OBJECTIVETo investigate the effects of radiosensitivity and X-ray dose on the expression of miR-7 in nasopharyngeal carcinoma (NPC) cells.

METHODSLow radiosensitive NPC cells CNE-1 and high radiosensitive NPC cells CNE-2 were exposed to 0, 2 and 8 Gy X-ray. The total RNAs of the cell lines were extracted 10 h after radiation for reverse transcription of miR-7 and 18S rRNA by stem-loop primer and random hexamers, respectively. The non-irradiated CNE-1 cells served as the control sample and the relative quantity of the expression level was calculated after real-time PCR using SyBR green.

RESULTSmiR-7 expression differed significantly between CNE-1 and CNE-2 cells (4.49-/+3.62 vs 1.29-/+1.10, F=135.483, P<0.001). The radiation dose also significantly affected the expression of miR-7 in NPC cells (F=39.565, P<0.001). CNE-1 cells with a 2 Gy exposure had the highest expression level of miR-7, while the non-irradiated CNE-1 cells had the lowest expression. CNE-2 cells exposed to 2 Gy X-ray had the lowest expression level of miR-7 and the non-irradiated CNE-2 cells had the highest.

CONCLUSIONRadiosensitivity and radiation dose of X-ray have significant effect on the expression of miR-7 in NPC cells, indicating that miR-7 plays an important role in radioresistance of NPC cells to X-ray, and suppressed miR-7 expression may elevate the radiosensitivity of NPC cells.

Apoptosis ; radiation effects ; Carcinoma ; Cell Line, Tumor ; Dose-Response Relationship, Radiation ; Gene Expression Regulation, Neoplastic ; radiation effects ; Humans ; MicroRNAs ; genetics ; Nasopharyngeal Neoplasms ; genetics ; Radiation Tolerance ; genetics ; X-Rays

The HL-60 cells were transfected with chk1 antisense and sense chain, and 24 h later subjected to irradiation. Twenty-four h after irradiation, the changes in the chk1 protein expression was assayed by Western blot, and the cell cycles and apoptosis rate detected by FCM. The irradiated apoptosis sensitivity was increased by antisense blocking of chk1 gene in HL-60 cell line with the apoptosis rate being 26.31%, significantly higher than that by the sense blocking (10.34%, 0.025 < P < 0.05). In HL-60 cells transfected with chk1 antisense chain, the G2/M phase arrest was attenuated and the cells in G2/M phase were accounted for 38.42%, significantly lower than those of the cells transfected with chkl sense chain (54.64%, 0.005 < P < 0.01). It was concluded that antisense blocking of chk1 gene could increase the apoptosis sensitivity to irradiation.
*Apoptosis/radiation effects ; Cell Cycle/radiation effects ; HL-60 Cells ; Oligonucleotides, Antisense/*genetics ; Protein Kinases/*genetics ; Protein Kinases/metabolism ; Radiation Tolerance/*genetics ; Transfection

BACKGROUND AND OBJECTIVEThe mRNA levels of 59 genes, detected by cDNA microarray, were up-regulated in the radioresistant human esophageal cacinoma cell line TE13R120 as compared with its parental cell line TE13 before and after radiation, and the expression of NRAGE gene showed a gradually up-regulating tendency. This study aimed to further detect the differences of NRAGE gene and protein expression and apoptosis between TE13R120 and TE13 cells, and to investigate the relationship between the NRAGE and the radioresistance of TE13R120 cells and its mechanism.

METHODSThe two cell lines were irradiated by ⁶⁰Co γ-ray at different conditions. Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry were used to detect the expression of NRAGE. Flow cytometry (FCM) was used to detect the cell apoptosis before and after irradiation.

RESULTSThe mRNA level of NRAGE was higher in TE13R120 cells than in TE13 cells before and after irradiation (before radiation: 0.25 ± 0.03 vs. 0.49 ± 0.03; 4 Gy 4 h: 0.31 ± 0.03 vs. 0.53 ± 0.02; 4 Gy 16 h: 0.32 ± 0.04 vs. 0.59 ± 0.04; 4 Gy 24 h: 0.36 ± 0.05 vs. 0.72 ± 0.04; 2 Gy 12 h: 0.32 ± 0.02 vs. 0.64 ± 0.04; 6 Gy 12 h: 0.36 ± 0.02 vs. 0.79 ± 0.05; 10 Gy 12 h: 0.46 ± 0.04 vs. 0.85 ± 0.01; P < 0.01), and the mRNA level of NRAGE was increased gradually with the increase of radiation dose and time in the two cell lines (P < 0.05 and P < 0.01). Western blot results showed no difference of NRAGE protein level in cytoplasm between TE13R120 cells and TE13 cells before and after irradiation, but its level in nuclei was higher in TE13R120 cells than in TE13 cells at different radiation time and dosages. Immunocytochemistry showed similar results as Western blot. FCM showed no significant difference in apoptosis rate between TE13R120 and TE13 cells before and after radiation.

