摘要
目的:探讨沉默四次跨膜蛋白1(Tspan1)基因表达对结直肠癌(CRC)细胞奥沙利铂(OXA)耐药的影响及作用机制。方法:收集31例OXA化疗敏感(OXA敏感组)和26例OXA化疗耐药(OXA耐药组)的CRC患者肿瘤组织,RT-qPCR和Western blot检测肿瘤组织中Tspan1 mRNA和蛋白表达水平。采用OXA浓度递增刺激法建立OXA耐药细胞株HCT116/OXA,MTT法检测耐药细胞株增殖活性,计算半数抑制浓度(IC_(50))和耐药指数(RI);RTqPCR和Western blot检测耐药细胞株及其亲本细胞中Tspan1 mRNA和蛋白表达水平。通过siRNA技术沉默HCT116/OXA细胞中Tspan1基因表达,RT-qPCR和Western blot检测转染后耐药细胞株中Tspan1 mRNA和蛋白表达水平。采用不同浓度OXA干预转染后的耐药细胞株,MTT法检测细胞增殖活性并计算IC_(50)值,流式细胞术检测细胞凋亡;免疫荧光实验检测自噬指标蛋白LC3B表达情况;Western blot检测细胞中LC3、beclin-1、P62等自噬蛋白的表达水平。结果:与OXA敏感组比较,OXA耐药组患者肿瘤组织中Tspan1 mRNA和蛋白表达水平升高(P<0.01)。不同浓度OXA处理48 h后,耐药细胞株HCT116/OXA及其亲本HCT116细胞的IC_(50)值分别为(101.6±2.7)和(16.0±0.5)mg/L,RI值为6.4。耐药细胞株HCT116/OXA中Tspan1表达水平较其亲本HCT116细胞显著升高(P<0.01)。Tspan1基因沉默可显著降低HCT116/OXA细胞中Tspan1 mRNA和蛋白表达水平及其对OXA的耐药性(P<0.05),提高OXA暴露下HCT116/OXA细胞的凋亡率(P<0.05),降低细胞中LC3B蛋白荧光强度及细胞中LC3-II/LC3-I蛋白比值和beclin-1蛋白表达水平(P<0.05),增加P62蛋白的表达(P<0.05)。结论:沉默Tspan1基因表达可通过抑制细胞自噬逆转结直肠癌细胞OXA耐药。
AIM:To examine the impact of inhibiting the expression of the tetraspanin 1(Tspan1)gene on the resistance of colorectal cancer(CRC)cells to oxaliplatin(OXA),as well as to elucidate the underlying mechanism.METHODS:This study involved the collection of cancer tissues from two groups of patients with CRC:one group comprising 31 patients who exhibited sensitivity to OXA chemotherapy(referred to as the OXA sensitive group),and the other group comprising 26 patients who showed resistance to OXA chemotherapy(referred to as the OXA resistant group).The mRNA and protein expression levels of Tspan1 in the cancer tissues were measured using quantitative reverse transcriptionpolymerase chain reaction(RT-qPCR)and Western blot analysis.To establish an OXA-resistant cell line,HCT116/OXA,the concentration of OXA was incrementally increased.The proliferation activity of the drug-resistant cell lines was assessed using the MTT assay,and the half inhibitory concentration(IC_(50))and drug resistance index(RI)were calculated.The mRNA and protein expression levels of Tspan1 were measured in both the drug-resistant HCT116/OXA cells and the parent HCT116 cells using RT-qPCR and Western blot analysis.Small interfering RNA(siRNA)technology was employed to silence the expression of the Tspan1 gene in HCT116/OXA cells,and the mRNA and protein expression levels of Tspan1 were measured in the transfected drug-resistant cell lines.Various concentrations of OXA were administered to the transfected drug-resistant cell lines,and the MTT assay was employed to assess cell proliferation activity and calculate the IC_(50) value.Cell apoptosis was examined using flow cytometry.The immunofluorescence intensity of the autophagy index LC3B was determined using an immunofluorescence assay,and the expression levels of autophagy-related proteins such as LC3,beclin-1,and P62 were evaluated using Western blot analysis.RESULTS:In comparison to the OXA sensitive group,the expression levels of Tspan1 mRNA and protein in the cancer tissues of patients in the OXA resistant group were significantly elevated(P<0.01).Likewise,the HCT116/OXA cells demonstrated higher mRNA and protein expression levels of Tspan1 compared to the HCT116 cells(P<0.01).The IC_(50) values of the drug-resistant cell line HCT116/OXA and its parent HCT116 cells were(101.6±2.7)and(16.0±0.5)mg/L,respectively,resulting in an RI value of 6.4 after 48 h of treatment with various OXA concentrations.The mRNA and protein expression levels of Tspan1 in the drug-resistant HCT116/OXA cell line was significantly greater than that in the parent HCT116 cell line(P<0.01).Silencing the expression of the Tspan1 gene significantly reduced the mRNA and protein expression levels of Tspan1 and reversed the drug resistance to OXA in HCT116/OXA cells(P<0.05).It also increased the rate of apoptosis in HCT116/OXA cells when exposed to OXA(P<0.05).Additionally,it led to a decrease in the fluorescence intensity of the LC3B protein,a downregulation of the LC3-II/LC3-I protein ratio and the expression level of beclin-1 protein in cells(P<0.05),and an increase in the expression of the P62 protein(P<0.05).CONCLUSION:Inhibiting autophagy through the silencing of Tspan1 gene expression effectively reversed OXA resistance in CRC cells.
作者
李孝平
周红见
高芳芳
李伟
LI Xiaoping;ZHOU Hongjian;GAO Fangfang;LI Wei(Department of Hepatobiliary Pancreatic Thyroid Gastrointestinal Surgery,Wuhan 430074,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2023年第8期1415-1421,共7页
Chinese Journal of Pathophysiology
基金
武汉市医学科研项目(No.WX20D26)。
作者简介
通讯作者:李伟,Tel:027-65399959,E-mail:lw523626@163.com。
