ABSTRACT
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system is a hotspot of gene editing and gene expression research, in which CRISPR/Cas13 system provides a new direction for RNA interference and editing. In this study, we designed and synthesized the corresponding gRNAs of CRISPR/Cas13a and CRISPR/Cas13b systems in non-homologous end joining (NHEJ) pathway, such as Ku70 and Lig4, and then detected the expression of ku70 and lig4 in HEK293T cells. The CRISPR/Cas13a system could efficiently knockdown the mRNA expression of ku70 and lig4 more than 50%, and CRISPR/Cas13b system also suppressed ku70 and lig4 about 92% and 76%, respectively. Also, CRISPR/Cas13a, b systems could down-regulate Ku70 and Lig4 proteins level to 68% and 53%, respectively. The study demonstrates that the CRISPR/Cas13 system could effectively knockdown the expression of RNA and protein in HEK293T cells, providing a new strategy for gene function and regulation research.
Subject(s)
Humans , CRISPR-Cas Systems , DNA Ligase ATP , Genetics , Gene Expression Regulation , Genetics , Gene Knockdown Techniques , HEK293 Cells , Ku Autoantigen , GeneticsABSTRACT
Objective To investigate expression change of Ku70 protein before and after radiotherapy in patients with cervical cancer, as well as its relationship with clinical characteristics. Methods Seventy cervical cancer patients who received three dimensional conformal radiotherapy combined with chemotherapy were registered. Specimens tissues were obtained from cervical tumors of irradiation dose of ≤10 Gy and ≥20 Gy group. Immunohistochemistry was used to detect the expression of Ku70 in 70 cases, and its relative expression was analyzed by image software. Results The relative expression levels of Ku70 in cervical cancer patients treated with radiation doses of ≥20 Gy group (1.31 ± 0.83) and ≤10 Gy group (0.71 ± 0.60) were significantly higher than that before radiotherapy (0.50 ± 0.19), and there was a significant difference (t=7.143, P 0.05). The expression of Ku70 in radiotherapy resistant group was significantly higher than that in radiotherapy sensitive group (0.55 ± 0.24 vs. 0.25 ± 0.17, t= 2.979, P< 0.01). Conclusions The expression of Ku70 protein in cervical cancer tissues is increased with the rise of radiotherapy dose. The expression of Ku70 in radiotherapy resistant group is significantly higher than that in radiotherapy sensitive group, indicating that Ku70 protein is correlated with the radiosensitivity, clinical stage, differential degree and tumor diameter.
ABSTRACT
Aim To explore the mechanism of insulin in combination with selenium preventing myocardial apoptosis in diabetic cardiomyopathy rats .Methods SD rats ( n =50 ) were randomly divided into five groups: control , diabetic cardiomyopathy ( DCM ) , DCM with insulin treatment , DCM with selenium treat-ment, and DCM with insulin and selenium combination treatment .The cell apoptosis was observed by TUNEL . The levels of Bcl-2, caspase-3, PARP, Cbl-b and p38 MAPK were examined by Western blot .The inter-actions of Cbl-b-p38 MAPK and Ku70-Bax were detec-ted by immunoprecipitation .Results Insulin in com-bination with selenium synergistically inhibited apopto-sis, up-regulated Cbl-b, down-regulated p38MAPK ex-pressions and increased the interactions of Cbl-b-p38MAPK and Ku70-Bax.Conclusion Insulin and selenium synergistically inhibit myocardial apoptosis by regulating Cbl-b-inhibited p38 MAPK and preventing Bax translocation .
