硫氧还蛋白相互作用蛋白通过抑制自噬途径促进结肠腺癌细胞的凋亡

Thioredoxin interacting protein promotes the occurrence of colon adenocarcinoma cell apoptosis by inhibiting autophagy pathway

目的:探究过表达硫氧还蛋白相互作用蛋白(thioredoxin interacting protein,TXNIP)对结肠腺癌细胞凋亡的影响及其机制。方法:收集我院30例结肠腺癌患者的结肠腺癌组织及癌旁组织,实时荧光定量PCR检测组织中TXNIP mRNA表达,免疫组织化学染色检测组织中自噬标志蛋白LC3-Ⅱ与凋亡标志蛋白Cleaved Caspase-3表达;将人结肠腺癌LoVo细胞随机分为对照组、阴性空载体组(LV-GFP)和TXNIP过表达组(LV-GFP-TXNIP),倒置荧光显微镜下观察细胞转染效率,收集转染72 h后的LoVo细胞,通过流式细胞术检测细胞凋亡,透射电子显微镜观察细胞自噬情况,免疫荧光染色检测细胞中LC3表达,Western blot检测细胞中自噬相关蛋白与凋亡相关蛋白表达变化。结果:与癌旁组织比较,结肠腺癌组织中TXNIP mRNA相对表达量显著降低(P<0.01),LC3-Ⅱ阳性表达率显著升高(P<0.05),Cleaved Caspase-3阳性表达率显著降低(P<0.05);慢病毒转染72 h后转染效率较高(P<0.05);与对照组比较,LV-GFP-TXNIP组LoVo细胞的凋亡率显著增加(P<0.05),细胞质较为致密,自噬体数目明显减少,LC3荧光染色减弱,Beclin-1、LC3-Ⅱ蛋白表达显著下降(P<0.05),而LC3-I、p62蛋白表达显著增加(P<0.05),同时,Bax、Cleaved Caspase-3蛋白表达显著增加(P<0.05),Bcl-2蛋白表达显著下降(P<0.05)。结论:在结肠腺癌细胞中过表达TXNIP可通过抑制自噬来促进细胞凋亡。

Objective:To explore the effect and mechanism of overexpression of thioredoxin interacting protein(TXNIP)on the apoptosis of colon adenocarcinoma cells.Methods:Collect colon adenocarcinoma and paracancerous tissues from 30 patients with colon adenocarcinoma in our hospital.Real-time fluorescence quantitative PCR detected the expression of TXNIP mRNA in the tissue.Immunohistochemical staining was used to detect the expression of autophagy marker protein LC3-Ⅱand apoptosis marker protein Cleaved Caspase-3 in tissues.Human colon adenocarcinoma LoVo cells were randomly divided into control group,negative empty vector group(LV-GFP)and TXNIP overexpression group(LV-GFP-TXNIP).Observe cell transfection efficiency under an inverted fluorescence microscope.Collect LoVo cells 72 h after transfection,detect cell apoptosis by flow cytometry.Transmission electron microscope observes the autophagy of cells.Immunofluorescence staining detected the expression of LC3 in cells.Western blot was used to detect changes in the expression of autophagy-related proteins and apoptosis-related proteins in cells.Results:Compared with adjacent tissues,the relative expression of TXNIP mRNA in the colon adenocarcinoma tumor tissue was significantly decreased(P<0.01),the positive expression rate of LC3-Ⅱwas significantly increased(P<0.05),and the positive expression rate of Cleaved Caspase-3 significantly reduced(P<0.05).72 h after lentivirus transfection,the transfection efficiency was higher(P<0.05).Compared with the control group,the apoptosis rate of LoVo cells in the LV-GFP-TXNIP group was significantly increased(P<0.05),the cytoplasm was denser,the number of autophagosomes was significantly reduced,the fluorescence staining of LC3 was weakened,and the expression of Beclin-1 and LC3-Ⅱproteins significantly decreased(P<0.05),while the expression of LC3-I and p62 protein increased significantly(P<0.05).At the same time,the protein expression of Bax and Cleaved Caspase-3 increased significantly(P<0.05),and the protein expression of Bcl-2 decreased significantly(P<0.05).Conclusion:Overexpression of TXNIP in colon adenocarcinoma cells can promote apoptosis by inhibiting autophagy.