Abstract: 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 RT-qPCR, 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.

Key words: SIRT1; FOXO1/Rab7 autophagy pathway; cholangiocarcinoma cells; 5-fluorouracil; drug resistance