ABSTRACT
AIM:To investigate the effect of insulin and selenium in combination on the apoptosis and the ex-pression of Ku70, acetylated Ku70, Bax and cytochrome C in myocardial cells of the rats with diabetic cardiomyopathy (DCM), and to explore the mechanism of insulin and selenium in their synergistic anti-DCM effect.METHODS:SD rats (n=50) were randomly grouped into control, DCM, DCM with insulin treatment (DCM+In) group, DCM with selenium treatment (DCM+Se) group, and DCM with insulin and selenium combination treatment (DCM+In+Se) group.Mito-chondrial membrane potential ( MMP) was measured by flow cytometry .The cell apoptosis was observed by terminal-deoxy-nucleotidyl transferase mediated nick end labeling (TUNEL).The levels of Ku70, Bax and cytochrome C were examined by Western blot .The acetylation status of Ku 70 was detected by co-immunoprecipitation .RESULTS: The rats in DCM group showed marked cell apoptosis compared with the control rats .The levels of Ku70 and acetylated Ku70 declined sig-nificantly compared with control group .Bax significantly translocated from cytoplasm into mitochondria and cytochrome C translocated from mitochondria into cytoplasm compared with control group .Compared with DCM +In group or DCM +Se group, insulin and selenium in combination significantly inhibited the apoptosis , down-regulated Ku70 and acetylated Ku70 levels, and prevented Bax and cytochrome C translocation .CONCLUSION: Insulin and selenium synergistically inhibits myocardial apoptosis by regulating Ku 70 acetylation and inhibiting Bax translocation .
ABSTRACT
ABSTRACT:Objective To investigate the roles of Ku70,epidermal growth factor receptor (EGFR),nuclear factor-kappa B (NF-κB)and Bcl-2 genes in the pathogenesis of Graves’disease.Methods Fine needle biopsies of 59 patients [M:F 1 6:43;mean age (44.66 ± 12.94)y)]diagnosed as having Graves’disease and 27 [M:F 6:21;mean age (44.64± 14.27)y]control normal tissues were collected.The mRNA expressions of EGFR,Ku70,NF-κB,and Bcl-2 were detected by Real-time polymerase chain reaction (RT-PCR).The Student’s t test was performed to analyze differences between patients with Graves’disease and control subjects.Pearson correlation analysis was applied to detect the relationship between gene mRNA expression and serological indexes.Results Compared with those of control subjects,the mRNA expression levels of EGFR (0.859 ±0.125 vs .1.752 ±0.660,P 0.05 ). Conclusion The signal transduction pathways mediated by Ku70,EGFR,NF-κB and Bcl-2 may participate in the occurrence and development of Graves’disease.
ABSTRACT
Objective To determine the effects of Sodium Nitroprusside ( SNP) , superoxide dismutase ( SOD) and catalase ( CAT ) on the expression of catalytic subunit of the DNA-dependent protein kinase ( DNA-PKcs ) and Ku70/80 heterodimer in cardiomycyte H9C2, as well as their expression in the myocardial tissues of SD rats . Methods H9C2 cells were co-cultured with SNP at concentreations of 10, 20 and 40 mmol/L for 6 hours, SD rats were injected with normal saline , SNP, SNP+SOD, SNP+CAT or SNP+SOD+CAT.Western blot and immuno-histochemistry assay were used to examine DNA-PKcs and Ku70/80 protein expression respectively .Results The expression of DNA-PKcs and Ku70/80 increased in H9C2 cells co-cultured with SNP when compared with control group, but they were be decreased when treated with SOD or/and CAT.The expression of DNA-Pkcs and Ku70/80 in myocardial tissues of experimental groups were higher than the control .Conclusions Radical scavengers may play a role as a protective effect for sodium nitroprusside related injury in cardiac myocytes .
