Objective: To determine the effect of tumor metastasis-associated gene 1 (MTA1) on the sensitivity of HeLa cells to radiotherapy, and to clarify its molecular mechanism. Methods: The transcriptome differences between MTA1 knocked down Hela cells and control cells were analyzed, and the differentially expressed genes (DEGs) was used to perform Gene-Set Enrichment Analysis (GSEA) and Gene Ontology (GO) cluster analysis. Flow cytometry was used to detect apoptosis in MTA1-overexpressed HeLa cells and control cells before and after 10 Gy X-ray irradiation. Cloning formation assay and real-time cellular analysis (RTCA) were used to monitor the cell proliferation before and after 2 Gy X-ray irradiation. To dissect the underlying molecular mechanisms of MTA1 affecting the sensitivity of radiotherapy, the proteins encoded by the DEGs were selected to construct a protein-protein interaction network, the expression of γ-H2AX was detected by immunofluorescence assay, and the expression levels of γ-H2AX, β-CHK2, PARP and cleaved caspase 3 were measured by western blot. Results: By transcriptome sequencing analysis, we obtained 649 DEGs, of which 402 genes were up-regulated in MTA1 knockdown HeLa cells and 247 genes were down-regulated. GSEA results showed that DEGs associated with MTA1 were significantly enriched in cellular responses to DNA damage repair processes. The results of flow cytometry showed that the apoptosis rate of MTA1 over-expression group (15.67±0.81)% after 10 Gy X-ray irradiation was significantly lower than that of the control group [(40.27±2.73)%, P<0.001]. After 2 Gy X-ray irradiation, the proliferation capacity of HeLa cells overexpressing MTA1 was higher than that of control cells (P=0.024). The numbers of colon in MTA1 over-expression group before and after 2 Gy X-ray irradiation were (176±7) and (137±7) respectively, higher than (134±4) and (75±4) in control HeLa cells (P<0.05). The results of immunofluorescence assay showed that there was no significant expression of γ-H2AX in MTA1 overexpressed and control HeLa cells without X-ray irradiation. Western blot results showed that the expression level of β-CHK2 in MTA1-overexpressing HeLa cells (1.04±0.06) was higher than that in control HeLa cells (0.58±0.25, P=0.036) after 10 Gy X-ray irradiation. The expression levels of γ-H2AX, PARP, and cleaved caspase 3 were 0.52±0.13, 0.52±0.22, and 0.63±0.18, respectively, in HeLa cells overexpressing MTA1, which were lower than 0.87±0.06, 0.78±0.12 and 0.90±0.12 in control cells (P>0.05). Conclusions: This study showed that MTA1 is significantly associated with radiosensitivity in cervical cancer HeLa cells. MTA1 over-expression obviously reduces the sensitivity of cervical cancer cells to X-ray irradiation. Mechanism studies initially indicate that MTA1 reduces the radiosensitivity of cervical cancer cells by inhibiting cleaved caspase 3 to suppress apoptosis and increasing β-CHK2 to promote DNA repair.
Apoptosis/genetics* ; Caspase 3/metabolism* ; Female ; HeLa Cells ; Humans ; Poly(ADP-ribose) Polymerase Inhibitors ; Radiation Tolerance/genetics* ; Repressor Proteins/metabolism* ; Trans-Activators/metabolism* ; Uterine Cervical Neoplasms/radiotherapy*

