Methylation-associated silencing of miR-193b improves the radiotherapy sensitivity of esophageal cancer cells by targeting cyclin D1 in areas with zinc deficiency

Abstract

BackgroundAlthough 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. MethodsRadioresistant 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. ResultsThe 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 suppressed 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). ConclusionsThe 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.