下调Derlin-1对鼻咽癌CNE-2Z细胞化疗敏感性的作用及机制
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株洲市中心医院

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湖南省卫生健康委科研计划项目;项目编号:202107010022


The effect and mechanism of down-regulation of Derlin-1 on the chemosensitivity of CNE-2Z cells in Nasopharyngeal carcinoma
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    摘要:

    目的 探讨下调Derlin-1对鼻咽癌CNE-2Z细胞化疗敏感性的作用及机制。方法 收集鼻咽癌组织及癌旁正常组织并检测组织中Derlin-1 mRNA含量;本实验分组:空白对照组(control组)、阴性对照组(NC组)、敲低组(si-Derlin-1组),四甲基偶氮唑蓝(MTT)法检测不同顺铂浓度时三组细胞的增殖; Annexin V-FITC/PI双染法检测顺铂浓度为5 μmol/L时,三组细胞的凋亡率;Transwell 检测顺铂浓度为5 μmol/L细胞侵袭迁移能力;实时荧光定量PCR(RT-qPCR)法、Western blot法检测Bcl-2、Bax、MMP2、MMP9 mRNA及蛋白相对表达量。结果 RT-qPCR结果显示,鼻咽癌癌组织中Derlin-1 mRNA相对表达量较癌旁正常组织显著减少(P<0.05); MTT法结果显示,与空白对照组相比,si-Derlin-1组细胞增殖率明显降低,差异具有统计学意义(P<0.05);Annexin V-FITC/PI双染法结果可见,当 DDP浓度为5 μmol/L时,si-Derlin-1组细胞凋亡率为(29.65±2.35)%,较control组(13.24±1.43)%、NC组(15.09±1.32)%明显升高,差异具有统计学意义(P<0.05); Transwell实验结果显示,当 DDP浓度为5 μmol/L时,si-Derlin-1组细胞迁移率、侵袭率分别为(20.15 ± 2.20)%、(22.33 ± 3.5)%,较对照组的(100 ± 1.3)%、(99 ± 2.4)%明显降低,差异具有统计学意义(P<0.05);RT-qPCR结果显示,相比对照组,si-Derlin-1组凋亡抑制因子Bcl-2 mRNA的表达量下降,凋亡诱导因子Bax mRNA表达量上调;si-Derlin-1组迁移相关因子MMP2与MMP9 mRNA相对表达量明显下降,差异均具有统计学意义(P值均<0.05);Western blot结果显示,相比对照组,si-Derlin-1组凋亡抑制蛋白Bcl-2的表达量下降,凋亡诱导蛋白Bax表达量上调;si-Derlin-1组迁移相关蛋白MMP2与MMP9蛋白相对表达量明显下降,差异均具有统计学意义(P值均<0.05)。结论 Derlin-1在鼻咽癌中表达上调,下调Derlin-1可提高鼻咽癌CNE-2Z细胞化疗敏感性,机制可能与促进细胞凋亡、抑制迁移有关。

    Abstract:

    Objective To investigate the effect and mechanism of down-regulation of Derlin-1 on the chemosensitivity of CNE-2Z cells in Nasopharyngeal carcinoma. Method Head and Neck Surgery of the hospital collected Nasopharyngeal carcinoma tissues and normal adjacent tissues and detected the Derlin-1 mRNA content in the tissues; the experimental groups: blank control group (control group), negative control group (NC group), knockdown group (si -Derlin-1 group), the tetramethylazazole blue (MTT) method detects the proliferation of the three groups of cells at different cisplatin concentrations; Annexin V-FITC/PI double staining method detects the cisplatin concentration at 5 μmol/L, The apoptotic rate of the three groups of cells; Transwell detects the cisplatin concentration of 5 μmol/L cell invasion and migration ability; real-time fluorescent quantitative PCR (RT-qPCR) method, Western blot method to detect Bcl-2, Bax, MMP2, MMP9 mRNA and protein Relative expression. Results RT-qPCR results showed that the relative expression of Derlin-1 mRNA in Nasopharyngeal carcinoma tissues was significantly lower than that in normal tissues adjacent to the cancer (P<0.05). the results of MTT method showed that si-Derlin-1 The cell proliferation rate in the group was significantly reduced, and the difference was statistically significant (P<0.05). the results of Annexin V-FITC/PI double staining method showed that when the DDP concentration was 5 μmol/L, the cell apoptosis rate in the si-Derlin-1 group It was (29.65±2.35)%, which was significantly higher than the control group (13.24±1.43)% and the NC group (15.09±1.32)%, and the difference was statistically significant (P<0.05). Transwell experiment results showed that when the DDP concentration was At 5 μmol/L, the cell migration rate and invasion rate of the si-Derlin-1 group were (20.15 ± 2.20)% and (22.33 ± 3.5)%, respectively, compared with (100 ± 1.3)% and (99 ± 2.4) in the control group. % Was significantly reduced, and the difference was statistically significant (P<0.05). RT-qPCR results showed that compared with the control group, the expression of apoptosis-inhibiting factor Bcl-2 mRNA in the si-Derlin-1 group decreased and the apoptosis-inducing factor Bax mRNA expression was up-regulated. the relative expression of migration-related factors MMP2 and MMP9 mRNA in the si-Derlin-1 group was significantly decreased and the difference was statistically significant (P<0.05). Western blot results showed that compared with the control group, si- The expression of apoptosis-inhibiting protein Bcl-2 decreased in Derlin-1 group and the expression of apoptosis-inducing protein Bax increased.The relative expression of migration-related proteins MMP2 and MMP9 in si-Derlin-1 group decreased significantly and the differences were statistically significant. (P values are all <0.05). Conclusion Derlin-1 is up-regulated in Nasopharyngeal carcinoma. Down-regulation of Derlin-1 can increase the chemosensitivity of CNE-2Z cells in Nasopharyngeal carcinoma. The mechanism may be related to promoting cell apoptosis and inhibiting migration.

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  • 收稿日期:2022-03-25
  • 最后修改日期:2022-05-05
  • 录用日期:2022-05-06
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