DICER 通过与 let-7i-5p 结合激活自噬并促进非小细胞肺癌对顺铂的耐药性。

DICER activates autophagy and promotes cisplatin resistance in non-small cell lung cancer by binding with let-7i-5p.

机构信息

Department of Oncology, Second People's Hospital of Jingmen, Jingmen, Hubei 448000, PR China.

Department of Traditional Chinese Medicine, Xiangyang Central Hospital, Affiliated Hospital of Hubei University of Arts and Science, Xiangyang, Hubei 441021, PR China.

出版信息

Acta Histochem. 2021 Oct;123(7):151788. doi: 10.1016/j.acthis.2021.151788. Epub 2021 Sep 17.

DOI:10.1016/j.acthis.2021.151788

PMID:34543777

Abstract

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.

摘要

目的

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 耐药性中的作用机制及其下游信号通路。

方法

建立 A549 顺铂（DDP）耐药株 A549/DDP。用 DICER 和 let-7i-5p 相关载体转染 A549/DDP 细胞，并给予自噬激活剂雷帕霉素处理。用 CCK-8 法和流式细胞术分别检测细胞活力和细胞凋亡。用透射电子显微镜观察自噬体的形成。通过 RT-qPCR 和 Western blot 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实验以确认 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 可靶向并负调控 let-7i-5p 的表达。DDP 处理可抑制 A549/DDP 细胞的活力并促进细胞凋亡。下调 A549/DDP 细胞中的 DICER 可降低细胞活力、LC3-II/LC3-I 比值和自噬体，并增加细胞凋亡率和 PI3K/AKT/mTOR 的磷酸化水平。雷帕霉素和 let-7i-5p 抑制剂处理可逆转下调 DICER 对 A549/DDP 细胞活力、LC3-II/LC3-I 比值、自噬体、细胞凋亡率和 PI3K/AKT/mTOR 磷酸化水平的影响。