Objective To investigate the fate and underlying mechanisms of G2 phase arrest in cancer cells elicited by ionizing radiation(IR).Methods Human melanoma A375 and 92-1 cells were treated with X-rays radiation or Aurora...Objective To investigate the fate and underlying mechanisms of G2 phase arrest in cancer cells elicited by ionizing radiation(IR).Methods Human melanoma A375 and 92-1 cells were treated with X-rays radiation or Aurora A inhibitor MLN8237(MLN)and/or p21 depletion by small interfering RNA(si RNA).Cell cycle distribution was determined using flow cytometry and a fluorescent ubiquitin-based cell cycle indicator(FUCCI)system combined with histone H3 phosphorylation at Ser10(p S10 H3)detection.Senescence was assessed using senescence-associated-β-galactosidase(SA-β-Gal),Ki67,andγH2AX staining.Protein expression levels were determined using western blotting.Results Tumor cells suffered severe DNA damage and underwent G2 arrest after IR treatment.The damaged cells did not successfully enter M phase nor were they stably blocked at G2 phase but underwent mitotic skipping and entered G1 phase as tetraploid cells,ultimately leading to senescence in G1.During this process,the p53/p21 pathway is hyperactivated.Accompanying p21 accumulation,Aurora A kinase levels declined sharply.MLN treatment confirmed that Aurora A kinase activity is essential for mitosis skipping and senescence induction.Conclusion Persistent p21 activation during IR-induced G2 phase blockade drives Aurora A kinase degradation,leading to senescence via mitotic skipping.展开更多
Objective To better understand the pathological causes of bone loss in a space environment, including microgravity, ionizing radiation, and ultradian rhythms.Methods Sprague Dawley （SD） rats were randomly divided in...Objective To better understand the pathological causes of bone loss in a space environment, including microgravity, ionizing radiation, and ultradian rhythms.Methods Sprague Dawley （SD） rats were randomly divided into a baseline group, a control group, a hindlimb suspension group, a radiation group, a ultradian rhythms group and a combined-three-factor group. After four weeks of hindlimb suspension followed by X-ray exposure and/or ultradian rhythms, biomechanical properties, bone mineral density, histological analysis, microstructure parameters, and bone turnover markers were detected to evaluate bone loss in hindlimbs of rats.Results Simulated microgravity or combined-three factors treatment led to a significant decrease in the biomechanical properties of bones, reduction in bone mineral density, and deterioration of trabecular parameters. Ionizing radiation exposure also showed adverse impact while ultradian rhythms had no significant effect on these outcomes. Decrease in the concentration of the turnover markers bone alkaline phosphatase （bALP）, osteocalcin （OCN）, and tartrate-resistant acid phosphatase-5b （TRAP-Sb） in serum was in line with the changes in trabecular parameters.Conclusion Simulated microgravity is the main contributor of bone loss. Radiation also results in deleterious effects but ultradian rhythms has no significant effect. Combined-three factors treatment do not exacerbate bone loss when compared to simulated microgravity treatment alone.展开更多
Objective miR-663 a has been reported to be downregulated by X-ray irradiation and participates in radiation-induced bystander effect via TGF-β1.The goal of this study was to explore the role of mi R-663 a during rad...Objective miR-663 a has been reported to be downregulated by X-ray irradiation and participates in radiation-induced bystander effect via TGF-β1.The goal of this study was to explore the role of mi R-663 a during radiation-induced Epithelium-to-mesenchymal transition(EMT).Methods TGF-β1 or IR was used to induce EMT.After mi R-663 a transfection,cell migration and cell morphological changes were detected and the expression levels of mi R-663 a,TGF-β1,and EMT-related factors were quantified.Results Enhancement of cell migration and promotion of mesenchymal changes induced by either TGF-β1 or radiation were suppressed by mi R-663 a.Furthermore,both X-ray and carbon ion irradiation resulted in the upregulation of TGF-β1 and downregulation of mi R-663 a,while the silencing of TGF-β1 by mi R-663 a reversed the EMT process after radiation.Conclusion Our findings demonstrate an EMT-suppressing effect by mi R-663 a via TGF-β1 in radiationinduced EMT.展开更多
目的原核表达及纯化带GST标签的人类乳头状瘤病毒E6相关蛋白E6AP,为研究泛素化修饰与多种疾病的机制关联提供实验基础。方法以人卵巢文库为模板,利用PCR技术扩增E6AP编码序列,将其插入pGEX KG GST载体中。利用DH5α感受态细胞鉴定重组...目的原核表达及纯化带GST标签的人类乳头状瘤病毒E6相关蛋白E6AP,为研究泛素化修饰与多种疾病的机制关联提供实验基础。方法以人卵巢文库为模板,利用PCR技术扩增E6AP编码序列,将其插入pGEX KG GST载体中。利用DH5α感受态细胞鉴定重组质粒并大量克隆。质粒转入大肠杆菌Rossate菌株,小量诱导表达。利用GST融合蛋白纯化磁珠提取并纯化GST E6AP融合蛋白,SDS PAGE电泳和Western印迹检测纯化效率。结果PCR技术成功获取大小约为2559 bp的E6AP编码序列,与pGEX KG GST载体连接,双酶切鉴定及公司测序结果显示GST E6AP载体构建成功。转入Rossate菌株后诱导质粒小量表达,获取并提纯带有GST标签的E6AP重组蛋白。SDS PAGE电泳和Western印迹结果显示E6AP蛋白纯化成功。结论成功构建重组质粒并表达GST E6AP蛋白,获取纯化E6AP蛋白,为进一步研究泛素化在疾病发生发展中的地位和作用奠定坚实基础。展开更多
