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通讯作者:王力群,副主任医师,Email:466167854@qq.com |
Exosome miR-182-5p increases chemosensitivity in gastric cancer cells by targeting the Cofilin1-PINK1-Parkin pathway |
投稿时间:2024-09-15 修订日期:2024-11-12 |
DOI: |
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中文关键词: 微小RNA-182-5p Cofilin1-PINK1-Parkin信号通路 胃癌 化疗敏感性 |
英文关键词:miRNA-182-5p Cofilin1-PINK1-Parkin signaling pathway gastric cancer chemotherapy sensitivity |
基金项目:福建省自然科学基金项目 (项目编号:2019J01539) ;福建省福州市临床重点专科学科建设计划(批准号:20220301) |
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中文摘要: |
目的 探讨外泌体miR-182-5p(exomiR-182-5p)促进胃癌(GC)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/耐药人癌症细胞株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+阴性组和miR-182-5p+CLF1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+CLF1组和miR-182-5p+阴性组分别将miR-182-5p mimics和pCDNA 3.1-CLF1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48h。利用双荧光素酶报告基因验证miR-182-5p和Cofilin 1转录基因(CLF1)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±2.95μg/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CLF1 mRNA水平显著降低(P<0.001)。双荧光素酶报告基因分析表明,CLF1 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+阴性组(P<0.05)。此外,与空白组和阴性组比较,miR-182-5p组和miR-182-5p+阴性组Cofilin1、PINK1和Parkin蛋白表达水平显著降低(P<0.05);而miR-182-5p+CFL1组Cofilin1、PINK1和Parkin蛋白表达水平较miR-182-5p+阴性组升高(P<0.05)。结论 exomiR-182-5p增加了耐药GC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: |
Objective To explore the effect of miR-182-5p (exomiR-182-5p) on the sensitivity of gastric cancer (GC) cells to chemotherapeutic drugs and its molecular mechanism. Methods The 5-fluorouracil (5-FU) sensitive/resistant human 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+negative group and miR-182-5p+CLF1 group. miR-182-5p group and the negative group were respectively transfected with miR-182-5p mimics or corresponding negative control sequence. miR-182-5p+CLF1 group and miR-182-5p+negative group were co-transfected with miR-182-5p mimics and pCDNA 3.1-CLF1 expression vector or control vector, respectively. After transfection, the exosomes were extracted and co-cultured with SGC-7901/5-FU cells for 48 hours. The interaction between miR-182-5p and Cofilin 1 transcriptional gene (CLF1) was verified by Luciferase reporter gene. Western blot method was used to detect the expression changes 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 μ g/mL),P<0.001. By PKH26 red-fluorescence labeling, SGC-7901-derived exosomes could be successfully absorbed by SGC-7901/5-FU receptor cells. Compared with the blank group and the negative group, the sensitivity of SGC-7901/5-FU cells in miR-182-5p group to 5-FU was increased, and IC50 decreased to 65.31 ± 3.94 μg/mL; meanwhile, the level of CLF1 mRNA was significantly decreased (P<0.001). Luciferase reporter gene analysis showed that CLF1 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+negative group (P<0.05). 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+negative group were significantly lower (P<0.05). The expression level of Cofilin1, PINK1 and Parkin protein in miR-182-5p+CFL1 group was higher than that in miR-182-5p+negative group (P<0.05). Conclusion ExomiR-182-5p increased the sensitivity of drug-resistant GC cells SGC-7901/5-FU to the chemotherapeutic drug 5-FU, and its mechanism may be related to the negative regulation of Cofilin1-PINK1-Parkin pathway signaling activity. |
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