| 吴芳华,赵若琴,陈 魁,等.外泌体miR-182-5p通过靶向Cofilin1-PINK1-Parkin通路增加胃癌细胞化疗敏感性[J].肿瘤学杂志,2025,31(8):703-710. |
| 外泌体miR-182-5p通过靶向Cofilin1-PINK1-Parkin通路增加胃癌细胞化疗敏感性 |
| Exosome miR-182-5p Enhances Chemosensitivity in Gastric Cancer Cells Through Targeting Cofilin1-PINK1-Parkin Pathway |
| 投稿时间：2024-09-15 |
| DOI：10.11735/j.issn.1671-170X.2025.08.B008 |
|
 |
| 中文关键词: 微小RNA-182-5p Cofilin1-PINK1-Parkin信号通路 胃肿瘤 外泌体 化疗敏感性 |
| 英文关键词:miRNA-182-5p Cofilin1-PINK1-Parkin signaling pathway gastric neoplasms exosome chemotherapy sensitivity |
| 基金项目:福建省自然科学基金项目 （2019J01539） ；福建省福州市临床重点专科学科建设计划（20220301） |
|
| 摘要点击次数: 210 |
| 全文下载次数: 96 |
| 中文摘要: |
| 摘 要：［目的］探讨外泌体miR-182-5p（exomiR-182-5p）促进胃癌细胞对化疗药物的敏感性及其分子机制。［方法］ 培养5-氟尿嘧啶（5-FU）敏感/耐药人胃癌细胞株SGC-7901和SGC-7901/5-FU作为研究对象，采用MTT法检测细胞的半数抑制浓度（IC50）。通过超速离心法提取SGC-7901细胞分泌的外泌体，并通过PKH26红色荧光探针验证SGC-7901/5-FU细胞对外泌体的摄取。将SGC-7901细胞分为空白组、阴性组、miR-182-5p组、miR-182-5p+CFL1阴性组和miR-182-5p+CFL1组。miR-182-5p组和阴性组分别转染miR-182-5p mimics或相应的阴性对照序列，miR-182-5p+CFL1组和miR-182-5p+CFL1阴性组分别将miR-182-5p mimics和pCDNA 3.1-CFL1表达载体/空载体共转染。转染后，提取外泌体，与SGC-7901/5-FU细胞共同培养48 h。利用双荧光素酶报告基因验证miR-182-5p和Cofilin1转录基因（CFL1）的相互作用。Western blot法检测Cofilin1-PINK1-Parkin通路相关蛋白表达变化。［结果］ SGC-7901/5-FU细胞对5-FU的IC50为（169.33±7.12） μg/mL，明显高于SGC-7901细胞［（19.57±2.95） μg/mL］（P＜0.001）。通过PKH26红色荧光标记，SGC-7901分泌的外泌体可被SGC-7901/5-FU受体细胞成功吸收。与空白组和阴性组相比，miR-182-5p组SGC-7901/5-FU细胞对5-FU的敏感性增强，其IC50降低至（65.31±3.94） μg/mL；同时CFL1 mRNA水平显著降低（P＜0.001）。双荧光素酶报告基因分析表明，CFL1 是miR-182-5p的直接作用靶点。在挽救实验中，miR-182-5p+CFL1组SGC-7901/5-FU细胞对5-FU的IC50值高于miR-182-5p组和miR-182-5p+CFL1阴性组（P＜0.001）。此外，与空白组和阴性组比较，miR-182-5p组和miR-182-5p+CFL1阴性组Cofilin1、PINK1和Parkin蛋白表达水平显著降低（P均＜0.001）；而miR-182-5p+CFL1组Cofilin1、PINK1和Parkin蛋白表达水平较miR-182-5p+CFL1阴性组升高（P均 ＜0.001）。［结论］exomiR-182-5p增加了耐药胃癌细胞SGC-7901/5-FU对化疗药物5-FU的敏感性，其作用机制可能与负调控Cofilin1-PINK1-Parkin通路信号的活性有关。 |
| 英文摘要: |
| Abstract： ［Objective］ To explore the effect of exosome miR-182-5p (exomiR-182-5p) on the sensitivity of gastric cancer cells to chemotherapeutic drugs and its molecular mechanism. ［Methods］ The 5-fluorouracil (5-FU) sensitive and resistant human gastric cancer cell lines SGC-7901 and SGC-7901/5-FU were cultured as the research objects. The half inhibitory concentration (IC50) of the cells was measured by MTT method. The exosomes derived by SGC-7901 cells were extracted by ultracentrifugation， and the uptake of exosomes by SGC-7901/5-FU cells was verified by PKH26 red fluorescence probe. SGC-7901 cells were divided into blank group， negative group， miR-182-5p group， miR-182-5p+CFL1 negative group and miR-182-5p+CFL1 group. The miR-182-5p and negative groups were transfected with miR-182-5p mimics or corresponding negative control sequence， respectively； the miR-182-5p+CFL1 group and miR-182-5p+CFL1 negative group were co-transfected with miR-182-5p mimics and pCDNA 3.1-CFL1 expression vector or control vector， respectively. After transfection， the exosomes were extracted and co-cultured with SGC-7901/5-FU cells for 48 h. The interaction between miR-182-5p and CFL1 gene was verified by luciferase reporter gene. Western blot method was used to detect the expression of Cofilin1-PINK1-Parkin pathway related proteins. ［Results］ The IC50 of SGC-7901/5-FU cells to 5-FU was (169.33±7.12) μg/mL， significantly higher than SGC-7901 cells ［(19.57±2.95) μg/mL］ (P＜0.001). By PKH26 red-fluorescence labeling， SGC-7901-derived exosomes were successfully absorbed by SGC-7901/5-FU receptor cells. Compared with the blank group and the negative group， the sensitivity to 5-FU of SGC-7901/5-FU cells in miR-182-5p group was increased， and IC50 decreased to (65.31±3.94) μg/mL； meanwhile， the level of CFL1 mRNA was significantly decreased (P＜0.001). Luciferase reporter gene analysis showed that CFL1 was the direct target of miR-182-5p. In the rescue experiment， IC50 of SGC-7901/5-FU cells in the miR-182-5p+CFL1 group to 5-FU was higher than that in the miR-182-5p group and the miR-182-5p+CFL1 negative group (all P＜0.001). In addition， compared with the blank group and negative group， the expression levels of Cofilin1， PINK1 and Parkin protein in miR-182-5p group and miR-182-5p+CFL1 negative group were significantly lower (all P＜0.001). The expression level of Cofilin1， PINK1 and Parkin protein in miR-182-5p+CFL1 group was higher than that in miR-182-5p+CFL1 negative group (P＜0.001). ［Conclusion］ Exosome miR-182-5p can enhance the sensitivity of drug-resistant gastric cancer cells SGC-7901/5-FU to 5-FU， which may be related to its negative regulation of Cofilin1-PINK1-Parkin signaling pathway activity. |
|
在线阅读
查看全文 查看/发表评论 下载PDF阅读器 |