Objective

Drug resistance is the main obstacle in the treatment of non-small cell lung cancer (NSCLC). This study aimed to explore the mechanism of DICER in NSCLC resistance and its downstream signaling pathways.

Methods

The A549 cisplatin (DDP)-resistant strain A549/DDP was established. A549/DDP cells were transfected with DICER- and let-7i-5p-related vectors, and treated with autophagy activator rapamycin. The cell viability and apoptosis were tested by CCK-8 assay and flow cytometry, respectively. The formation of autophagosomes was observed with a transmission electron microscopy. RT-qPCR and Western blot assay were conducted to detect expression levels of DICER, let-7i-5p, autophagy-related proteins, and the PI3K/AKT/mTOR pathway-related proteins. The dual luciferase reporter gene assay was implemented to confirm the targeted binding of DICER and let-7i-5p.

Results

DICER was highly expressed in DDP-resistant NSCLC tissues and cells, and DICER could target and negatively regulate the expression of let-7i-5p. DDP treatment could inhibit the viability and promote cell apoptosis of A549/DDP cells. Downregulation of DICER in A549/DDP cells exhibited a decrease of cell viability, a decreased ratio of LC3-II/LC3-I and autophagosomes, together with an elevation of cell apoptosis rate and the phosphorylation levels of PI3K/AKT/mTOR. Treatment of rapamycin and let-7i-5p inhibitor reversed the effects of downregulated DICER in cell viability, ratio of LC3-II/LC3-I, autophagosomes, cell apoptosis rate and the phosphorylation levels of PI3K/AKT/mTOR in A549/DDP cells.

Conclusion

Our research suggests that DICER promotes autophagy and DDP resistance in NSCLC through downregulating let-7i-5p, and inhibits the activation of PI3K/AKT/mTOR pathway.

中文翻译：

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DICER 通过与 let-7i-5p 结合激活自噬并促进非小细胞肺癌顺铂耐药

客观的

耐药性是非小细胞肺癌（NSCLC）治疗的主要障碍。本研究旨在探讨DICER在NSCLC耐药中的作用机制及其下游信号通路。

方法

建立了A549顺铂（DDP）抗性菌株A549/DDP。用 DICER 和 let-7i-5p 相关载体转染 A549/DDP 细胞，并用自噬激活剂雷帕霉素处理。分别通过CCK-8法和流式细胞仪检测细胞活力和凋亡。用透射电子显微镜观察自噬体的形成。采用 RT-qPCR 和蛋白质印迹法检测 DICER、let-7i-5p、自噬相关蛋白和 PI3K/AKT/mTOR 通路相关蛋白的表达水平。实施双荧光素酶报告基因测定以确认 DICER 和 let-7i-5p 的靶向结合。

结果

DICER在DDP耐药的NSCLC组织和细胞中高表达，DICER可以靶向并负调控let-7i-5p的表达。DDP处理可以抑制A549/DDP细胞的活力并促进细胞凋亡。A549/DDP 细胞中 DICER 的下调表现出细胞活力的降低、LC3-II/LC3-I 和自噬体的比例降低，以及细胞凋亡率和 PI3K/AKT/mTOR 磷酸化水平的升高。雷帕霉素和 let-7i-5p 抑制剂的治疗逆转了下调的 DICER 对细胞活力、LC3-II/LC3-I 比率、自噬体、细胞凋亡率和 A549/DDP 细胞中 PI3K/AKT/mTOR 磷酸化水平的影响.

结论

我们的研究表明，DICER 通过下调 let-7i-5p 促进 NSCLC 自噬和 DDP 耐药，并抑制 PI3K/AKT/mTOR 通路的激活。