Cancer Institute, Fourth Hospital of Hebei Medical University, Shijiazhuang, China.
Cancer Institute, Fourth Hospital of Hebei Medical University, Shijiazhuang, China.
Radiother Oncol. 2020 Sep;150:104-113. doi: 10.1016/j.radonc.2020.06.022. Epub 2020 Jun 21.
Although radiotherapy is an important treatment mode for esophageal cancer (EC), the outcome remains unsatisfactory due to radioresistance, and the key cause of radiotherapy resistance is a change in the cell cycle. Zinc deficiency (ZD) has a significant influence on the cell cycle, and this effect is a common phenomenon in areas with a high incidence of esophageal cancer.
Radioresistant sub-cell lines were established by exposing esophageal cancer cells to nine rounds of X-ray irradiation at a dose of 2 Gy. The cells were treated with a range of different concentrations of zinc and overexpression of miR-193b. And proliferation, colony formation, cell cycle and apoptosis assays were then conducted. Luciferase reporter assays were performed to confirm direct interactions between miR-193b and ZRT/IRT-like protein 5 (ZIP5) and between miR-193b and Cyclin D1. Analysis of clinical and follow-up data was performed using data obtained from 75 patients from Cixian, a well-known high incidence area of esophageal cancer. All these patients are unable to tolerate surgery due to their advanced age or advanced stage, and serum specimens were obtained before the patients received therapy.
The cell cycle of radioresistant cells is blocked in G0/G1 phase (from 50% to 68%). The expression level of Cyclin D1 was decreased and miR-193b was increased in radioresistant cells (P < 0.001). ZD decreased the proportion of cells in G0/G1 phase both in EC (from 50% to 32%) and radioresistant (from 68% to 47%) cells. And the radioresistance of these two cells were decreased. ZD increased the expression of Cyclin D1 (P < 0.001) and inhibited the level of miR-193b (P < 0.001). Up-regulation of miR-193b recovered the proportion of cells in G0/G1 phase and the radioresistance, meanwhile, recovered the altered expression levels of Cyclin D1 and miR-193b of these two cells by ZD. ZD enhanced DNMT activity both in EC (32%) and radioresistant (26%). And miR-193b was hypermethylated both in EC and radioresistant cells. MiR-193b supp ressed Cyclin D1 expression by targeting the 3'UTR of Cyclin D1 mRNA. The expression level of miR-193b in the serum of patients was correlated with the disease control rate (DCR) and had a good diagnostic value for distinguishing DCR of EC patients (AUC = 0.710, 95%CI: 0.580-0.839, P = 0.003). And the level of miR-193b was correlated with overall survival (OS) (HR = 0.208, 95%CI: 0.094-0.464).
The methylation-mediated silencing of miR-193b in EC cells due to ZD increased the expression of ZIP5, and the overexpression of ZIP5 increased the intracellular zinc levels to maintain zinc homeostasis. Meanwhile, the silencing of miR-193b increased the sensitivity of radiotherapy by promoting the expression of Cyclin D1.
尽管放疗是食管癌（EC）的重要治疗模式，但由于放射抗性，其疗效仍不理想，放射抗性的关键原因是细胞周期的改变。锌缺乏（ZD）对细胞周期有显著影响，这种现象在食管癌高发地区很常见。
通过对食管癌细胞进行 9 轮 2Gy 的 X 射线照射，建立放射抗性亚细胞系。用一系列不同浓度的锌和 miR-193b 的过表达处理细胞。然后进行增殖、集落形成、细胞周期和凋亡检测。荧光素酶报告实验证实 miR-193b 与 ZRT/IRT-like protein 5（ZIP5）之间以及 miR-193b 与 Cyclin D1 之间存在直接相互作用。使用从著名的食管癌高发地区磁县的 75 名患者获得的数据进行临床和随访数据分析。所有这些患者由于年龄较大或晚期而无法耐受手术，并在接受治疗前获得了血清标本。
放射抗性细胞的细胞周期在 G0/G1 期被阻断（从 50%到 68%）。放射抗性细胞中 Cyclin D1 的表达水平降低，miR-193b 的表达水平升高（P<0.001）。ZD 降低了 EC（从 50%到 32%）和放射抗性（从 68%到 47%）细胞中 G0/G1 期细胞的比例。这两种细胞的放射抗性都降低了。ZD 增加了 Cyclin D1 的表达（P<0.001）并抑制了 miR-193b 的水平（P<0.001）。上调 miR-193b 恢复了 G0/G1 期细胞的比例和放射抗性，同时恢复了 ZD 对这两种细胞中 Cyclin D1 和 miR-193b 表达的改变。ZD 增强了 EC（32%）和放射抗性（26%）中的 DNMT 活性。miR-193b 在 EC 和放射抗性细胞中均发生高度甲基化。miR-193b 通过靶向 Cyclin D1 mRNA 的 3'UTR 抑制 Cyclin D1 的表达。患者血清中 miR-193b 的表达水平与疾病控制率（DCR）相关，对区分 EC 患者的 DCR 具有良好的诊断价值（AUC=0.710，95%CI：0.580-0.839，P=0.003）。并且 miR-193b 的水平与总生存期（OS）相关（HR=0.208，95%CI：0.094-0.464）。
由于 ZD 导致的 EC 细胞中 miR-193b 的甲基化沉默增加了 ZIP5 的表达，而过表达的 ZIP5 增加了细胞内锌水平以维持锌平衡。同时，miR-193b 的沉默通过促进 Cyclin D1 的表达提高了放疗的敏感性。
