实用医学杂志 ›› 2023, Vol. 39 ›› Issue (9): 1072-1078.doi: 10.3969/j.issn.1006⁃5725.2023.09.002

• 基础研究 • 上一篇    下一篇

Tspan1通过诱导细胞自噬拮抗奥沙利铂诱导的结直肠癌细胞凋亡

李孝平 周红见 高芳芳 李伟    

  1. 武汉市第三医院肝胆胰甲状腺胃肠外科(武汉 430047)

  • 出版日期:2023-05-10 发布日期:2023-05-10
  • 通讯作者: 李伟 E⁃mail:lw523626@163.com
  • 基金资助:
    武汉市医学科研项目(编号:WX20D26) 

Tspan1 antagonizes oxaliplatin⁃induced apoptosis in colorectal cancer cells by inducing cellular autophagy

LI Xiaoping,ZHOU Hongjian,GAO Fangfang,LI Wei.   

  1. Department of Hepatobiliary Pancreatic Thyroid Gastrointes⁃ tinal Surgery,Wuhan 430074,China
  • Online:2023-05-10 Published:2023-05-10
  • Contact: LI Wei E⁃mail:lw523626@163.com

摘要:

目的 探讨四次跨膜蛋白 1(Tspan1)过表达对奥沙利铂(OXA)诱导的人结直肠癌细胞系 HCT116 细胞凋亡的影响并分析其可能作用机制。方法 将 Tspan1 过表达质粒及其阴性对照空载质粒分 别转染至 HCT116 细胞中,qRT⁃PCR 和 Western blot 法检测胞中 Tspan1 mRNA 及其蛋白表达水平。采用 14.15 μg/mL OXA 或联合 5 mmol/L 自噬抑制剂 3⁃MA 干预转染后 HCT116 细胞,MTT 检测细胞增殖活性;流 式细胞术检测细胞凋亡水平;免疫荧光检测细胞中 LC⁃3B 蛋白表达情况;Western blot 法检测细胞中自噬 相关蛋白 LC⁃3、Beclin1 和 p62 蛋白表达水平。结果 Tspan1 过表达可显著上调 HCT116 细胞中 Tspan1 mRNA 和蛋白表达水平,降低HCT116细胞对OXA 的敏感性,抑制OXA 诱导的HCT116细胞凋亡,并增强细 胞中 LC3B 蛋白荧光强度,上调细胞中 LC⁃3II/LC⁃3I 比值及 Beclin1 蛋白表达水平,下调 p62 蛋白表达水平。 然而,联合 3⁃MA 处理可逆转 Tspan1 过表达对 OXA 诱导 HCT116 细胞凋亡的抑制作用,提高细胞对 OXA 的 敏感性。结论 Tspan1 过表达可拮抗 OXA 诱导的 HCT116 细胞凋亡,其机制可能是通过诱导细胞自噬来实现的。

关键词:

结直肠癌, 四次跨膜蛋白1, 奥沙利铂, 自噬, 凋亡

Abstract:

Objective To investigate the effect of tetraspanin 1(Tspan1)overexpression on oxaliplatin (OXA)⁃induced apoptosis in human colorectal cancer cell line HCT116 and to analyze its possible mechanism of action. Methods HCT116 cells were transfected with Tspan1 overexpression plasmid and its negative control vec⁃ tor plasmid. The expression levels of Tspan1 mRNA and protein were detected by qRT⁃PCR and Western blot. The transfected HCT116 cells were treated with 14.15 μg/mL OXA or combined with 5 mmol/L autophagy inhibitor 3⁃ MA. MTT assay was used to detect cell proliferation. Flow cytometry was used to detect cell apoptosis. The expres⁃ sion of LC⁃3B protein was detected by immunofluorescence. The expression levels of autophagy⁃related proteins LC⁃ 3,Beclin1 and P62 were detected by Western blot. Results Overexpression of Tspan1 significantly up⁃regulated Tspan1 mRNA and protein expression levels in HCT116 cells,reduced the sensitivity of HCT116 cells to OXA,in⁃ hibited OXA⁃induced apoptosis of HCT116 cells,and enhanced the fluorescence intensity of LC3B protein,up⁃reg⁃ ulated the protein expression level of Beclin1 and the ratio of LC⁃3II/LC⁃3I,down⁃regulated the protein expression level of p62,and antagonized the induction of apoptosis by OXA. However,combined treatment with 3⁃MA re⁃ versed the inhibitory effect of Tspan1 overexpression on OXA ⁃induced apoptosis of HCT116 cells,and increased the sensitivity of cells to OXA. Conclusion Overexpression of Tspan1 antagonizes OXA ⁃ induced apoptosis in HCT116 cells by a mechanism that may be mediated through the induction of cellular autophagy.

Key words:

colorectal cancer, tetraspanin 1, oxaliplatin, autophagy, apoptosis