Downregulation of high mobility group box 1 modulates telomere homeostasis and increases the radiosensitivity of human breast cancer cells
Downregulation of high mobility group box 1 modulates telomere homeostasis and increases the radiosensitivity of human breast cancer cells
复制标题
高迁移率族盒 1 的下调可调节端粒稳态并增加人乳腺癌细胞的放射敏感性
DOI：
10.3892/ijo.2014.2793
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发表时间：
2015-03-01
影响因子：
5.2
通讯作者：
Zhou, Yunfeng
中科院分区：
文献类型：
--
作者：
Ke, Shaobo;Zhou, Fuxiang;Zhou, Yunfeng
高迁移率组盒1 （HMGB1）在肿瘤细胞中的功能包括补充端粒DNA和维持细胞不朽。人端粒酶逆转录酶（hTERT）与肿瘤细胞放射敏感性呈负相关。我们的目的是阐明HMGB1、端粒稳态和MCF-7细胞放射敏感性之间的关系。在这项研究中，我们建立了稳定转染的对照（MCF-7-NC）和HMGB1敲低（MCF-7-shHMGB1）细胞系。实时荧光定量PCR检测HMGB1 mRNA表达及相对端粒长度。用克隆法检测放射敏感性。western blot检测蛋白表达。采用PCR-ELISA法检测端粒酶活性。CCK-8法检测细胞增殖能力。流式细胞术检测细胞周期和凋亡情况。免疫荧光法检测DNA损伤灶。shrna介导的HMGB1表达下调增加了MCF-7细胞的放射敏感性，减少了hTERT和cyclin Dl的积累。此外，MCF-7细胞中HMGB1的下调抑制了端粒酶活性和细胞增殖，同时增加了细胞凋亡的程度。HMGB1的下调通过改变端粒结合蛋白如TPP1 （PTOP）、TRF1和TRF2的水平来调节端粒稳态。这种下调也抑制了ATM和ATR信号通路。目前的数据表明，HMGB1的敲低打破了端粒稳态，增强了放射敏感性，抑制了人乳腺癌细胞DNA损伤的修复。这些结果提示HMGB1可能是人乳腺癌的潜在放疗靶点。
The functions of the high mobility group box 1 (HMGB1) in tumor cells include replenishing telomeric DNA and maintaining cell immortality. There is a negative correlation between human telomerase reverse transcriptase (hTERT) and radiosensitivity in tumor cells. Our aim was to elucidate the relationship among HMGB1, telomere homeostasis and radiosensitivity in MCF-7 cells. In this study, we established stably transfected control (MCF-7-NC) and HMGB1 knockdown (MCF-7-shHMGB1) cell lines. The expression of HMGB1 mRNA and the relative telomere length were examined by real-time PCR. Radiosensitivity was detected by clonogenic assay. The protein expressions were determined by western blot analysis. The telomerase activity was detected by PCR-ELISA. Proliferation ability was examined by CCK-8 assay. Cell cycle and apoptosis were examined by flow cytometry. DNA damage foci were detected by immunofluorescence. ShRNA-mediated downregulation of HMGB1 expression increased the radiosensitivity of MCF-7 cells, and reduced the accumulation of hTERT and cyclin Dl. Moreover, knockdown of HMGB1 in MCF-7 cells inhibited telomerase activity and cell proliferation, while increasing the extent of apoptosis. Downregulation of HMGB1 modulated telomere homeostasis by changing the level of telomere-binding proteins, such as TPP1 (PTOP), TRF1 and TRF2. This downregulation also inhibited the ATM and ATR signaling pathways. The current data demonstrate that knockdown of HMGB1 breaks telomere homeostasis, enhances radiosensitivity, and suppresses the repair of DNA damage in human breast cancer cells. These results suggested that HMGB1 might be a potential radiotherapy target in human breast cancer.