Objective: To study the effect of circ-WHSC1 on the growth, metastasis and radiosensitivity of nasopharyngeal carcinoma cells and its molecular mechanism. Methods: Cancerous tissues and adjacent tissues were collected from 23 patients with nasopharyngeal carcinoma, and real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expression levels of circ-WHSC1, miR-338-3p, and ELAVL1 mRNA. Western blot was used to detect the expression of ELAVL1 protein. Nasopharyngeal carcinoma cells 5-8F and SUNE1 were divided into si-NC group, si-circ-WHSC1 group, pCD5-ciR group, circ-WHSC1 group, anti-miR-NC group, anti-miR-338-3p group, miR-NC group, miR-338-3p group, si-circ-WHSC1+ anti-miR-NC group, si-circ-WHSC1+ anti-miR-338-3p group, miR-338-3p+ pcDNA group, miR-338-3p+ ELAVL1 group. Tetramethylazolium salt colorimetric method (MTT) was used to detect cell viability. Clone formation test was used to detect cell clone formation and cell radiosensitivity. Flow cytometry was used to detect cell apoptosis. Transwell was used to detect cell migration and invasion. Dual luciferase assay was used to detect the targeting relationship between circ-WHSC1 and miR-338-3p, miR-338-3p and ELAVL1. The SUNE1 cells stably transfected with sh-circ-WHSC1 were injected into nude mice and irradiated with radiation, and then the tumor volume and weight of mice were detected. Results: The expressions of circ-WHSC1 (1.57±0.94 vs 3.78±1.18, 1.00±0.10 vs 1.64±0.14/2.00±0.21/2.81±0.26/3.36±0.34) and ELAVL1 (1.28±0.74 vs 3.36±0.77, 1.00±0.08 vs 2.51±0.19/3.27±0.27) in nasopharyngeal carcinoma tissues and cells were increased, and the expression of miR-338-3p (3.13±0.96 vs 1.37±0.98, 1.00±0.08 vs 0.48±0.08/0.38±0.07) was decreased (P<0.05). After knockdown of circ-WHSC1, the activity of nasopharyngeal carcinoma cells was decreased [(100.00±8.00)% vs (51.33±8.62)%, (100.00±10.10)% vs (41.02±7.31)%], the number of clone-forming cells was decreased (101.00±8.54 vs 50.33±8.02, 114.00±14.10 vs 42.33±10.01), the rate of apoptosis was increased [(5.37±1.20)% vs (18.3±1.01)%, (6.5±1.18)% vs (22.43±1.40)%], and the numbers of migration (136.00±13.00 vs 72.33±9.50, 154.00±14.10 vs 62.67±11.50) and invasion (113.67±11.59 vs 60.67±9.07, 124.33±15.57 vs 50.33±9.01) were decreased; after different doses of radiation, the cell survival score was decreased (0.23±0.04 vs 0.06±0.01, 0.32±0.07 vs 0.05±0.02) (P<0.05). Circ-WHSC1 targeted and negatively regulated miR-338-3p. Inhibition of miR-338-3p affected the effect of knockdown of circ-WHSC1 on the proliferation, apoptosis, migration, invasion and radiosensitivity of nasopharyngeal carcinoma cells. MiR-338-3p targeted and negatively regulated ELAVL1; ELAVL1 overexpression affected the effects of miR-338-3p on the proliferation, apoptosis, migration, invasion and radiosensitivity of nasopharyngeal carcinoma cells. After the cells stably transfected with sh-circ-WHSC1 were injected into nude mice, the tumor volume [(884.67±95.63)mm(3) vs (487.33±76.51)mm(3)] and weight [(899.01±88.54)mg vs (558.67±75.04) mg] of the nude mice were reduced; after further irradiation, the tumor volume [(395.00±73.50)mm(3) vs 243.13±42.51)mm(3)] and weight[ (452.33±67.30)mg vs (211.09±57.51)mg] of the nude mice were reduced (P<0.05). Circ-WHSC1 regulated the expression of ELAVL1 by targeting miR-382. Conclusion: Knockdown of circ-WHSC1 can inhibit the growth and metastasis of nasopharyngeal carcinoma cells by targeting miR-338-3p/ELAVL1 axis, and enhances the radiosensitivity of nasopharyngeal carcinoma cells.
Mice ; Animals ; Nasopharyngeal Carcinoma/radiotherapy* ; Mice, Nude ; MicroRNAs/genetics* ; Antagomirs ; Cell Line, Tumor ; Radiation Tolerance/genetics* ; Nasopharyngeal Neoplasms/pathology* ; Cell Proliferation/genetics* ; Gene Expression Regulation, Neoplastic

