell survival rate of 0.50, 1.00 and 2.00 g/L groups gradually decreased, and the differences were statistically significant ( P<0.05). Compared with blank group, the tumor weight and volume of mice in different doses of matrine group were significantly decreased, and the differences were statistically significant ( P<0.05). Conclusion:Matrine can down-regulate the expression of CyclinD and Bcl-2, up-regulate the expression of Bax, promote the apoptosis of MuM2B human melanoma cells, inhibit cell proliferation, and enhance cell radiosensitivity.

9.
Zhonghua zhong liu za zhi ; (12): 879-891, 2023.
Article in Chinese | WPRIM | ID: wpr-1045816

ABSTRACT

Objective: To investigate the effect of hsa_circ_0000392 (circ_0000392) on the radiosensitivity of cervical cancer cells and explore its potential mechanism. Methods: Cervical cancer tissues and adjacent normal tissues of 42 patients with cervical cancer who were confirmed pathologically for the first time in Huaihe Hospital of Henan University from 2016 to 2019 were collected. According to the patients' response to radiotherapy, the cancer tissues were divided into radio-sensitive tissues and radio-resistant tissues. The expressions of circ_0000392, miR-145-5p, and CRKL in radiation-sensitive, radiation-resistant cervical cancer tissues and Hela, SiHa cells were detected by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. SiRNA circ_0000392, miR-145-5p mimic, miR-145-5p inhibitor, pcDNA 3.1-CRKL and its negative control were transfected into HeLa and Siha cells, respectively. After radiation induction, the survival fraction of cells was detected by clone formation assay, apoptosis was detected by flow cytometry, and the expressions of apoptosis-related proteins Bax and Bcl-2 and ERK pathway protein p-ERK1/2 and ERK1/2 were detected by western blot. The targeting relationship between circ_0000392, miR-145-5p and CRKL was verified by dual luciferase reporter gene assay. The effect of circ_0000392 on radiotherapy sensitivity of cervical cancer in vivo was observed in the tumor formation experiment in nude mice. Results: circ_0000392 and CRKL were upregulated in radiation-resistant tissues and cancer cells of cervical cancer, while miR-145-5p was downregulated. The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+ 6 Gy group were (78.67±10.97) and (71.00±9.54), respectively, which were lower than those in si-Ctrl+ 6 Gy group [(176.00±22.27) and (158.33±17.56), respectively]. The apoptosis rates were (41.55±3.40)% and (31.41±3.29)%, respectively, which were higher than those in si-Ctrl+ 6 Gy group [(15.91±1.37)% and (13.70±1.89)%, P<0.05]. The protein expression of Bax was higher than that of si-Ctrl+ 6 Gy group, and the protein expressions of Bcl2 was lower than those of si-Ctrl+ 6 Gy group. The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+ miR-145-5p inhibitor+ 6 Gy group were (171.33±25.01) and (137.00±21.66), higher than those in si-circ_0000392#1+ inhibitor NC+ 6 Gy group [(84.67±17.79) vs (71.00±11.00), P<0.05]. The apoptosis rates were (17.41±2.58) % and (15.96±1.25) %, lower than those of si-circ_0000392 #1+ inhibitor NC+ 6 Gy [(40.29±2.92)% and (30.82±2.34)%, respectively, P<0.05]. The expression of Bax protein was lower than that of si-circ_0000392#1+ inhibitor NC+ 6 Gy group, and the expressions of Bcl2 protein were higher than those of si-circ_0000392#1+ inhibitor NC+ 6 Gy group. Circ_0000392 can target miR-145-5p, and CRKL is the downstream target gene of miR-145-5p. The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+ 6 Gy group were (74.33±10.02) and (66.00±12.17), respectively, which were lower than those of mimic NC+ 6 Gy group [(197.67±17.21) vs (157.67±11.59), respectively, P<0.05]. The apoptosis rates were (45.58±2.16)% and (32.10±3.55)%, higher than those of mimic NC+ 6 Gy group [(15.85±2.45)% and (13.99±1.69)%, respectively, P<0.05]. The expression of Bax protein was higher than that of the mimic NC+ 6 Gy mimic group, and the expression of Bcl2 protein was lower than that of the mimic NC+ 6 Gy group. The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+ pcDNA-CRKL+ 6 Gy group were (158.00±15.88) and (122.33±13.65), respectively, which were higher than those of miR-145-5p mimic+ pcDNA+ 6 Gy group [(71.33±8.02) vs (65.67±12.22), P<0.05]. The apoptosis rates were (19.50±3.45)% and (17.04±0.94)%, respectively, which were lower than those of miR-145-5p mimic+ pcDNA+ 6 Gy group [(44.33±2.36)% and (32.05±2.76)%, respectively, P<0.05]. The expression of Bax protein was lower than that of miR-145-5p mimic+ pcDNA group+ 6 Gy group, and the expression of Bcl2 protein was higher than that of miR-145-5p mimic+ pcDNA+ 6 Gy group. Sh-circ_0000392 group had smaller tumor volume and decreased tumor weight (P<0.05). The relative mRNA expression levels of circ_0000392, miR-145-5p and CRKL and the relative protein expression levels of CRKL, Bcl-2 and p-ERK1/2 were decreased, while the relative expression level of Bax protein was increased (P<0.05). Conclusion: Circ_0000392 could enhance the radiosensitivity of cervical cancer cells, and its mechanism may be related to the regulation of CRKL/ERK signaling pathway by targeting miR-145-5p, which provides a new reference for enhancing the radiosensitivity of cervical cancer cells.


