Zheng Shurong, Fu Weida, Huang Qidi, Zhou Jieyu, Lu Kangkang, Gu Junwei, Ma Ruimin, Guo Guilong
Department of Breast Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China.
Department of Breast Surgery, Hangzhou Hospital of Traditional Chinese Medicine, Hangzhou, China.
Clin Exp Pharmacol Physiol. 2023 Jun;50(6):431-442. doi: 10.1111/1440-1681.13758. Epub 2023 Mar 14.
Paclitaxel (PTX) resistance is a key cause of chemotherapy failure in patients with triple negative breast cancer (TNBC). The aim of this study is to investigate the effect and mechanism of long non-coding RNA (lncRNA) on the PTX resistance of TNBC cells through autophagy. MDA-MB-231 cells are used to induce the PTX-resistant TNBC cell line MDA-MB-231.PR (MDR) by increasing dose intermittently. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the mRNA levels of phosphoinositide-3-kinase class 3 (PIK3C3), miR-361-5p and lncRNA PRKCQ-AS1 in the cells, and Western blot analysis was used to detect the protein expressions of PIK3C3, autophagy-related, drug-resistant and apoptosis-related genes. MDC staining detected the formation of autophagic vacuoles. The interactions between miR-361-5p and PIK3C3 and between lncRNA PRKCQ-AS1 and miR-361-5p were verified by dual-luciferase assay. Cell viability, apoptosis, migration and invasion were assessed by performing MTT, flow cytometry assay, and transwell assay. The mRNA level of miR-361-5p and the autophagy and drug resistance levels of TNBC PTX-resistant cells were significantly up-regulated. miR-361-5p could target autophagy-related gene PIK3C3, and overexpression of miR-361-5p could down-regulate PIK3C3 protein expression and autophagy level and PTX resistance of MDR cells. LncRNA PRKCQ-AS1 was selected through bioanalysis, and miR-361-5p could target lncRNA PRKCQ-AS1. In addition, lncRNA PRKCQ-AS1 level was up-regulated in TNBC PTX-resistant cells, and knockdown of lncRNA PRKCQ-AS1 could weaken autophagy and drug resistance level and could promote cell apoptosis. Overexpression of lncRNA PRKCQ-AS1 reversed the pro-apoptotic effect and down-regulation of autophagy and resistance levels was induced by miR-361-5p. In vivo experiments were performed to verify the role of lncRNA PRKCQ-AS1. We demonstrate that down-regulation of lncRNA PRKCQ-AS1 weakened PTX resistance and promoted cell apoptosis by miR-361-5p/PIK3C3 mediated autophagy.
紫杉醇（PTX）耐药是三阴性乳腺癌（TNBC）患者化疗失败的关键原因。本研究旨在探讨长链非编码RNA（lncRNA）通过自噬对TNBC细胞PTX耐药的影响及机制。通过间歇性增加剂量，用MDA-MB-231细胞诱导出PTX耐药的TNBC细胞系MDA-MB-231.PR（MDR）。采用定量实时聚合酶链反应（qRT-PCR）验证细胞中磷酸肌醇-3-激酶3类（PIK3C3）、miR-361-5p和lncRNA PRKCQ-AS1的mRNA水平，并用蛋白质免疫印迹分析检测PIK3C3、自噬相关、耐药和凋亡相关基因的蛋白表达。MDC染色检测自噬空泡的形成。通过双荧光素酶报告基因实验验证miR-361-5p与PIK3C3以及lncRNA PRKCQ-AS1与miR-361-5p之间的相互作用。通过MTT实验、流式细胞术检测和Transwell实验评估细胞活力、凋亡、迁移和侵袭。miR-361-5p的mRNA水平以及TNBC PTX耐药细胞的自噬和耐药水平显著上调。miR-361-5p可靶向自噬相关基因PIK3C3，miR-361-5p过表达可下调PIK3C3蛋白表达、自噬水平以及MDR细胞的PTX耐药性。通过生物分析筛选出lncRNA PRKCQ-AS1，miR-361-5p可靶向lncRNA PRKCQ-AS1。此外，TNBC PTX耐药细胞中lncRNA PRKCQ-AS1水平上调，敲低lncRNA PRKCQ-AS1可减弱自噬和耐药水平，并可促进细胞凋亡。lncRNA PRKCQ-AS1过表达逆转了miR-361-5p诱导的促凋亡作用以及自噬和耐药水平的下调。进行体内实验验证lncRNA PRKCQ-AS1的作用。我们证明lncRNA PRKCQ-AS1的下调通过miR-361-5p/PIK3C3介导的自噬减弱了PTX耐药性并促进了细胞凋亡。
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