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*

Objective: To explore the impacts of miR-18a overexpression or depression on the radiosensitivities of nasopharyngeal carcinoma cell line CNE1 and CNE2 and underlying mechanisms. Methods: CNE1 and CNE2 were transfected with miR-18a mimics, inhibitor and the corresponding control vectors. qRT-PCR and western blot were used to determine the ataxia telangiectasia mutated (ATM) expressions in CNE1 and CNE2. CNE1 and CNE2 with stably expressing miR-18a and miR-18a siRNA were constructed. Methyl thiazolyl tetrazolium (MTT) assay was used to detect the impacts of the miR-18a overexpression or depression combined with irradiation on the cell growth. Flow cytometry was used to detect the cell apoptosis and cell cycle. Colony formation assay was used to evaluate the raodiosensitivities of cells. Acridine orange (AO) staining and western blot were used respectively to test the autophagy and the expressions of related proteins. Independent samples t test was used to compare the mean value between groups by using SPSS 16.0. Results: ATM mRNA was decreased significantly in CNE1 and CNE2 cells transfected with 100 or 200 nmol/L miR-18a mimics for 48 hours (CNE1: RQ=0.174±0.139 and 0.003±0.001, t=9.939 and 19 470.783;CNE2: RQ=0.024±0.008 and 0.019±0.012, t=270.230 and 137.746, respectively, all P<0.001). ATM proteins were also decreased after transfected with 100 or 200 nmol/L miR-18a mimics for 72 hours. While in the cells transfected with 100 and 200 nmol/L miR-18a inhibitor for 48 hours, the expressions of ATM mRNA were upregulated significantly (CNE1: RQ=9.419±2.495 and 2.500±1.063, t=-4.427 and -41.241; CNE2: RQ=7.210±0.171 and 115.875±15.805, t=-62.789 and -12.589, all P<0.05), and the expressions of ATM proteins increased after transfected for 72 hours. The growth of cells with miR-18a overexpression plus 4 Gy irradiation were obviously inhibited compared to that of cells with the 4Gy irradiation alone; while the growth of miR-18a-inhibited cells increased compared to that of cells with 4 Gy irradiation alone (all P<0.05). CNE1 transfected with 100 nmol/L miR-18a mimics plus 4 Gy irradiation showed the higher apoptosis rate than the cells with 4 Gy irradiation alone ((22.9±2.1)% vs. (16.3±1.0)%, t=-4.870, P<0.01). Compared to the cells with 4 Gy irradiation alone, miR-18a-overexpressed cells plus 4 Gy irradiation decreased their percentages in G1 phases ((20.2±3.0)% vs. (29.8±4.4)%, t=3.119) and G2/M phases ((21.5±0.9)% vs. (33.4±3.1)%, t=6.410, P<0.05 for both), and increased their percentages in S phases ((56.7±4.9)% vs. (36.8±6.4)%, t=-4.246, P<0.05), and these cells possessed less colony number after exposure to different doses of irradiation, more autophagy-lysosome number, and more expressions of LC3 proteins (all P<0.05). There were no significant differences in the expressions of p62 expressions between different groups of cells. Conclusion: Overexpression of miR-18a can enhance the radiosensitivities of NPC cells by targeting ATM to abrogate G1/S, G2/M arrest and to induce autophagy and apoptosis.
Apoptosis ; Autophagy ; Cell Line, Tumor ; Cell Proliferation ; G2 Phase Cell Cycle Checkpoints ; Humans ; MicroRNAs/genetics* ; Nasopharyngeal Carcinoma/genetics* ; Nasopharyngeal Neoplasms/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

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

OBJECTIVETo detect the expression of miR-124 in colorectal carcinoma (CRC) cells and tissue specimens and analyze its association with the radiosensitivity of the cells.

METHODSThe expression of miR-124 in CRC cell lines and tissues were detected using qRT-PCR. The effect of miR-124 in modulating cell radiosensitivity was assessed in CRC cells with miRNA-124 overexpression and miRNA-124 knockdown, and bioinformatics prediction and dual luciferase reporter system were employed to identify the direct target of miR-124.

RESULTSs miR-124 expression was down-regulated in CRC cell lines and tissues. CRC cells over-expressing miR-124 showed an obviously enhanced radiosensitivity, whereas miR-124 knockdown resulted in a reduced radiosensitivity of the cells. Bioinformatics prediction and dual luciferase reporter system verified PRRX1 as a direct target of miR-124, which regulated the radiosensitivity of CRC cells by directly inhibiting PRRX1.

CONCLUSIONmiR-124 can enhance the radiosensitivity of CRC cells by directly targeting PRRX1, which provides a target for improving the therapeutic effect of radiotherapy of CRC.

Cell Line, Tumor ; Colorectal Neoplasms ; pathology ; radiotherapy ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Luciferases ; MicroRNAs ; genetics ; metabolism ; Radiation Tolerance

OBJECTIVETo investigate the effect of RNA interference of IgG gene on the radiosensitivity of the human prostate cancer PC3 cell line.

METHODSPC3 cells were trasnfected via lipofectamine by the shRNA vector FCGR1AshRNA targeting the Fc segment of IgG, using NCshRNA as the negative control. Q-PCR and Western blotting were used to analyze the expression of IgG in the trasnfected cells. The cells were then exposed to ⁶⁰Co γ ray at 0, 2, 4, 6, 8, 10 Gy, and the cell proliferation was evaluated by MTS and the cells apoptosis estimated by flow cytometry at 12, 24 and 48 h.

