Objective: To investigate the mechanism by which SIRT1 silencing reduces 5-fluorouracil (5-FU) resistance of cholangiocarcinoma cells and the role of FOXO1/Rab7 autophagy pathway in mediating this effect.
Methods: Human cholangiocarcinoma HCCC-9810 cells were treated with 50, 100, 150, and 200 μg/mL 5-FU to construct a 5-FU-resistant cell model, whose expressions of SIRT1, FOXO1 and Rab7 were detected with immunofluorescence assay, Western blotting and RTqPCR, and the expression levels of autophagy related proteins (Beclin1, LC3, and p62) were detected with Western blotting. The 5-FU resistant cells were transfected with a SIRT1 siRNA, and the changes in 5-Fu resistance and migration ability of the cells were evaluated using CCK-8 assay and wound healing assay; The changes in FOXO1 and Rab7 mRNA levels and protein expressions of SIRT1, FOXO1, Rab7, Beclin1, LC3 and P62 were detected with RT-qPCR and Western blotting.
Results: Treatments with 5-FU at 50, 100, 150, and 200 μg/mL all inhibited the proliferation of HCCC-9810 cells. Immunofluorescence assay revealed significantly enhanced SIRT1 expression in 5-FU-resistant HCC-9810 cells, and Western blotting also showed significantly up-regulated protein expressions of SIRT1, Rab7, P62, FOXO1 and Beclin 1 (P < 0.001) and an increased LC3II/LC3I ratio in the cells (P < 0.001). The mRNA levels of SIRT1, Rab7 and FOXO1 were also up-regulated in 5-Fu-resistant cells (P < 0.05). SIRT1 silencing significantly attenuated 5-FU resistance and migration ability of HCCC-9810 cells, and obviously decreased the protein expressions of SIRT1, Rab7, P62, FOXO1 and Beclin1 and the LC3II/LC3I ratio as well (P < 0.001). FOXO1 and Rab7 mRNA levels were significantly decreased in 5-FU-resistant HCC-9810 cells after SIRT1 silencing (P < 0.05).
Conclusion: Silencing SIRT1 attenuates 5-FU resistance in HCC-9810 cells by inhibiting the activation of the FOXO1/Rab7 autophagy pathway.
目的: 探讨SIRT1靶向FOXO1/Rab7自噬通路调节胆管癌细胞5-氟尿嘧啶（5-FU）耐药的机制。
方法: 不同浓度（50、100、150、200 μg/mL）的5-FU处理HCCC-9810细胞构建HCCC-9810/5-FU耐药细胞模型，免疫荧光检测细胞中SIRT1表达情况，Western blot和q-PCR检测SIRT1、FOXO1、Rab7表达水平，Western blot检测自噬相关蛋白Beclin1、LC3、p62表达水平。SIRT1 siRNA和NC siRNA转染HCCC-9810/5-FU耐药细胞，CCK-8检测细胞耐药性，划痕实验检测细胞迁移能力，RT-qPCR检测FOXO1、Rab7 mRNA水平，Western blot检测SIRT1、FOXO1、Rab7、Beclin1、LC3、p62蛋白表达水平。
结果: 不同浓度（50、100、150、200 μg/mL）的5-FU均能抑制HCCC-9810细胞增殖。与对照组相比，HCCC-9810/5-FU耐药细胞中SIRT1荧光强度略有增强；SIRT1、Rab7、p62、FOXO1和Beclin 1蛋白表达上调（P < 0.01），LC3Ⅱ/LC3Ⅰ比值增加（P < 0.001）；SIRT1、Rab7和FOXO1 mRNA水平上调（P < 0.05）。沉默SIRT1表达后，HCCC-9810细胞的5-FU耐药性降低，细胞迁移能力降低，SIRT1（P < 0.01）、Rab7（P < 0.05）、p62（P < 0.05）、FOXO1（P < 0.01）和Beclin1（P < 0.001）蛋白表达降低，LC3Ⅱ/LC3Ⅰ比值降低（P < 0.001）；FOXO1和Rab7 mRNA水平降低（P < 0.05）。
结论: 沉默SIRT1的表达抑制FOXO1/Rab7自噬通路激活。
Keywords: 5-fluorouracil; FOXO1/Rab7 autophagy pathway; SIRT1; cholangiocarcinoma cells; drug resistance.