CONCLUSIONNRAGE may play an important role in the radiation responses of the two cell lines, and may participate in the formation of radioresistance of TE13R120 cells by changing its subcellular localization, but its relationship with cell apoptosis has not been confirmed.

Antigens, Neoplasm ; genetics ; metabolism ; radiation effects ; Apoptosis ; radiation effects ; Cell Line, Tumor ; radiation effects ; Cobalt Radioisotopes ; Esophageal Neoplasms ; metabolism ; pathology ; Humans ; Neoplasm Proteins ; genetics ; metabolism ; radiation effects ; RNA, Messenger ; metabolism ; radiation effects ; Radiation Tolerance ; Radiotherapy Dosage ; Time Factors ; Up-Regulation

OBJECTIVE: To explore the effect of mmu-circRNA_016901 on the regulation of radiation injury of bone marrow stem cells and its mechanism. METHODS: Bone marrow stem cells were exposed to different dose of X-ray, then the expression level of mmu-circRNA_016901 in bone marrow cells treated with different doses of X-ray was detected. The luciferase reporter gene assay was used to confirm that miRNA1249-5p is the target of mmu-circRNA_016901, and RNA Binding Protein Immunoprecipitation was used to confirm that TGF-β3 is the targeted on miRNA1249-5p，the expression of TGF-β3 and cell proliferation were detected after the expression of mmu-circRNA_01690 was regulated. RESULTS: When the irradiation dose＜6 Gy, there were significant difference in the expression of mmn-circRNA-016901 after the bone marrow mesenchymal stem cells were treated by different doses of irradiation, which showed a statistically significant (P＜0.05). The luciferase reporter gene detection and co-immunoprecipitation experiments confirmed that Mmu-circRNA_016901 could binds to miRNA1249-5p specifically, and overexpression of mmu-circRNA_016901 could regulate miRNA1249-5p negatively, which resulted in a significant increase in TGF-β3 expression and promoting of cell proliferation. CONCLUSION mmu-circRNA_016901 affects the expression of TGF-β3 through miRNA1249-5p, and thus participates in the regulation of the radiation damage mechanism of bone marrow mesenchymal stem cells.
Bone Marrow Cells ; Mesenchymal Stem Cells ; RNA, Circular ; genetics ; Radiation Tolerance

OBJECTIVES: Radiotherapy is one of the main therapies for colorectal cancer, but radioresistance often leads to radiotherapy failure. To improve the radioresistance, we explore the effect of oligomycin A, the H METHODS: The effects of different concentrations of oligomycin A on the survival rate and glycolysis of HT29 colorectal cancer cells at different time points were investigated via MTT and glycolysis assay. siRNA-PFK1 was synthesized in vitro and transfected into HT29 cells. The effects of oligomycin A on radiosensitivity of HT29 colorectal cancer cells were measured via MTT and colony formation assay. Western blotting was used to detect the effect of oligomycin A on the expression of glycolytic enzyme PFK1. We compared difference between the effects of siRNA-PFK1 group and oligomycin A combined with siRNA-PFK1 group on cell survival and glycolysis. After 4 Gy X-ray irradiation, the effects of cell survival and glycolysis between the siRNA-PFK1 group and the oligomycin A combined with siRNA-PFK1 group were compared. RESULTS: Compared with the 0 μmol/L oligomycin A group, the cell survival rate of HT29 cells treated with 4 μmol/L oligomycin A was significantly increased ( CONCLUSIONS Oligomycin A can promote the radioresistance of HT29 colorectal cancer cells, which may be related to up-regulation of the PFK1 expression and increase of cell glycolysis.
Cell Line, Tumor ; Colorectal Neoplasms/genetics* ; HT29 Cells ; Humans ; Oligomycins/pharmacology* ; Radiation Tolerance