RESULTSMTS assay showed that ⁶⁰Co γ ray significantly inhibited the proliferation of PC3 cells transfected with FCGR1AshRNA as compared with NCshRNA-transfected and blank control cells (P<0.05). Flow cytometry showed that the cell apoptosis rate was significantly higher in FCGR1AshRNA group than in NCshRNA and blank control groups at 48 h after γ ray exposure (P<0.05). At 12, 24 and 48 h after 6 Gy radiation, the cells in FCGR1AshRNA group showed a significantly lowered proliferation rate and an increased apoptosis rate (P<0.05).

CONCLUSIONThe shRNA targeting IgG gene can significantly enhance the sensitivity of PC3 cells to radiation. The combination of RNA interference targeting IgG gene with radiotherapy may be more effective in the treatment of prostate cancer.

Apoptosis ; Cell Line, Tumor ; radiation effects ; Cell Proliferation ; Humans ; Immunoglobulin G ; genetics ; Male ; Prostatic Neoplasms ; pathology ; RNA Interference ; RNA, Small Interfering ; Radiation Tolerance ; Transfection

OBJECTIVETo study the radiobiological characteristics of a HepG2 cell line with mitochondrial DNA (mtDNA) deletion.

METHODSHepG2 cells were cultured in a medium containing ethidium bromide, acetylformic acid and uracil. The HepG2 cell line with mtDNA deletion (ρ(0)HepG2 cells) were acquired after 30 subcultures by limited dilution cloning. The cell survival was then observed in the absence of acetylformic acid and uracil, and the total mtDNA deletion in the cells was confirmed by PCR. The radiosensitivity of HepG2 and ρ(0)HepG2 cells was evaluated by exposure to gradient doses of 6 MV X ray irradiation. The cell apoptosis was assessed following a 2 Gy X-ray exposure with Hochest33342 staining, and the invasiveness of ρ(0)HepG2 cells was measured by Transwell assay.

RESULTSHepG2 cells could survive 30 subcultures in the presence of ethidium bromide, and massive cell death occurred after removal of acetylformic acid and uracil from the medium. PCR confirmed total mtDNA deletion from ρ(0)HepG2 cells, whose α/β value was significantly lower than that of HepG2 cells. ρ(0)Hep-G2 cells showed an obviously lowered cell apoptosis rate following X-ray exposure with enhanced cell invasiveness.

CONCLUSIONHepG2 cells can be induced by ethidium bromide into ρ(0)HepG2 cells with an increased radiation resistance, anti-apoptosis ability and cell invasiveness.

Apoptosis ; Culture Media ; chemistry ; DNA, Mitochondrial ; genetics ; Ethidium ; chemistry ; Hep G2 Cells ; radiation effects ; Humans ; Radiation Tolerance ; genetics ; Sequence Deletion ; X-Rays

OBJECTIVE: To study the relationship between the radiotherapy resistance and autophagy. To provide a theoretiacal basis for drugs that regulate autophagy to improve radiotherapy sensitivity. METHOD: Flow cytometry (FCM) was performed to analyze the distribution of the cell cycle of CNE2 and CNE2/DDP cells under the action of X radiation. The expression of autopagy-specific gene Beclin1 and microtubule-associated protein light chain 3β (MAPLC3β) in CNE2 and CNE2/DDP cells was determined by real time PCR and Immumofluorescence staining. RESULT: CNE2/DDP and their parental CNE2 cells produced the G2-M phase arrest under the action of X radiation. With the radiation dose increasing,The cells which in the G2-M phase were more and more (P<0. 05). The G2-M phase arrest in CNE2/DDP cells was more obvious than in CNE2 cells (P<0. 05). The expression of Beclin1 and MAPLC3β in CNE2 and CNE2/DDP cells increased under the action of X radiation. What's more, the raise was more and more obvious with the increase of the irradiation dose(P<0. 05). The expression levels of Beclin1 and MAPLC3β in CNE2/DDP was lower than that in CNE2 cells (P<0. 05). CONCLUSION Autophagic cell death may be the one manner of death in nasopharyngeal carcinoma CNE2 and CNE2/DDP cells under the action of X radiation. The radiation resistance of CNE2/DDP cells may be related to the low expression of autophagy-related genes.
Apoptosis Regulatory Proteins ; genetics ; Autophagy ; Beclin-1 ; Carcinoma ; Cell Cycle ; Cell Line, Tumor ; radiation effects ; Dose-Response Relationship, Radiation ; Humans ; Membrane Proteins ; genetics ; Microtubule-Associated Proteins ; genetics ; Nasopharyngeal Carcinoma ; Nasopharyngeal Neoplasms ; genetics ; Radiation Tolerance ; X-Rays