Subject(s)
Animals , Mice , Female , Humans , Uterine Cervical Neoplasms/radiotherapy , bcl-2-Associated X Protein/genetics , Mice, Nude , Proto-Oncogene Proteins c-bcl-2/genetics , Apoptosis , MicroRNAs/genetics , Cell Proliferation , Cell Line, Tumor
10.
Article in Chinese | WPRIM | ID: wpr-1017765

ABSTRACT

Objective To investigate the effects of microRNA(miR)-432-5p on the proliferation,metasta-sis and radiosensitivity of gastric cancer cells and the targeted regulatory relationship with DEAD-box helicase 41(DDX41)in this process.Methods The expressions of miR-432-5p and DDX41 in different gastric cancer cells were detected by quantitative real-time PCR(qRT-PCR).HGC-27 cells were transfected and divided into NC group(not transfected with plasmid),miR-NC group(transfected with miR-432-5p mimics negative con-trol),miR-432-5p group(transfected with miR-432-5p mimics),miR-432-5p+pcDNA-NC group(transfection miR-432-5p and pcDNA-DDX41 negative control)and miR-432-5p+pcDNA-DDX41 group(transfection miR-432-5p mimics and pcDNA-DDX41).After 48 h,qRT-PCR was used to detect the expression of miR-432-5p and DDX41 in each group of cells,MTT assay was used to detect the proliferation activity of each group of cells,Transwell assay was used to detect the migration ability of each group of cells,colony formation assay and flow cytometry were used to detect the radiosensitivity of each group of cells.The dual luciferase reporter gene assay was used to verify the targeting relationship between miR-432-5p and DDX41.The tumor growth of nude mice was detected,and the expression of DDX41 protein in tumor tissues was detected by Western blot.Results Compared with normal cells,cancer of the stomach cells miR-432-5p levels were decreased(P<0.05),DDX41 mRNA levels were increased(P<0.05).Compared with NC group and miR-NC group,the cell survival rate and DDX41 mRNA level in miR-432-5p group were decreased,the number of migrating cells was decreased(P<0.05),the miR-432-5p level was increased,and the radiosensitivity was enhanced(P<0.05).Overexpression of DDX41 reversed the effects of miR-432-5p on proliferation,migration and radiosensitivity of gastric cancer cells(P<0.05).Compared with NC group and miR-NC group,the volume and weight of tumor tissue and the expression of DDX41 protein in miR-432-5p group were significantly decreased(P<0.05).Con-clusion miR-432-5p through targeted cut DDX41 to inhibit the proliferation and metastasis of gastric cancer cells,enhance cell radiation sensitivity.

11.
Chinese Pharmacological Bulletin ; (12): 1668-1674, 2023.
Article in Chinese | WPRIM | ID: wpr-1013714

ABSTRACT

Aim To investigate the radiosensitizing effect of the PARP inhibitor Olaparib on MCF-7 breast cancer model and to monitor the radiosensitizing effect of Olaparib by F-Fluoroerythronitroimidazole (F-FETNIM) PET/CT. Methods MCF-7 breast cancer model was established and divided into control group, Olaparib group, irradiation group and Olaparib + irradiation group according to random number table method; tumor volume was measured to calculate tumor inhibition rate and survival time of tumor-bearing mice was counted.

12.
Article in Chinese | WPRIM | ID: wpr-984568

ABSTRACT

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.

