miR-193a-3p在食管癌细胞放射抵抗作用的初步研究

A preliminary study of the role of miR-193a-3p in radioresistance of esophageal cancer cells

目的：探讨miR-193a-3p在食管鳞癌放射耐受性机制中的作用。方法通过6 MV X射线对4个食管癌细胞系进行照射，采用MTT法检测出相对敏感系及耐受系细胞；茎环引物实时定量PCR法检测miR-193a-3p、miR-155、miR-22-3p在2个细胞中的表达，miR-193a-3p作为表达差异较为明显的 microRNA 被挑选进行下一步研究。分别合成并转染 miR-193a-3p 的 mimic （3PM）或antagomiR （3PA）序列及小干扰RNA （si-LOXL4）以提高或抑制其在细胞中的表达水平，MTT法和流式细胞分析术检测miR-193a-3p及其下游基因LOXL4对于放射敏感性的影响。结果筛选出相对放射敏感细胞系（ KYSE510）及耐受细胞系（ KYSE410）；miR-193a-3p在两系细胞中的表达水平差异显著高于miR-155、miR-22-3p （1.00∶21.13）；KYSE510细胞中转染mimic提高其表达后，与对照组相比，其放射敏感性降低，细胞凋亡比例显著下降11.10％（P＜0.05），而 KYSE410细胞中转染 antagomiR后，其敏感性增加（ P＜0.05）。作为miR-193a-3p的下游基因，LOXL4的表达抑制也受到miR-193a-3p的调控，转染si-LOXL4降低其表达，与对照组相比放射敏感性也降低，细胞凋亡比例下降7.07％（ P＜0.05）。结论 miR-193a-3p可能通过调控基因LOXL4促进食管癌细胞放射耐受性。

Objective To investigate the role of miR-193a-3p in the radioresistance of esophageal squamous cell carcinoma （ ESCC） . Methods MTT assay was used to identify the cell lines with the highest radiosensitivity and radioresistance in four esophageal cancer cell lines exposed to irradiation of 6 MV X-ray. Stem-loop quantitative real-time PCR was used to measure the expression levels of miR-193a-3p, miR-155, and miR-22-3P in the two cell lines. Further studies were performed on miR-193a-3p because of the substantial difference in its expression between the two cell lines. The mimic （3PM） and antagomiR （3PA） of miR-193a-3p as well as siRNA （ si-LOXL4） were synthesized and transfected into cells to elevate and inhibit miR-193a-3p expression. MTT assay and flow cytometry were used to evaluate the effects of miR-193a-3p and its downstream gene LOXL4 on radiosensitivity. Results KYSE510 and KYSE410 were characterized as cell lines with the highest radiosensitivity and radioresistance, respectively. miR-193a-3p had a substantially larger difference in expression between the two cell lines than miR-155 or miR-22-3P （1. 00 ∶ 21. 00）. Transfection of 3PM resulted in elevated expression of miR-193a-3p in KYSE510, which had a significantly lower radiosensitivity and a significantly reduced apoptosis ratio by 11. 01% compared with the control group （ P〈0. 05） . KYSE410 transfected with 3PA had a significantly higher radiosensitivity （ P〈0. 05） . The expression of LOXL4, a downstream gene of miR-193a-3p, was negatively correlated with miR-193a-3p expression. Transfection with si-LOXL4 inhibited the expression of LOXL4, which resulted in a significantly lower radiosensitivity and a significantly reduced apoptosis ratio by 7. 07% compared with the control group （ P〈0. 05） . Conclusions miR-193a-3p promotes the radioresistance of esophageal cancer cells probably by regulation of LOXL4.