Preoperative neoadjuvant chemoradiotherapy, combined with total mesorectal excision, has become the standard treatment for advanced localized rectal cancer (RC). However, the biological complexity and heterogeneity of tumors may contribute to cancer recurrence and metastasis in patients with radiotherapy-resistant RC. The identification of factors leading to radioresistance and markers of radiosensitivity is critical to identify responsive patients and improve radiotherapy outcomes. MicroRNAs (miRNAs) are small, endogenous, and noncoding RNAs that affect various cellular and molecular targets. miRNAs have been shown to play important roles in multiple biological processes associated with RC. In this review, we summarized the signaling pathways of miRNAs, including apoptosis, autophagy, the cell cycle, DNA damage repair, proliferation, and metastasis during radiotherapy in patients with RC. Also, we evaluated the potential role of miRNAs as radiotherapeutic biomarkers for RC.
Humans ; MicroRNAs/metabolism* ; Neoplasm Recurrence, Local ; Rectal Neoplasms/pathology* ; Neoadjuvant Therapy ; Radiation Tolerance/genetics*

OBJECTIVETo investigate the effect of multidrug resistance-associated protein 4 (MRP4) expression on the radiosensitivity of colorectal carcinoma cell lines in vitro.

METHODSThe vector of shRNA for RNA interference was constructed and then transfected into HCT116 cell line to steadily down-regulate the expression of MRP4. HCT116 cells were divided into 3 groups including the CON group(non-transfected), NC group (negative control virus was added), and KD group (RNAi target was added for transfection). To test the effectiveness of RNA interference, real-time polymerase chain reaction and Western blot were used to measure the expression pattern of MRP4 at both mRNA and protein levels, respectively. For the examination of the effect of RNA interference of MRP4 on the radiosensitivity, flow cytometry was used to calculate the rate of apoptotic cells 24 h after 4 Gy radiation. Proliferation of the cells was measured via MTT assay at different time points.

RESULTSShRNA plasmid was successfully constructed. Transfection of this constructed vector into HCT116 cell line caused steady silencing of MRP4 expression (HCT116-KD). MRP4 mRNA and protein expression were significantly down-regulated following RNA interference(P<0.05). Twenty-four hours after radiation, the apoptosis rate of KD cell line was (71.7±0.8)%, significantly higher than that in the CON group [(56.1±0.9)%] and NC group[(59.8±0.8)%](P<0.05). Fourty-eight hours and 72 hours after radiation, the proliferation was significantly inhibited in KD cells compared to the control groups(P<0.05).

CONCLUSIONSExpression of MRP4 is closely related to radio-tolerance of colorectal carcinoma. Down-regulation of MRP4 expression by RNA interference enhances radiosensitivity of colorectal carcinoma cell lines in vitro. MRP4 may be an effective molecular marker for predicting the radiosensitivity of colorectal carcinoma.

Colorectal Neoplasms ; genetics ; metabolism ; Down-Regulation ; HCT116 Cells ; Humans ; Multidrug Resistance-Associated Proteins ; genetics ; RNA Interference ; Radiation Tolerance ; genetics

BACKGROUNDCockayne syndrome (CS) is a rare human genetic disorder characterized by increased UV sensitivity, developmental abnormalities and premature aging. Cells isolated from individuals with CS have a defect in transcription-coupled DNA repair. Despite the repair defect, there is no any increased risk of spontaneous or UV-induced cancer for CS individuals. The strategy of RNA interfering was used here to explore the potential radiosensitizing and anticancer activity of targeting CS group B (CSB) gene.

METHODSThe vectors encoding CSB-specific siRNAs were constructed by inserting duplex siRNA encoding oligonucleotides into the plasmid P(silencer TM 3.1). The cell lines expressing the CSB-siRNA were generated from HeLa cells transfected with the above vectors. Colony-forming ability was used to assay cell survival. Cell cycle was analyzed by FACScan flow cytometry. The apoptosis was measured by detecting the accumulation of sub-G(1) population as well as by fluorescence staining assay. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to semi-quantify mRNA expression. Protein level was detected by Western blotting analysis.