ABSTRACT
Objeceive To compare the expression of Ku70 and Bax in hippocampus CA1 between normal rats and diabetic rats following cerebral ischemia/reperfusion, so as to explore the roles of Ku70 and Bax in aggravating cerebral ischemial reperfusion mjury nn diabetes. Meehods Totally 72 male SD rats were divided into 3 groups randomly: sham operated (SO) group, normoglycemia global cerebral ischemia/reperfusion (NCI) group, and diabetic global cerebral ischemia/reperfusion (DCI) group. The rats in DCI group were treated by streptozodn (STZ) and improved Puliinelii's foue-vessel occlusion method to estabiish diabetic global cerebral ischemia/reperfusion modll. Andanimals in NCI group were not gvven STZ, and other treatments were iimllar to those in the DCI group. Animals in the SO group only had blood vessels exposed. Changes of neuron pathology in hippocampus CA1 were observed by H-E stammg under iight microscopy at 1, 6, 24, and 48 h after lschemial reperfusion; expresioon of Ku70 was detected by immunohistochemistry and Western blotting analyils, and expresioon of Bax was detected by immunohistochemistry. Resules The neuron structure in hippocampus CA1 of rats was damaged nnNCI group, and the density of survival neuronswas significanely lower than that nn the SO group at all studied time ponnts(P<0. 05); the neuron structure damage nn DCI group was more severe, and the demity of sunival neurons was signiticanely lower than that of the NCI group at all studied time points(P<0. 05). The expresioon of Ku70 at 1 and 6 h nn NCI group was signiticanely higher than that in the SO group (P<0. 05), and that at 24 and 48 h was signiticanely lower than that nn the SO group(P<0. 05); Ku70 expression nn DCI group was significanely lower than that nn the NCI group at all studied time ponnts(P<0. 05). Bax expresioon in NCI group was signiticanely higher than that in the SO group at all studied time points(P<0. 05), and that in DCI group was signiticanely higher than that in the NCI group at all studied time ponnts (P<0. 05). Conclusion Diabetes can aggravate global cerebral ischemia/reperfusion injury, which may be related to Ku70 Expression decrease and Bax Expression Increase.
ABSTRACT
Objective To investigate the effect of C225 on DNA repair and molecular pathways in CL187 colorectal cancer cells after irradiated by 125Ⅰ radioactive seeds.Methods In the experiment involved were four groups:control group,100 nmol/L C225 treatment group,125Ⅰ radioactive seeds continuous low-dose rate irradiation group and C225 combined with 125Ⅰ radioactive seeds continuous lowdose rate irradiation group.Cells were collected at 48 h after 4 Gy irradiation,and γH2AX foci/cell and γH2AX foci positive cells were counted with immunofluorescence.At the same time,DNA repair proteins were detected by Western blot.Cells were lyzed immediately after 4 Gy irradiation,and changs in EGFR downstream signaling molecules were detected by Western blot.Results Compared with 125Ⅰ seeds irradiated cells,cells treated with C225 and 125Ⅰ seeds irradiation showed more γH2AX foci per cell (t =8.0,P =0.05),and more γH2AX foci positive cells (t =6.8,P ＜ 0.05) and less expression of Ku70 (t =6.6,P ＜ 0.05) and DNA-PKcs (t =5.6,P ＜ 0.05).Combined with 125Ⅰ-CLDR irradiation,C225 reduced cellular EGFR level(t =4.9,P ＜0.05) and inhibited the activation of Akt(t =5.5,P ＜0.05).Conclusions In the condition of 125Ⅰ seeds irradiation,C225 reduced the expression of Ku70 and DNA-PKcs,inhibited the activation of Akt and attenuated the DNA damage repair capacity in CL187 colorectal cancer cells.
ABSTRACT
The present study investigated the enhanced radiosensitivity of U-251 cells induced by sodium butyrate (NaB) and its possible mechanisms.Increased radiosensitivity of U251 cells was examined by clonogenic cell survival assays.The expression of Ku70 mRNA and protein was detected by using RT-PCR and Western blotting respectively.γ-H2AX foci were measured at different time points after ionizing irradiation alone or combined with NaB treatment.The results showed that cell survival rate was significantly reduced,both D0 and Dq values were decreased (D0:1.43 Gy vs.1.76 Gy; Dq:1.22 Gy vs.2.05 Gy) after the combined treatment as compared with irradiation alone,and sensitivity enhancing ratio (SER) reached 1.23.The average number ofγ-H2AX foci per cell receiving the combined treatment was significantly increased at different time points,and the expression levels of Ku70mRNA and protein were suppressed by NaB in a dose-dependent manner.It was concluded that enhanced radiosensitivity induced by NaB involves an inhibited expression of Ku70 and an increase in γ-H2AX foci,which suggests decreased ability in DSB repair.
