[沉默SIRT1通过抑制FOXO1/Rab7自噬途径降低胆管癌细胞对5-氟尿嘧啶的耐药性]
[Silencing SIRT1 reduces 5-fluorouracil resistance of cholangiocarcinoma cells by inhibiting the FOXO1/Rab7 autophagy pathway].
作者信息
Xin C, Wang X, Li X, Chen Y, Wang X, Ning J, Yang S, Wang Z
机构信息
Department of Gastroenterology, Affiliated Hospital of Southwest Medical University, Luzhou 646000, China.
Department of Anesthesiology, Affiliated Hospital of Southwest Medical University, Luzhou 646000, China.
出版信息
Nan Fang Yi Ke Da Xue Xue Bao. 2023 Mar 20;43(3):454-459. doi: 10.12122/j.issn.1673-4254.2023.03.16.
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 ( < 0.001) and an increased LC3II/LC3I ratio in the cells ( < 0.001). The mRNA levels of SIRT1, Rab7 and FOXO1 were also up-regulated in 5-Fu-resistant cells ( < 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 ( < 0.001). FOXO1 and Rab7 mRNA levels were significantly decreased in 5-FU-resistant HCC-9810 cells after SIRT1 silencing ( < 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
探讨沉默SIRT1降低胆管癌细胞5-氟尿嘧啶（5-FU）耐药性的机制以及FOXO1/Rab7自噬途径在介导该效应中的作用。
方法
用50、100、150和200μg/mL的5-FU处理人胆管癌HCCC-9810细胞以构建5-FU耐药细胞模型，采用免疫荧光法、蛋白质印迹法和RTqPCR检测SIRT1、FOXO1和Rab7的表达，并用蛋白质印迹法检测自噬相关蛋白（Beclin1、LC3和p62）的表达水平。用SIRT1 siRNA转染5-FU耐药细胞，采用CCK-8法和伤口愈合试验评估细胞对5-Fu的耐药性和迁移能力的变化；用RT-qPCR和蛋白质印迹法检测FOXO1和Rab7 mRNA水平以及SIRT1、FOXO1、Rab7、Beclin1、LC3和P62的蛋白表达变化。
结果
50、100、150和200μg/mL的5-FU处理均抑制了HCCC-9810细胞的增殖。免疫荧光法显示5-FU耐药的HCC-9810细胞中SIRT1表达显著增强，蛋白质印迹法也显示细胞中SIRT1、Rab7、P62、FOXO1和Beclin 1的蛋白表达显著上调（<0.001），且细胞中LC3II/LC3I比值增加（<0.001）。5-Fu耐药细胞中SIRT1、Rab7和FOXO1的mRNA水平也上调（<0.05）。沉默SIRT1显著减弱了HCCC-9810细胞对5-FU的耐药性和迁移能力，并明显降低了SIRT1、Rab7、P62、FOXO1和Beclin1的蛋白表达以及LC3II/LC3I比值（<0.001）。沉默SIRT1后，5-FU耐药的HCC-9810细胞中FOXO1和Rab7 mRNA水平显著降低（<0.05）。
结论
沉默SIRT1通过抑制FOXO1/Rab7自噬途径的激活来减弱HCC-9810细胞对5-FU的耐药性。
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