Radiotherapy is commonly used to treat many cancers, and their sensitivity to radiation is crucial for favorable outcomes. This study investigated whether the immediate early response 5 (IER5) protein affects Cdc25B protein expression in HeLa cells after gamma irradiation. IER5 was knocked down using RNA interference (RNAi) in HeLa cells irradiated with 2 or 4 Gy of gamma rays. The mRNA and protein expression levels were subsequently determined via qRT–PCR and western blotting. The distribution of cells during the cell cycle was also determined using flow cytometry. IER5 protein levels were successfully reduced with RNAi. Variations in IER5 levels led to differences in Cdc25B mRNA and protein levels. Moreover, IER5 affected the proportion of cells in the G2 phase, which is regulated mainly by Cdc25B. Pearson correlation analysis was conducted on the expression levels of IER5 and Cdc25B in HeLa cells and the IER5‐silenced HeLa (siIER5‐HeLa) cell line at various time points after exposure to 4 Gy of gamma rays, and a negative correlation was detected between IER5 and Cdc25B expression levels, with correlation coefficients of −0.686 and −0.663, respectively. Additionally, variations in IER5 levels led to differences in the expression levels of p53, NF‐YB, and p300, which may be putative transcriptional regulators of Cdc25B. These results suggest that IER5 plays a negative role in regulating Cdc25B expression, which may involve interactions with the transcriptional regulators p53, NF‐YB, and p300.

Introduction:Traditional methods for determining radiation dose in nuclear medicine include the Monte Carlo method, the discrete ordinate method, and the point kernel integration method. This study presents a new mathematical model for predicting the radiation dose rate in the vicinity of nuclear medicine patients. Methods:A new algorithm was created by combining the physical model of "cylinder superposition" of the human body with integral analysis to assess the radiation dose rate in the vicinity of nuclear medicine patients. Results:The model accurately predicted radiation dose rates within distances of 0.1-3.0 m, with a deviation of less than 11% compared to observed rates. The model demonstrated greater accuracy at shorter distances from the radiation source, with a deviation of only 1.55% from observed values at 0.1 m. Discussion:The model proposed in this study effectively represents the spatial and temporal distribution of the radiation field around nuclear medicine patients and demonstrates good agreement with actual measurements. This model has the potential to serve as a radiation dose rate alert system in hospital environments.

Cervical cancer (CC) is a type of pelvic malignant tumor that severely threatens women's health. Current evidence suggests that IER5, as a potential radiosensitizer, promotes irradiation-induced apoptosis in CC tissues in patients undergoing chemoradiotherapy. IER5 has been shown to be involved in the G2/M-phase transition. In the present study, we used Cdc25B as the breakthrough point to explore the underlying mechanism of IER5 in the cell cycle regulation of radiation-damaged HeLa cells. IER5 was evidently upregulated after irradiation, but Cdc25B was significantly downregulated. In monoclonal IER5-silenced HeLa cells, irradiation-induced downregulation of Cdc25B was attenuated. The effect of irradiation on Cdc25B promoter activity was determined by dual-luciferase reporter assays. The response elements on the Cdc25B promoter related to irradiation were predicted by JASPAR. These conserved sequences were mutated individually or in combination by splicing-by-overlap extension PCR, and their function was confirmed by dual-luciferase reporter assays. The enrichment efficiency of transcription factors after irradiation was determined by chromatin immunoprecipitation (ChIP) assay. Both Sp1/Sp3 and NF-YB binding sites were involved in irradiation-mediated regulation of Cdc25B. IER5 was involved in irradiation-mediated regulation of Cdc25B through the NF-YB binding site. Furthermore, ChIP assays showed that IER5 bound to the Cdc25B promoter, and the binding of IER5 to the Cdc25B promoter region in irradiation-induced HeLa cells induced the release of the coactivator p300 through interaction with NF-YB. Taken together, these findings indicate that IER5 is the transcriptional repressor that accelerates the downregulation of Cdc25B expression after irradiation.

Radiation resistance is the most common challenge for improving radiotherapy. The mechanisms underlying the development of radioresistance remain poorly understood. This study aims to explore the role of LINC00460 in ionizing radiation-induced radioresistance as well as the mechanisms by which LINC00460 is regulated by radiation exposure. The expression of LINC00460 was measured. Cell proliferation and colony formation were measured in HCT116 cells after treatment by radiation. The development of epithelial-mesenchymal transition (EMT) was determined with or without knockdown LINC00460 expression using western blot analysis. Transcription activity was determined using a series of LINC00460-promoter luciferase reporter gene vectors. LINC00460 expression was significantly higher in HCT116 cells, relative to other cell types, with LINC00460 expression significantly affecting HCT116 cell proliferation. Suppression of LINC00460 inhibits EMT development in HCT116 cells via regulation of ZEB1 expression. Furthermore, LINC00460 expression was induced by irradiation via the activation of c-jun transcription factor-binding element located on the LINC00460 promoter. LINC00460 was shown to play a crucial role in EMT-associated progression of colorectal cancer, indicating that LINC00460 may be an indicator or new potential therapeutic target for colorectal cancer radiosensitization.

