迷迭香酸通过下调 FOXO4 靶向 miR-6785-5p 降低胃癌细胞对 5-氟尿嘧啶的耐药性。

Rosmarinic acid reduces the resistance of gastric carcinoma cells to 5-fluorouracil by downregulating FOXO4-targeting miR-6785-5p.

机构信息

Department of Integrated TCM & Western Medicine, Jiangsu Cancer Hospital & Jiangsu Institute of Cancer Research & The Affiliated Cancer Hospital of Nanjing Medical University, Nanjing, Jiang Su, 210000.

Department of Oncology, The First Affiliated Hospital of Soochow University, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, Nan Jing Medical University Affiliated Cancer Hospital, Suzhou, Jiang Su, 210000.

出版信息

Biomed Pharmacother. 2019 Jan;109:2327-2334. doi: 10.1016/j.biopha.2018.10.061. Epub 2018 Nov 30.

DOI:10.1016/j.biopha.2018.10.061

PMID:30551491

Abstract

OBJECTIVE

Chemoresistance has been a major problem in cancer chemotherapy. The present study aimed to investigate the effect of Rosmarinic acid (RA) on chemoresistance to 5-Fu and its molecular mechanism in gastric carcinoma.

METHODS

CCK8 cell proliferation and apoptosis assay were used to evaluate the effect of RA on chemoresistance to 5-Fu in GC cells. RNA microarray was used to identify miRNA involved. Expression level of miRNA in GC cells was determined by RT-PCR. Down- or up-regulating of miRNA in the GC cells was performed by transfection of RNA interference or expression vectors in the GC cells. Double luciferase reporter assay was used to verify miRNA target genes. Expression of P-glycoprotein and Bax was analyzed with Western blot.

RESULTS

RA treated SGC7901/5-Fu cells showed significant increased chemosensitivity to 5-Fu. The IC50 of 5-Fu was significantly reduced in RA treated SGC7901/5-Fu cells (70.43 ± 1.06 μg/mL) compared to untreated SGC7901/5-Fu cells (208.6 ± 1.09 μg/mL) (P < 0.05). Apoptosis rate was significantly increased in RA+5-Fu treated SGC7901/5-Fu cells compared to 5-FU treatment alone (P < 0.01). Two miRNAs, namely miR-642a-3p and miR-6785-5p, were identified to be involved in the chemo-sensitizing effect of RA in the SGC7901/5-Fu cells. RA treated SGC7901/5-Fu cells showed reduced expression levels of miR-642a-3p and miR-6785-5p compared to untreated SGC7901/5-Fu cells (P < 0.05). Down- or up-regulation of miR-6785-5p increased or reduced chemosensitivity of gastric carcinoma cells to 5-Fu, respectively. RA treated SGC7901/5-Fu and the SGC7901/5-Fu-Si cells showed significantly increased FOXO4 expression (P < 0.01). Double luciferase reporter assay confirmed miR-6785-5p directly targets FOXO4 to regulate its expression. RA significantly reduced P-gp expression and increased Bax expression in SGC7901/5-Fu and the SGC7901/5-Fu-Si cells (P < 0.05).

CONCLUSION

RA enhances chemosensitivity of resistant gastric carcinoma SGC7901 cells to 5-Fu by downregulating miR-6785-5p and miR-642a-3p and increasing FOXO4 expression. These study suggest the potential for RA as a multidrug resistance-reversing agent in GC.

摘要

目的

化疗耐药性一直是癌症化疗中的一个主要问题。本研究旨在探讨迷迭香酸（RA）对胃癌细胞对 5-Fu 化疗耐药性的影响及其分子机制。

方法

CCK8 细胞增殖和凋亡实验用于评估 RA 对 GC 细胞对 5-Fu 耐药性的影响。使用 RNA 微阵列鉴定涉及的 miRNA。通过 RT-PCR 测定 GC 细胞中 miRNA 的表达水平。通过转染 RNA 干扰或表达载体在 GC 细胞中下调或上调 miRNA。双荧光素酶报告实验验证 miRNA 靶基因。Western blot 分析 P-糖蛋白和 Bax 的表达。

结果

RA 处理的 SGC7901/5-Fu 细胞对 5-Fu 的化疗敏感性显著增加。与未经处理的 SGC7901/5-Fu 细胞（208.6±1.09μg/mL）相比，RA 处理的 SGC7901/5-Fu 细胞中 5-Fu 的 IC50 明显降低（70.43±1.06μg/mL）（P<0.05）。与单独使用 5-FU 治疗相比，RA+5-Fu 处理的 SGC7901/5-Fu 细胞中的凋亡率显著增加（P<0.01）。鉴定出两种 miRNA，即 miR-642a-3p 和 miR-6785-5p，参与了 RA 在 SGC7901/5-Fu 细胞中的化疗增敏作用。与未经处理的 SGC7901/5-Fu 细胞相比，RA 处理的 SGC7901/5-Fu 细胞中 miR-642a-3p 和 miR-6785-5p 的表达水平降低（P<0.05）。下调或上调 miR-6785-5p 分别增加或降低胃癌细胞对 5-Fu 的化疗敏感性。RA 处理的 SGC7901/5-Fu 和 SGC7901/5-Fu-Si 细胞中 FOXO4 表达明显增加（P<0.01）。双荧光素酶报告实验证实 miR-6785-5p 可直接靶向 FOXO4 调节其表达。RA 显著降低 SGC7901/5-Fu 和 SGC7901/5-Fu-Si 细胞中 P-糖蛋白的表达并增加 Bax 的表达（P<0.05）。