RESULTSTwo constructs encoding CSB-specific siRNA were generated, both of them resulted in remarkable suppression on CSB expression in HeLa cells, and led to an increased sensitivity to (gamma-ray and UV light. siRNA-mediated silencing of CSB decreased cell proliferation rate, increased spontaneous apoptosis as well as the occurrence of UV- or cisplatin-induced apoptosis by 2 to 3.5 fold. A significant S phase blockage and a remarkable reduction of G(1) population were induced in control HeLa cells at 18 hours after being exposed to 10 J/m(2) of UV light. The S phase blockage was also observed in UV-irradiated CSB-siRNA transfected HeLa cells, but the extent of increased S phase population was lower than that in the UV-irradiated control cells. No or a relative weak reduction on G(1) phase population was observed in UV-irradiated CSB-siRNA transfected HeLa cells. In addition, siRNA-mediated silencing of CSB promoted the elimination of G(2)/M phase cells after UV light radiation.

CONCLUSIONSsiRNA-mediated silencing of CSB causes cells to proliferate more slowly, sensitize cells to genotoxicants, and modify UV radiation-induced cell cycle changes. siRNA-mediated inactivation of CSB could be an attractive strategy for ameliorating cancer therapy, which can be fulfilled via the combination of gene therapy and sensitization of radiotherapy or chemotherapy.

Apoptosis ; radiation effects ; Cell Cycle ; radiation effects ; Cell Proliferation ; radiation effects ; Cisplatin ; pharmacology ; Cockayne Syndrome ; genetics ; Gene Silencing ; Genetic Therapy ; HeLa Cells ; radiation effects ; Humans ; RNA, Small Interfering ; genetics ; Radiation Tolerance ; Ultraviolet Rays

OBJECTIVETo investigate the effect of knocking-down microRNA-221 (miR-221) expression on the radiosensitivity of human colorectal carcinoma cells.

METHODSHuman colorectal carcinoma-derived cell line Caco2 was transfected with miR-221 antisense oligonucleotides (anti-miR-221) via Lipofectamine 2000. Real-time quantitative PCR was performed to detect the expression of miR-221 and PTEN mRNA in Caco2 cells. The changes in the protein expression of PTEN in the transfected cells were detected by Western blotting. The cell death after transfection and irradiation was detected by flow cytometry.

RESULTSTransfection with anti-miR-221 caused a significant reduction in miR-221 expression (P<0.05) and up-regulated PTEN protein expression (P<0.05) in Caco2 cells. The percentage of cell death was significantly increased in anti-miR-221 group and anti-miR-221 with irradiation group (P<0.01). Anti-miR-221 significantly enhanced the radiosensitivity of Caco2 cells, which was partially reversed by PTEN-siRNA.

CONCLUSIONAnti-miR-221 can enhance the radiosensitivity of colorectal carcinoma cells by up-regulating the expression of PTEN.

Caco-2 Cells ; radiation effects ; Colorectal Neoplasms ; genetics ; metabolism ; Humans ; MicroRNAs ; genetics ; metabolism ; PTEN Phosphohydrolase ; metabolism ; RNA, Messenger ; genetics ; Radiation Tolerance ; Transfection ; Up-Regulation

OBJECTIVETo screen long non-coding RNA (lncRNA) associated with radiosensitivity in colorectal carcinoma cell lines.

METHODSColony formation assay was performed in colorectal cancer cell lines HT29, SW480, RKO, Lovo and HCT116 after irradiation with different radiation doses. Radiation sensitivity of these 5 cell lines was detected through survival fraction at 2 Gy (SF2 value). High-throughput lncRNA chip was used to screen lncRNA genes with expression differences more than 2 folds among SW480, RKO and Lovo. Further experiment on the expression differences of lncRNAs selected was conducted by realtime PCR.

RESULTSThe radiosensitivity order of these 5 cell lines from low to high (SF2 value from high to low) was HT29 (0.83 ± 0.03), SW480 (0.69 ± 0.02), RKO(0.53 ± 0.02), Lovo (0.47 ± 0.05), HCT116 (0.32 ± 0.03) (P < 0.01). Five lncRNAs associated with radiation sensitivity were screened. Among them, expression levels of R05532, NR_015441, and NR_033374 were positively correlated with radiation resistance(all P < 0.01), and expression levels of the other 2 lncRNAs, NR_073156 and AA745020, were not correlated with radiation resistance of colorectal cancer cells (both P>0.05).

CONCLUSIONSlncRNA R05532, NR_015441 and NR_033374 may be used as the predictive marker of radiosensitivity of colorectal cancer cells. Higher expression of these genes shows radiation resistance.

Cell Line, Tumor ; Colorectal Neoplasms ; genetics ; radiotherapy ; Humans ; Oligonucleotide Array Sequence Analysis ; RNA, Long Noncoding ; genetics ; Radiation Tolerance