南方医科大学学报

南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (1): 187-193.doi: 10.12122/j.issn.1673-4254.2024.01.22

• • 上一篇    下一篇

LncRNA SOX2OT靶向SIRT1/自噬通路增强胆管癌细胞5-FU耐药

辛 辰，王笑影，李 响，陈 宇，王 雪，宁佳曦，杨 适，王忠琼   

  1. 西南医科大学附属医院消化内科，麻醉科，四川 泸州 646000
  • 发布日期:2024-01-24

LncRNA SOX2OT enhances 5-fluorouracil resistance of cholangiocarcinoma cells by promoting autophagy via up-regulating SIRT1 expression

XIN Chen, WANG Xiaoying, LI Xiang, CHEN Yu, WANG Xue, NING Jiaxi, YANG Shi, WANG Zhongqiong   

  1. Department of Gastroenterology, Department of Anesthesiology, Affiliated Hospital of Southwest Medical University, Luzhou 646000, China
  • Published:2024-01-24

RichHTML

21

PDF

266

摘要： 目的 探讨LncRNA SOX2OT靶向SIRT1/自噬通路调节胆管癌细胞5-FU耐药的机制。方法 将未用5-FU处理HCCC-9810细胞和不同浓度（50、100、150、200 μg/mL）5-FU处理的HCCC-9810细胞分为对照组和模型组，qRT-PCR检测LncRNASOX2OT、SIRT1 mRNA表达水平，Western blot检测SIRT1、Beclin1、LC3和p62蛋白表达。pcDNA3.1-SOX2OT和pcDNA3.1-NC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，qRT-PCR检测LncRNA SOX2OT、SIRT1 mRNA表达水平，Western blot检测SIRT1、Beclin1、LC3和p62表达。在以上基础上做了Rescue 实验，将OV-SOX2OT（2 μg/孔）和si-NC（75 pmol/孔）、OV-SOX2OT（2 μg/孔）和si-SIRT1（75 pmol/孔）分别共转染HCCC-9810/5-FU耐药细胞，分组为OV-SOX2OT+si-NC组和OV-SOX2OT+si-SIRT1组，以证明LncRNA SOX2OT 通过SIRT1影响自噬，并由此影响胆管癌细胞对5-FU的耐药性。RNA Pulldown验证SOX2OT与SIRT1的靶向结合关系。结果 不同浓度的5-FU均能抑制HCCC-9810细胞增殖（P<0.05）。与对照组相比，模型组SIRT1、Beclin1（P<0.001）和p62（P<0.01）蛋白表达、LC3Ⅱ/LC3Ⅰ比值（P<0.001）、SIRT1和LncRNA SOX2OT mRNA 水平（P<0.05）均明显增加。与 OV-NC 组相比，OV-SOX2OT 组细胞迁移能力、SIRT1、Beclin1（P<0.001）和p62（P<0.05）蛋白表达、LC3Ⅱ/LC3Ⅰ比值（P<0.001）、SIRT1和LncRNA SOX2OT mRNA（P<0.05）水平均明显增加。沉默SIRT1表达导致LncRNA SOX2OT过表达的HCCC-9810细胞对5-FU的耐药性显著降低。与OV-SOX2OT+si-NC 组相比，OV-SOX2OT+si-SIRT1 组 SIRT1（P<0.001）、p62 和 Beclin1（P<0.01）蛋白表达、LC3Ⅱ/LC3Ⅰ比值（P<0.01）、SIRT1 mRNA（P<0.05）水平均明显降低。RNA Pulldown 检测结果显示 SOX2OT 能够直接与 SIRT1 结合。结论 LncRNA SOX2OT通过上调SIRT1表达促进自噬来增强胆管癌HCCC-9810细胞5-FU耐药性。

关键词: LncRNA SOX2OT；SIRT1；自噬；胆管癌细胞；5-FU耐药

Abstract: Objective To investigate the role of SIRT1/autophagy pathway in mediating the regulatory effect of lncRNA SOX2OT on 5-fluorouracil (5-FU) resistance in cholangiocarcinoma cells. Methods HCCC-9810 cells were used to construct a 5-FU-resistant cell model (HCCC-9810/5-FU cells), and the expression levels of lncRNA SOX2OT and SIRT1 mRNA and the protein expressions of SIRT1, Beclin1, LC3 and P62 were detected with qRT-PCR and Western blotting. The effects of transfection with a SOX2OT mimic on drug resistance and cell migration of HCCC-9810/5-FU cells were detected using CCK-8 assay and wound healing assay, and the changes in expressions of SOX2OT, SIRT1, Beclin1, LC3 and P62 were detected. Rescue experiment was performed by co- transfection of HCCC-9810/5-FU cells with both a SOX2OT-overexpressing plasmid and si-SIRT1 to confirm the role of SIRT1 in SOX2OT-mediated regulation of 5-FU resistance. A RNA pulldown assay was used to verify the targeted binding between SOX2OT and SIRT1. Results The proliferation of HCCC-9810 cells was significantly inhibited after treatment with different concentrations of 5-FU (P<0.05). The 5-FU-resistant cells showed significantly increased protein expressions of SIRT1, Beclin1 and p62, an increased LC3Ⅱ/LC Transfection of the resistant cells with SOX 3Ⅰ ratio, and enhanced expressions of SIRT1 mRNA and SOX2OT (P<0.05). 2OT mimic significantly enhanced cell migration and increased the protein expressions of SIRT1, Beclin1 and p62, the LC3Ⅱ/LC3Ⅰ ratio, and expression levels of SIRT1 mRNA and SOX2OT (P<0.05), and these changes were obviously attenuated by SIRT1 knockdown, which also resulted in lowered 5-FU resistance of the cells without significantly affecting the expression level of SOX2OT (P>0.05). RNA pulldown assay suggested that SOX2OT could directly bind to SIRT1. Conclusion LncRNA SOX2OT enhances 5-FU resistance in HCCC-9810 cells by promoting autophagy through up-regulating SIRT1 expression.

Key words: LncRNA SOX2OT; SIRT1; autophagy; cholangiocarcinoma cells; 5-FU resistant