ABSTRACT
Objective: To investigate the therapeutic effects of radioisotope labeled Ku70 antisense oligodeoxynuclecotide (ASODNs) on thyroid carcinoma implanted in nude mice and the related mechanism. Methods: The Ku70 ASODNs labeled with 131I was used to treat the tumor-bearing mouse model derived from thyroid carcinoma TT cells. The tumor-forming rate, mortality rate and tumor growth were observed and calculated after treatment with 131I-ASODNs, 131I-Na, ASODNs or NS (normal saline). Annexin V/PI assay was used to examine the apoptosis of tumor cells by flow cytometry. Western blotting analysis was performed to determine the protein expressions of Ku70, proliferating cell nuclear antigen (PCNA, a cell proliferation marker) and Bcl-2(a cell apoptosis marker). Results: After treatment with 131I-ASODNs, the Ku70 protein level was down-regulated in the tumor tissues and the growth of tumor was inhibited. The tumor volume, tumor-forming rate and mortality rate were significantly decreased in 131I-ASODNs group than in the NS control group (P<0.01). The tumor volume of 131I-ASODNs group was also significantly smaller than that in the 131I-Na group (P<0.05); the apoptosis rate of 131I- ASODNs group (35.6%) was significantly higher than that of the ASODNs group (10.4%), NS group (9.2%) and 131I-Na group (26.6%) (P<0.05). Further investigation found that PCNA and Bcl-2 protein levels in 131I-ASODNs group were lower than those in NS and ASODNs groups. Conclusion: The Ku70 131I-ASODNs can effectively inhibit the growth of thyroid carcinoma and promote apoptosis of TT tumor cells, which might be related to the down-regulation of Ku70 and the changes of PCNA and Bcl-2 signal pathway.
ABSTRACT
Objective: To study the effect of β-elemene combined with radiotherapy on the expression of KU70 mRNA and apoptosis of lung adenocarcinoma cell line A549. Methods: A549 cells were divided into the control group (c), irridation group (IR), β-elemene group (0.1×IC50 and 0.2×IC50 I.e. 10 μg/mL and 20pg/mL β-elemene) and β-elemene combined with irridation group (0.1×IC50 + IR and 0.2×IC50 + IR). IC50 was obtained through MTT method and cell survival rate was analyzed by colony formation test. Cell cycle and apoptosis were detected with flow cytometry. The expression level of KU70 mRNA was detected by RT-PCR. Results: MTT assay showed that IC50 value of A549 cells was 120 μg/mL. The number of cell clones and survival rate of β-elemene groups were declined significantly. The radiosensitivity of A549 cells can be enhanced by β-elemene. The flow cytometry confirmed that the ratio of G_2/M and apoptosis were significantly in-creased under the effect of β-elemene, statistically different from those in the control group (P <0.05). The expression level of KU70 mRNA in β-elemene with radiotherapy group was declined significantly, statistically different from those in the con-trol group (P<0.05). Cendusion: The rediosensitization effect of β-elemene on A549 cells is associated with induction of cell apoptosis and down-regulation of KU70 mRNA expression.
ABSTRACT
Objective:To investigate the effect of Ku70 silencing on the expression of apoptosis-related genes in human NSLC cell line,A549 and drug-resistant A549DDP.Methods:Expression of Ku70mRNA in human A549 cells was examined by RT-PCR.Expression of apoptosis-related genes was examined by PCR-Array assay.Results: The Ku70mRNA was higher in A549DDP cells than in A549 cells.The expression of BAX,TP53 and TP73 was significantly increased whereas the expression of TNFRSF1A and BFAR decreased in siRNA-Ku70 A549DDP cells when compared to those of A549DDP cells.Conclsion:The amount of Ku70 mRNA is higher in A549DDP cells,suggesting that Ku70 is important for drug-resistance; Silencing Ku70 might reverse the drug-resistance in A549DDP cells through regulation of expression of apoptofic genes.
