Li Tiezhi, Zhang Helin, Wang Zhichao, Gao Shaolin, Zhang Xu, Zhu Haiyong, Wang Na, Li Honglin
Department of Thoracic Surgery, The Second Hospital of Hebei Medical University, Shijiazhuang, China.
Department of Pediatrics, The First Hospital of Hebei Medical University, Shijiazhuang, China.
Cancer Cell Int. 2022 Mar 15;22(1):120. doi: 10.1186/s12935-022-02505-1.
Autophagy has been found to be involved in the multidrug resistance (MDR) of cancers, but whether it is associated with resistance of small cell lung cancer (SCLC) has not been studied. Here, we hypothesized that a potential autophagy-regulating miRNA, miR-199a-5p, regulated cisplatin-resistant SCLC.
We validated the MDR of H446/EP using CCK-8 and LDH. We tested the binding of miR-199a-5p to p62 using the Dual-Luciferase assay and validated the association of miR-199a-5p and p62 in SCLC samples. We overexpressed (OE) and knocked down (KD) miR-199a-5p in H446 and H446/EP and determined the expression of miR-199a-5p, autophagy-related proteins, and the formation of autophagolysosomes using QPCR, western blotting, and MDC staining respectively. These results were validated in an orthotopic H446 mouse model of SCLC.
H446/EP was resistant to cisplatin, etoposide, paclitexal, epirubicin, irinotecan, and vinorelbine. Exposure of cisplatin at 5 μg/ml for 24 h increased LC3II/LC3I, ATG5, p62, and the formation of autophagolysosomes in H446 cells, but not in H446/EP cells. The expression of miR-199a-5p was up-regulated in H446/EP compared to H446. MiR-199a-5p directly targeted the p62 gene. The expression of miR-199a-5p and p62 were correlated in SCLC samples. In H446 and H69PR, the OE of miR-199a-5p increased LC3II/LC3I, p62, and the formation of autophagolysosomes, but not ATG5, while the KD of miR-199a-5p decreased p62, but did not affect LC3II/LC3I, ATG5, and the formation of autophagolysosomes. In H446/EP, the OE of miR-199a-5p decreased p62 only. These results were generally consistent to results in the animal tumor samples.
The regulation of autophagy by the miR-199a-5p/p62 axis was a potential mechanism of small cell lung cancer cisplatin resistance.
自噬已被发现与癌症的多药耐药性（MDR）有关，但它是否与小细胞肺癌（SCLC）的耐药性相关尚未得到研究。在此，我们假设一种潜在的自噬调节性微小RNA，即miR-199a-5p，对顺铂耐药的小细胞肺癌具有调节作用。
我们使用CCK-8和LDH验证了H446/EP的多药耐药性。我们使用双荧光素酶测定法检测miR-199a-5p与p62的结合，并在小细胞肺癌样本中验证miR-199a-5p与p62的相关性。我们在H446和H446/EP中过表达（OE）和敲低（KD）miR-199a-5p，并分别使用QPCR、蛋白质印迹法和MDC染色确定miR-199a-5p、自噬相关蛋白的表达以及自噬溶酶体的形成。这些结果在小细胞肺癌的原位H446小鼠模型中得到验证。
H446/EP对顺铂、依托泊苷、紫杉醇、表柔比星、伊立替康和长春瑞滨耐药。5μg/ml顺铂处理24小时可增加H446细胞中LC3II/LC3I、ATG5、p62的表达以及自噬溶酶体的形成，但对H446/EP细胞无此作用。与H446相比，H446/EP中miR-199a-5p的表达上调。miR-199a-5p直接靶向p62基因。在小细胞肺癌样本中，miR-199a-5p和p62的表达相关。在H446和H69PR中，miR-199a-5p的过表达增加了LC3II/LC3I、p62的表达以及自噬溶酶体的形成，但不影响ATG5，而miR-199a-5p的敲低降低了p62的表达，但不影响LC3II/LC3I、ATG5以及自噬溶酶体的形成。在H446/EP中，miR-199a-5p的过表达仅降低了p62的表达。这些结果与动物肿瘤样本中的结果总体一致。
miR-199a-5p/p62轴对自噬的调节是小细胞肺癌顺铂耐药的潜在机制。
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