摘要
目的探讨miR-129-5p靶向抑制高迁移率族蛋白B1(HMGB1)对甲状腺髓样细胞MZ-CRC-1放射敏感性的影响机制。方法建立抗辐射细胞株MZ-CRC-1/R;克隆形成实验分析细胞存活分数;qRT-qPCR检测miR-129-5p在MZ-CRC-1和MZ-CRC-1/R细胞中的表达;噻唑蓝(MTT)法检测细胞活力;流式细胞术检测细胞凋亡;双荧光酶报告基因实验验证miR-129-5p与高迁移率族蛋白B1 (HMGB1)之间的靶向关系;Western blot检测HMGB1和p-AKt的蛋白表达。结果与MZ-CRC-1细胞相比,MZ-CRC-1/R细胞的细胞存活分数显著提高(t=3.038、4.330、4.885、4.568,P<0.05);细胞活力增加(t=3.637、7.734、11.896、14.522,P<0.05);与MZ-CRC-1细胞(1.00±0.06)相比,miR-129-5p在MZ-CRC-1/R细胞中的表达(0.26±0.03)显著降低(t=19.107,P<0.05);与miR-NC-inhibitor组细胞相比,miR-129-5p-inhibitor组细胞的细胞活力显著增加(t=5.156、6.005、9.649,P<0.05),细胞凋亡率降低(t=8.659,P<0.05)。与miR-NC组细胞相比,miR-129-5p mimic组细胞的细胞活力显著降低(t=3.118、5.034、6.005、7.488,P<0.05),细胞凋亡率升高(t=6.362,P<0.05);过表达miR-129-5p可抑制HMGB1及p-AKt信号通路的表达(t=9.325、10.614, P<0.05);与miR-129-5p inhibitor组细胞相比,miR-129-5p inhibitor+si-HMGB1组细胞的细胞凋亡率显著升高(t=6.700,P<0.05);与miR-129-5p mimic组细胞相比,miR-129-5p mimic + si-HMGB1组细胞的细胞凋亡率显著降低(t=7.073,P<0.05)。结论miR-129-5p可靶向抑制HMGB1增加甲状腺髓样细胞MZ-CRC-1的放射敏感性。
Objective To explore whether MiR-129-5p participates in radiosensitivity of medullary thyroid cell MZ-CRC-1 by inhibiting the gene expression of high mobility group protein B1 (HMGB1). Methods The radioresistant cell line MZ-CRC-1/R was established from MZ-CRC-1. Cell survival fraction was analyzed by colony formation assay. The expressions of miR-129-5p in MZ-CRC-1 and MZ-CRC-1/R cells were detected by qRT-PCR. Cell viability was determined by MTT assay. Cell apoptosis was measured by flow cytometry. Dual-luciferase reporter assay was performed to confirm the relationship between miR-129-5p and HMGB1. Besides, the protein expressions of HMGB1 and p-AKt were evaluated by western blot. Results Compared with that of MZ-CRC-1 cells, the survival fraction of MZ-CRC-1/R cells was significantly increased (t=3.038, 4.330, 4.885, 4.568, P<0.05), the cell viability of MZ-CRC-1/R cells was also increased (t=3.637, 7.734, 11.896, 14.522, P<0.05), and the expression of miR-129-5p(0.26±0.03) was significantly decreased in MZ-CRC-1/R cells(1.00±0.06)(t=19.107, P<0.05). Compared with miR-NC-inhibitor group, cell viability was promoted and cell apoptosis was blocked in the miR-129-5p-inhibitor group (t=5.156, 6.005, 9.649, 8.659, P<0.05). Moreover, miR-129-5p mimic suppressed cell viability and enhanced cell apoptosis after irradiation (t=3.118, 5.034, 6.005, 7.488, 6.362, P<0.05). Overexpression of miR-129-5p inhibited the protein expressions of HMGB1 and p-AKt (t=9.325, 10.614, P<0.05). In addition, HMGB1 depletion rescued cell apoptosis that was reduced by miR-129-5p inhibitor in MZ-CRC-1 cells (t=6.700, P<0.05), while HMGB1 overexpression attenuated the effect of miR-129-5p upregulation on MZ-CRC-1/R cells (t=7.073, P<0.05). Conclusions miR-129-5p increased the radiosensitivity of medullary thyroid-like cell MZ-CRC-1 by inhibiting HMGB1.
作者
闫培
张振华
刘建波
连利霞
丁超
Yan Pei;Zhang Zhenhua;Liu Jianbo;Lian Lixia;Ding Chao(Department of Thyroid Surgery, Henan Provincial People′s Hospital, Zhengzhou 450000, China;Department of Radiotherapy, Henan Provincial People′s Hospital, Zhengzhou 450000, China)
出处
《中华放射医学与防护杂志》
CAS
CSCD
北大核心
2019年第7期493-499,共7页
Chinese Journal of Radiological Medicine and Protection