基金supported by the Science and Technology Research Project of Gansu Province[20JR5RA555 and145RTSA012]the Natural Science Foundation of Shaanxi Province[2020JQ-541]+1 种基金the National Natural Science Foundation of China[31870851 and 12175289]the Youth Innovation Promotion Association CAS[2021415]
文摘Objective To investigate the fate and underlying mechanisms of G2 phase arrest in cancer cells elicited by ionizing radiation(IR).Methods Human melanoma A375 and 92-1 cells were treated with X-rays radiation or Aurora A inhibitor MLN8237(MLN)and/or p21 depletion by small interfering RNA(si RNA).Cell cycle distribution was determined using flow cytometry and a fluorescent ubiquitin-based cell cycle indicator(FUCCI)system combined with histone H3 phosphorylation at Ser10(p S10 H3)detection.Senescence was assessed using senescence-associated-β-galactosidase(SA-β-Gal),Ki67,andγH2AX staining.Protein expression levels were determined using western blotting.Results Tumor cells suffered severe DNA damage and underwent G2 arrest after IR treatment.The damaged cells did not successfully enter M phase nor were they stably blocked at G2 phase but underwent mitotic skipping and entered G1 phase as tetraploid cells,ultimately leading to senescence in G1.During this process,the p53/p21 pathway is hyperactivated.Accompanying p21 accumulation,Aurora A kinase levels declined sharply.MLN treatment confirmed that Aurora A kinase activity is essential for mitosis skipping and senescence induction.Conclusion Persistent p21 activation during IR-induced G2 phase blockade drives Aurora A kinase degradation,leading to senescence via mitotic skipping.
基金supported by the International Science&Technology Cooperation Program of China[No.2015DFR30940]the Science and Technology Research Project of Gansu Province[No.145RTSA012 and No.17JR5RA307]
文摘Objective To better understand the pathological causes of bone loss in a space environment, including microgravity, ionizing radiation, and ultradian rhythms.Methods Sprague Dawley （SD） rats were randomly divided into a baseline group, a control group, a hindlimb suspension group, a radiation group, a ultradian rhythms group and a combined-three-factor group. After four weeks of hindlimb suspension followed by X-ray exposure and/or ultradian rhythms, biomechanical properties, bone mineral density, histological analysis, microstructure parameters, and bone turnover markers were detected to evaluate bone loss in hindlimbs of rats.Results Simulated microgravity or combined-three factors treatment led to a significant decrease in the biomechanical properties of bones, reduction in bone mineral density, and deterioration of trabecular parameters. Ionizing radiation exposure also showed adverse impact while ultradian rhythms had no significant effect on these outcomes. Decrease in the concentration of the turnover markers bone alkaline phosphatase （bALP）, osteocalcin （OCN）, and tartrate-resistant acid phosphatase-5b （TRAP-Sb） in serum was in line with the changes in trabecular parameters.Conclusion Simulated microgravity is the main contributor of bone loss. Radiation also results in deleterious effects but ultradian rhythms has no significant effect. Combined-three factors treatment do not exacerbate bone loss when compared to simulated microgravity treatment alone.
基金funded by the National Natural Science Foundation of China[no.U1932208,YANG Kai]the Science and Technology Research Project of Gansu Province[no.17JR5RA307 and 145RTSA012,WANG Ju Fang]the Science and Technology Research Project of Gansu Province[no.21JR7RA108,DING Nan]
文摘Objective miR-663 a has been reported to be downregulated by X-ray irradiation and participates in radiation-induced bystander effect via TGF-β1.The goal of this study was to explore the role of mi R-663 a during radiation-induced Epithelium-to-mesenchymal transition(EMT).Methods TGF-β1 or IR was used to induce EMT.After mi R-663 a transfection,cell migration and cell morphological changes were detected and the expression levels of mi R-663 a,TGF-β1,and EMT-related factors were quantified.Results Enhancement of cell migration and promotion of mesenchymal changes induced by either TGF-β1 or radiation were suppressed by mi R-663 a.Furthermore,both X-ray and carbon ion irradiation resulted in the upregulation of TGF-β1 and downregulation of mi R-663 a,while the silencing of TGF-β1 by mi R-663 a reversed the EMT process after radiation.Conclusion Our findings demonstrate an EMT-suppressing effect by mi R-663 a via TGF-β1 in radiationinduced EMT.