ABSTRACT
The gradual loss of telomeric DNA can contribute to replicative senescence and thus, having longer telomeric DNA is generally considered to provide a longer lifespan. Maintenance and stabilization of telomeric DNA is assisted by binding of multiple DNA-binding proteins, including those involved in double strand break (DSB) repair. We reasoned that declining DSB repair capacity and increased telomere shortening in aged individuals may be associated with decreased expression of DSB repair proteins capable of telomere binding. Our data presented here show that among the DSB repair proteins tested, only the expression of Ku70 and Mre11 showed statistically significant age-dependent changes in human lymphocytes. Furthermore, we found that expressions of Ku70 and Mre11 are statistically correlated, which indicate that the function of Ku70 and Mre11 may be related. All the other DSB repair proteins tested, Sir2, TRF1 and Ku80, did not show any significant differences upon aging. In line with these data, people who live in the regional community (longevity group), which was found to have statistically longer average life span than the rest area, shows higher level of Ku70 expression than those living in the neighboring control community. Taken together, our data show, for the first time, that Ku70 and Mre11 may represent new biomarkers for aging and further suggest that maintenance of higher expression of Ku70 and Mre11 may be responsible for keeping longer life span observed in the longevity group.
Subject(s)
Middle Aged , Humans , Aged, 80 and over , Aged , Adult , Telomere/genetics , Longevity , DNA-Binding Proteins/metabolism , DNA Repair/genetics , DNA/genetics , Cellular Senescence/physiology , CD4-Positive T-Lymphocytes/metabolism , Antigens, Nuclear/metabolism , Aging/physiologyABSTRACT
PURPOSE: The objective of this study was to determine whether the expressions of the two components of DNA-dependent protein kinase, Ku70 and DNA-PKcs, influence the response to radiotherapy (RT) and outcome of treatment of non-disseminated nasopharyngeal carcinoma (NPC) in patients who received definitive RT. MATERIASL AND METHODS: Sixty-six patients with NPC who were treated with radiotherapy alone or with concurrent chemotherapy between June 1995 and December 2001 were divided into groups based on the levels of immunoreactivity for Ku70 and DNA-PKcs in pretreatment biopsy specimens. The over-expression of Ku70 or DNA-PKcs groups included patients whose biopsy specimens showed at least 50% immunopositive tumor cells; patients in which less than 50% of the tumor cells in the biopsy tissues were immunopositive were placed in the low Ku70 and DNA-PKcs groups. The immunoreactivities for Ku70 and DNA-PKcs were retrospectively compared with the sensitivity of the tumor to radiation and the patterns of therapy failure. Univariate analyses were performed to determine the prognostic factors that influenced locoregional control of NPC. RESULTS: The five-year locoregional control rate was significantly higher in the low Ku70 group (Ku(-)) (85%) than in the high Ku70 group (Ku(+)) (42%) (p=0.0042). However, there were no differences in the metastases-free survival rates between the two groups (Ku70 (+), 82%; Ku70 (-), 78%; p=0.8672). Univariate analysis indicated that the over-expression of Ku70 surpassed other well-known predictive clinocopathologic parameters as an independent prognostic factor for locoregional control. Eighteen of 22 patients who had locoregional recurrences of the tumor displayed an over-expression of Ku70. No significant association was found between the level of DNA-PKcs expression and the clinical outcome. CONCLUSION: Our data suggest that the level of Ku70 expression can be used as a molecular marker to predict the response to RT and the locoregional control after RT and concurrent chemotherapy in patients with non-disseminated NPC.