13.
Zhonghua zhong liu za zhi ; (12): 879-891, 2023.
Article in Chinese | WPRIM | ID: wpr-1046139

ABSTRACT

Objective: To investigate the effect of hsa_circ_0000392 (circ_0000392) on the radiosensitivity of cervical cancer cells and explore its potential mechanism. Methods: Cervical cancer tissues and adjacent normal tissues of 42 patients with cervical cancer who were confirmed pathologically for the first time in Huaihe Hospital of Henan University from 2016 to 2019 were collected. According to the patients' response to radiotherapy, the cancer tissues were divided into radio-sensitive tissues and radio-resistant tissues. The expressions of circ_0000392, miR-145-5p, and CRKL in radiation-sensitive, radiation-resistant cervical cancer tissues and Hela, SiHa cells were detected by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot. SiRNA circ_0000392, miR-145-5p mimic, miR-145-5p inhibitor, pcDNA 3.1-CRKL and its negative control were transfected into HeLa and Siha cells, respectively. After radiation induction, the survival fraction of cells was detected by clone formation assay, apoptosis was detected by flow cytometry, and the expressions of apoptosis-related proteins Bax and Bcl-2 and ERK pathway protein p-ERK1/2 and ERK1/2 were detected by western blot. The targeting relationship between circ_0000392, miR-145-5p and CRKL was verified by dual luciferase reporter gene assay. The effect of circ_0000392 on radiotherapy sensitivity of cervical cancer in vivo was observed in the tumor formation experiment in nude mice. Results: circ_0000392 and CRKL were upregulated in radiation-resistant tissues and cancer cells of cervical cancer, while miR-145-5p was downregulated. The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+ 6 Gy group were (78.67±10.97) and (71.00±9.54), respectively, which were lower than those in si-Ctrl+ 6 Gy group [(176.00±22.27) and (158.33±17.56), respectively]. The apoptosis rates were (41.55±3.40)% and (31.41±3.29)%, respectively, which were higher than those in si-Ctrl+ 6 Gy group [(15.91±1.37)% and (13.70±1.89)%, P<0.05]. The protein expression of Bax was higher than that of si-Ctrl+ 6 Gy group, and the protein expressions of Bcl2 was lower than those of si-Ctrl+ 6 Gy group. The clone formation numbers of Hela and SiHa cells in si-circ_0000392#1+ miR-145-5p inhibitor+ 6 Gy group were (171.33±25.01) and (137.00±21.66), higher than those in si-circ_0000392#1+ inhibitor NC+ 6 Gy group [(84.67±17.79) vs (71.00±11.00), P<0.05]. The apoptosis rates were (17.41±2.58) % and (15.96±1.25) %, lower than those of si-circ_0000392 #1+ inhibitor NC+ 6 Gy [(40.29±2.92)% and (30.82±2.34)%, respectively, P<0.05]. The expression of Bax protein was lower than that of si-circ_0000392#1+ inhibitor NC+ 6 Gy group, and the expressions of Bcl2 protein were higher than those of si-circ_0000392#1+ inhibitor NC+ 6 Gy group. Circ_0000392 can target miR-145-5p, and CRKL is the downstream target gene of miR-145-5p. The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+ 6 Gy group were (74.33±10.02) and (66.00±12.17), respectively, which were lower than those of mimic NC+ 6 Gy group [(197.67±17.21) vs (157.67±11.59), respectively, P<0.05]. The apoptosis rates were (45.58±2.16)% and (32.10±3.55)%, higher than those of mimic NC+ 6 Gy group [(15.85±2.45)% and (13.99±1.69)%, respectively, P<0.05]. The expression of Bax protein was higher than that of the mimic NC+ 6 Gy mimic group, and the expression of Bcl2 protein was lower than that of the mimic NC+ 6 Gy group. The clone formation numbers of Hela and SiHa cells in miR-145-5p mimic+ pcDNA-CRKL+ 6 Gy group were (158.00±15.88) and (122.33±13.65), respectively, which were higher than those of miR-145-5p mimic+ pcDNA+ 6 Gy group [(71.33±8.02) vs (65.67±12.22), P<0.05]. The apoptosis rates were (19.50±3.45)% and (17.04±0.94)%, respectively, which were lower than those of miR-145-5p mimic+ pcDNA+ 6 Gy group [(44.33±2.36)% and (32.05±2.76)%, respectively, P<0.05]. The expression of Bax protein was lower than that of miR-145-5p mimic+ pcDNA group+ 6 Gy group, and the expression of Bcl2 protein was higher than that of miR-145-5p mimic+ pcDNA+ 6 Gy group. Sh-circ_0000392 group had smaller tumor volume and decreased tumor weight (P<0.05). The relative mRNA expression levels of circ_0000392, miR-145-5p and CRKL and the relative protein expression levels of CRKL, Bcl-2 and p-ERK1/2 were decreased, while the relative expression level of Bax protein was increased (P<0.05). Conclusion: Circ_0000392 could enhance the radiosensitivity of cervical cancer cells, and its mechanism may be related to the regulation of CRKL/ERK signaling pathway by targeting miR-145-5p, which provides a new reference for enhancing the radiosensitivity of cervical cancer cells.


Subject(s)
Animals , Mice , Female , Humans , Uterine Cervical Neoplasms/radiotherapy , bcl-2-Associated X Protein/genetics , Mice, Nude , Proto-Oncogene Proteins c-bcl-2/genetics , Apoptosis , MicroRNAs/genetics , Cell Proliferation , Cell Line, Tumor
14.
Article in Chinese | WPRIM | ID: wpr-992079

ABSTRACT

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.

15.
Article in Chinese | WPRIM | ID: wpr-993043

ABSTRACT

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.

16.
Article in Chinese | WPRIM | ID: wpr-993044

ABSTRACT

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.

17.
Article in Chinese | WPRIM | ID: wpr-993149

ABSTRACT

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.

18.
Article in Chinese | WPRIM | ID: wpr-993188

ABSTRACT

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.

19.
Article in Chinese | WPRIM | ID: wpr-993199

ABSTRACT

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.

20.
Article in Chinese | WPRIM | ID: wpr-993213

ABSTRACT

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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