目的:利用昆虫杆状病毒表达系统表达早期快速反应基因5(IER5)蛋白,为后续蛋白质结晶及探索蛋白质三级结构提供线索.方法:以HeLa细胞cDNA为模板扩增出IER5基因片段,构建重组转移质粒pFastBac1-IER5;重组转移质粒经双酶切及测序鉴定后转化至感受态细胞DH10Bac,以获得重组的穿梭质粒rBacmid-IER5;将重组穿梭质粒感染Sf9细胞,待细胞出现明显病变时收集重组杆状病毒;用间接免疫荧光、SDS-PAGE及Western blot以及对表达产物进行分析鉴定.结果:重组转移质粒pFastBac1-IER5经双酶切后得到与预期相同的2条条带;重组穿梭质粒rBacmid-IER5经PCR鉴定在3300 bp左右出现一条特异性条带;间接免疫荧光结果提示IER5蛋白在Sf9细胞中得到表达;SDS-PAGE结果显示,表达产物的相对分子质量约为48 k,Western blot结果表明表达产物能与IER5抗体特异性结合.结论:利用昆虫-杆状病毒表达系统成功表达了人IER5蛋白,获得了体外表达重组IER5蛋白的相对分子质量、溶解性等物理化学特性.

X射线晶体衍射技术是一种非常重要的测定蛋白质晶体结构的方法.近几十年,虽然各种新的实验方法和通过计算机预测蛋白质结构域的方法层出不穷,但是测定蛋白质结构主要还是通过X射线晶体衍射技术.随着同步辐射装置和X射线自由电子激光的发展,X射线在测定蛋白质结构中的作用越来越重要.本文就X射线测定蛋白质晶体空间结构的技术进展进行综述,介绍了其原理和发展,简述了同步辐射和X射线自由电子激光的研究现状,为理解用X射线测定蛋白质结构的机制,从原子水平上了解生命物质奠定基础,并提出今后X射线光源的发展趋势.

早反应基因5(IER5)属于早期反应基因,因其在多种肿瘤细胞中均发挥着抑制肿瘤细胞增殖的作用而受到广泛关注.在受到外界刺激(热压力、血清、电离辐射等)时,肿瘤细胞内的IER5会过表达从而使细胞作出一系列应激性反应如G2期周期阻滞和凋亡等.在临床放化疗中IER5的表达量可以作为一种潜在的肿瘤放化疗后诱导细胞凋亡的生物标记物,并和肿瘤组织大小相关,对今后放化疗方案的改进具有重要的指导意义.

目的 构建人细胞分裂周期25家族蛋白B(Cdc25B)基因启动子(promoter)荧光素酶报告基因载体pGL3-Cdc25B-promoter,并检测其转录活性.方法 应用欧洲启动子在线数据库(EPD)获得人Cdc25B基因5′端非编码区处包含转录起始位点在内的-2000～+100的DNA序列.以正常人全血基因组DNA为模板,利用PCR扩增该序列,并插入到pGL3-Basic荧光素酶报告基因载体上.通过设计不同引物,进一步构建系列缺失体,共获得7个不同长度启动子序列的报告基因载体.将其与内参质粒pRL-TK瞬时共转染HeLa细胞,通过双荧光素酶报告基因活性测定实验检测不同缺失体的转录活性.利用定点突变及RNA干扰(RNAi)探究预测的转录因子对Cdc25B转录活性的影响.结果 构建的人Cdc25B基因启动子荧光素酶报告基因载体pGL3-Cdc25B-promoter经酶切鉴定及测序结果比对完全正确,将其转染HeLa细胞后,检测到荧光素酶高表达(P<0.05).启动子系列缺失分析显示-160～-53片段包含基本核心启动子,与生物信息学的预测结果一致.定点突变及RNA干扰显示NF-YB转录因子对Cdc25B起正调控作用.结论 成功构建了7个具有转录活性的人Cdc25B启动子缺失片段,确定了NF-YB是Cdc25B转录的一个重要调控因子,为进一步将其用于抗肿瘤药物的筛选与评价奠定了基础.

宫颈癌治疗以手术切除辅以放化疗作为首选治疗方法,但是上述治疗手段创伤较大,不良反应也较严重,甚至可能剥夺年轻患者的生育功能.近年来随着分子生物学的发展,研究者试图从基因水平寻找对辐射敏感的放疗靶点,以最高效的杀灭肿瘤,同时最大限度地保护正常组织,从而提高放疗效果,减少放疗副作用.本文主要就宫颈癌辐射敏感性相关的多种基因及其表达产物展开综述,为研究放疗抵抗的分子机制,预测宫颈癌放射治疗预后效果奠定基础,并为肿瘤的放疗增敏治疗提供新思路.