miR-193a-3p在食管癌细胞放射抵抗作用的初步研究

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。

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

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.