Subject(s)
Humans , Biopsy , DNA-Activated Protein Kinase , Drug Therapy , Immunohistochemistry , Radiotherapy , Recurrence , Retrospective Studies , Survival RateABSTRACT
PURPOSE: DNA-dependent protein kinase (DNA-PK) is a serine/threonine kinase consisting of a 470 kDa catalytic subunit (DNA-PKcs) and a heterodimeric regulatory complex, called Ku, which is composed of 70 kDa (Ku 70) and 86 kDa (Ku 80) proteins. The DNA-PK has been shown to play a pivotal role in rejoining DNA double-strand-breaks (dsb) in mammalian cells. The purpose of this study is to examine the relationship between the level of Ku expression and radiation sensitivity. METHODS AND MATERIALS: Nine head and neck, cancer cell lines showed various intrinsic radiation sensitivities. Among the nine, AMC-HN-3 cell was the most sensitive for X-ray irradiation and AMC-HN-9 cell was the most resistance. The most sensitive and resistant cell lines were selected and the test sensitivity of radiation and expression of Ku were measured. Radiation sensitivity was obtained by colony forming assay and Ku protein expression using Western blot analysis. RESULTS: Ku80 increased expression by radiation, wheres Ku70 did not. Overexpression of Ku80 protein increased radiation resistance in AMC-HN9 cell line. There was a correlation between Ku80 expression and radiation resistance. Ku80 was shown to play an important role in radiation damage response. CONCLUSION: Induction of Ku80 expression had an important role in DNA damage repair by radiation. Ku80 expression may be an effective predictive assay of radiosensitivity on head and neck cancer.
Subject(s)
Blotting, Western , Catalytic Domain , Cell Line , DNA , DNA Damage , DNA-Activated Protein Kinase , Head and Neck Neoplasms , Head , Neck , Phosphotransferases , Radiation ToleranceABSTRACT
Recent studies indicated that cancer cells become resistant to ionizing radiation (IR) and chemotherapy drugs by enhanced DNA repair of the lesions. Therefore, it is expected to increase the killing of cancer cells and reduce drug resistance by inhibiting DNA repair pathways that tumor cells rely on to escape chemotherapy. There are a number of key human DNA repair pathways which depend on multimeric polypeptide activities. For example, Ku heterodimer regulatory DNA binding subunits (Ku70/Ku80) on binding to double strand DNA breaks (DSBs) are able to interact with 470-kDa DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and are essential for DNA-dependent protein kinase (DNA-PK) activity. It has been known that DNA-PK is an important factor for DNA repair and also is a sensor-transmitting damage signal to downstream targets, leading to cell cycles arrest. Our ultimate goal is to develop a treatment of breast tumors by targeting proteins involved in damage-signaling pathway and/or DNA repair. This would greatly facilitate tumor cell cytotoxic activity and programmed cell death through DNA damaging drug treatment. Therefore, we designed a domain of Ku80 mutants that binds to Ku70 but not DNA end binding activity and used the peptide in co-therapy strategy to see whether the targeted inhibition of DNA-PK activity sensitized breast cancer cells to irradiation or chemotherapy drug. We observed that the synthesized peptide (HNI-38) prevented DNA-PKcs from binding to Ku70/Ku80, thus resulting in inactivation of DNA-PK activity. Consequently, the peptide treated cells exhibited poor to no DNA repair, and became highly sensitive to IR or chemotherapy drugs, and the growth of breast cancer cells was inhibited. Additionally, the results obtained in the present study also support the physiological role of resistance of cancer cells to IR or chemotherapy.
Subject(s)
Humans , Breast Neoplasms , Catalytic Domain , Cell Cycle , Cell Death , DNA , DNA Breaks, Double-Stranded , DNA Repair , DNA-Activated Protein Kinase , Drug Resistance , Drug Therapy , Homicide , Radiation, Ionizing , United NationsABSTRACT
Objective:To investigate the relationship between Ku70 mRNA expression in non-neoplasm,in normal appearing pulmonary tissues,and in lung cancer tissue of pre-or post-chemotherapy and drug resistance.Methods:Ku70 mRNA in the lung tissues was measured by reverse transcription polymerase chain reaction (RT-PCR).The samples were extracted form 26 non-neoplasm, normal appearing pulmonary tissues and 56 lung cancer tissues.Results:Non-neoplasm, normal appearing pulmonary tissue group expressed Ku70 mRNA lower than lung cancer tissue group(P