目的：探讨miR-216b-5p 对食管癌Eca109 细胞顺铂（DDP）耐药性的影响及其作用机制。方法:采用qPCR 法检测miR-216b-5p 在食管癌细胞TE-1、KYSE-150、Eca109 和耐药细胞Eca109/DDP 中的表达水平。利用脂质体转染技术分别将miR-216b-5p mimic 及mimic NC、自噬相关蛋白5（ATG5）过表达质粒转染到Eca109/DDP 细胞中，用CCK-8、EdU 法和FCM分别检测转染后细胞的增殖和凋亡；mRFP-eGFP-LC3双荧光标记实验检测mRFP-eGFP-LC3慢病毒感染后各组细胞自噬发生情况， WB法检测自噬相关蛋白LC3、Beclin 1和P62 表达。用荧光素酶报告基因实验验证miR-216b-5p与ATG5的靶向关系，WB法检测ATG5的表达。建立裸鼠Eca109/DDP细胞移植瘤模型，观察miR-216b-5p过表达对移植瘤生长的影响。结果：miR-216b-5p在TE-1、KYSE-150、Eca109 和Eca109/DDP 细胞中均呈低表达（均P<0.05）。过表达miR-216b-5p可显著抑制Eca109/DDP 细胞的增殖并诱导凋亡（均P<0.05），减少细胞中自噬小体数量（P<0.05），下调LC3Ⅱ/LC3Ⅰ比值和Beclin 1蛋白水平、上调P62 蛋白水平（均P<0.05）。双荧光素酶报告基因实验证实miR-216b-5p靶向并负调控ATG5的表达（P<0.05），过表达ATG5 可使miR-216b-5p mimic 对Eca109/DDP 细胞增殖、自噬的抑制作用和凋亡的诱导作用明显减弱（均P<0.05），自噬相关蛋白P62 表达降低、LC3Ⅱ/LC3Ⅰ比值和Beclin 1 表达升高（均P<0.05）。荷瘤实验结果表明，miR-216b-5p 过表达可显著抑制裸鼠移植瘤的生长（P<0.05）。结论：miR-216b-5p过表达可逆转食管癌Eca109/DDP细胞对DDP的耐药性，其机制可能与靶向负调控ATG5表达并影响细胞自噬有关。

Objective: To investigate the effect of miR-216b-5p on cisplatin (DDP) resistance in esophageal cancer Eca109 cells and its mechanism. Methods: The expression levels of miR-216b-5p in esophageal cancer cells TE-1, KYSE-150, Eca109 and drug resistant Eca109/DDP cells were detected by qPCR. The miR-216b-5p mimics, mimic NC and autophagy related protein 5 (ATG5) over-expressed plasmids were transfected into Eca109/DDP cells by liposome transfection technique. The proliferation and apoptosis of transfected Eca109/DDP cells were detected by CCK-8, EdU methods and flow cytometry, respectively. The occurrence of autophagy in each group of cells after transfection with mRFP-eGFP-LC3 lentivirus was examined by mRFP-eGFP-LC3 dual fluorescent labeling assay, and the expression of autophagy-related markers, LC3, Beclin 1 and P62, was detected by WB. The targeting relationship between miR-216b-5p and ATG5 was verified by luciferase reporter gene assay, and the expression of ATG5 was detected by Western blotting. A nude mouse Eca109/DDP cell transplanted tumor model was established to observe the effect of miR-216b-5p over-expression on the growth of transplanted tumor. Results: miR-216b-5p was lowly expressed in TE-1, KYSE-150, Eca109 and Eca109/DDP cells (all P<0.05). Over-expression of miR-216b-5p significantly inhibited the proliferation and induced apoptosis of Eca109/DDP cells (both P<0.05), reduced the number of autophagosomes in transfected cells (P<0.05), and down-regulated LC3Ⅱ/LC3Ⅰratio and Beclin 1 protein level, but up-regulated P62 protein level (all P<0.05). Luciferase reporter gene assay confirmed that miR-216b-5p negatively regulated ATG5 expression (P<0.05). Over-expression of ATG5 could significantly weaken the effects of miR-216b-5p mimic on suppressing proliferation, autophagy and inducing apoptosis of Eca109/DDP cells (all P<0.05), and reduce the expression of autophagy related protein P62, but increase the LC3Ⅱ/LC3Ⅰratio and Beclin 1 protein level (all P<0.05). The tumor bearing experiments showed that over-expression of miR-216b-5p could significantly inhibit the growth of transplanted tumors in nude mice (P<0.05).Conclusion: Over-expression of miR-216b-5p can reverse DDP resistance in Eca109/DDP cells, and the mechanism may be related to its negative regulation of autophagy-related gene ATG5 to affect cell autophagy.

浙江省中医药科技计划项目（No. 2021ZB078）；浙江省大学生创新创业项目（No. 202113023001）；浙江省医药卫生科技计划项目（No. 2022RC124）；杭州医学院基本科研业务费项目（No. KYQN202001，No. KYYB202003）