限制蛋氨酸通过下调 circ-CDK13 水平促进胃癌耐药细胞对顺铂的敏感性。

Methionine restriction promotes cisplatin sensitivity of gastric cancer resistant cells by down-regulating circ-CDK13 level.

机构信息

Department of General Surgery, The Second Affiliated Hospital of Nanchang University, Nanchang, 330006, Jiangxi Province, China.

出版信息

Exp Cell Res. 2024 Nov 1;443(1):114315. doi: 10.1016/j.yexcr.2024.114315. Epub 2024 Oct 31.

DOI:10.1016/j.yexcr.2024.114315

PMID:39488295

Abstract

BACKGROUND

Methionine restriction (MR) is a research direction in the treatment of gastric cancer (GC). The aim of this study was to investigate the molecular mechanism of MR on enhancing cisplatin (DDP) sensitivity of drug-resistant GC cells.

METHODS

Twenty pairs of GC tissues and adjacent normal gastric mucosa tissues were collected. DDP-resistant cell lines (KATO/DDP and MKN45/DDP), mouse model of GC and GC patient-derived organoid (PDO) models were established. Lentivirus-mediated METase overexpression was used for MR. Cell viability and apoptosis were detected by MTT assay and flow cytometry. Western blotting was used to detect multi-drug resistance-1 (MDR1), MDR-associated protein 1 (MRP1) eukaryotic initiation factor 4A-Ⅲ (EIF4A3), and METase protein expressions. The levels of circRNAs were detected by qRT-PCR. Tumor volume and weight were measured. The proliferation of tumor cells was detected by immunohistochemical staining.

RESULTS

The differentially expressed circRNAs of GC were screened in Gene Expression Omnibus database. MR in KATO/DDP and MKN45/DDP cells significantly down-regulated circ-CDK13 level. Overexpression of circ-CDK13 significantly inhibited apoptosis of sensitive cells (KATO III and MKN45). Interference with circ-CDK13 significantly promoted apoptosis of drug-resistant cells (KATO/DDP and MKN45/DDP). MR enhanced the DDP sensitivity of GC resistant cells, GC PDO and GC mice by down-regulating circ-CDK13. EIF4A3 binds to the downstream flanking sequence of circ-CDK13, and interference with EIF4A3 reduces circ-CDK13 levels, but does not affect CDK13. The expressions of circ-CDK13 and EIF4A3 in GC clinical samples were increased and positively correlated. Simultaneously overexpression of METase and EIF4A3 in resistant cells inhibited apoptosis, and further interference with circ-CDK13 reversed this effect.

CONCLUSION

MR inhibits circ-CDK13 level by down-regulating EIF4A3, thereby increasing the sensitivity of GC drug-resistant cells to DDP.

摘要

背景

甲硫氨酸限制（MR）是胃癌（GC）治疗的一个研究方向。本研究旨在探讨 MR 增强耐药 GC 细胞对顺铂（DDP）敏感性的分子机制。

方法

收集 20 对 GC 组织及其相邻正常胃黏膜组织。建立 DDP 耐药细胞系（KATO/DDP 和 MKN45/DDP）、GC 小鼠模型和 GC 患者来源类器官（PDO）模型。采用慢病毒介导的 METase 过表达进行 MR。通过 MTT 检测和流式细胞术检测细胞活力和凋亡。Western blot 检测多药耐药-1（MDR1）、多药耐药相关蛋白 1（MRP1）、真核起始因子 4A-Ⅲ（EIF4A3）和 METase 蛋白表达。通过 qRT-PCR 检测 circRNA 水平。测量肿瘤体积和重量。通过免疫组化染色检测肿瘤细胞增殖。

结果

从 Gene Expression Omnibus 数据库中筛选出 GC 的差异表达 circRNA。KATO/DDP 和 MKN45/DDP 细胞中的 MR 显著下调 circ-CDK13 水平。过表达 circ-CDK13 显著抑制敏感细胞（KATO III 和 MKN45）的凋亡。干扰 circ-CDK13 显著促进耐药细胞（KATO/DDP 和 MKN45/DDP）的凋亡。MR 通过下调 circ-CDK13 增强 GC 耐药细胞、GC PDO 和 GC 小鼠对 DDP 的敏感性。EIF4A3 与 circ-CDK13 的下游侧翼序列结合，干扰 EIF4A3 可降低 circ-CDK13 水平，但不影响 CDK13。GC 临床样本中 circ-CDK13 和 EIF4A3 的表达增加且呈正相关。同时过表达耐药细胞中的 METase 和 EIF4A3 抑制凋亡，进一步干扰 circ-CDK13 逆转了这一效应。