下调内质网相关降解蛋白-1对鼻咽癌CNE-2Z细胞化疗敏感性的作用及机制

The effect and mechanism of down-regulation of Derlin-1 on the chemosensitivity of nasopharyngeal carcinoma cell CNE-2Z

摘要:

目的 探讨下调内质网相关降解蛋白(Derlin-1)对鼻咽癌CNE-2Z细胞株化疗敏感性的作用及机制。方法 收集10例鼻咽癌患者鼻咽癌组织及癌旁正常组织并通过实时荧光定量PCR(RT-qPCR)法检测组织中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)。RT-qPCR法检测鼻咽癌Derlin-1 mRNA的表达；采用四甲基偶氮唑蓝(MTT)法检测不同顺铂浓度时3组细胞的增殖；Annexin V-FITC/PI双染法检测顺铂浓度为5 μmol/L时3组细胞的凋亡率；Transwell检测顺铂浓度为5 μmol/L时3组细胞的侵袭迁移能力；实时荧光定量PCR(RT-qPCR)法、Western blot法检测Bcl-2、Bax、基质金属蛋白酶(MMP)2、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，差异具有统计学意义(P<0.05)；si-Derlin-1组Derlin-1 mRNA较control组、NC组明显下降，差异具有统计学意义(P<0.05)MTT法结果显示，与control组、NC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(绿色荧光)/PI(红色荧光)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(100±1.3)%、(99±2.43)%以及NC组(96.72±3.22)%、(94.44±1.21)%明显降低，差异具有统计学意义(F_迁移率=1080.610，P=0.000)、(F_侵袭率=848.590，P=0.000)；RT-qPCR结果显示，相比control组、NC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均<0.05)；Western blot结果显示，相比control组、NC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 explore effect and mechanism of down-regulation of Derlin-1 on the chemosensitivity of nasopharyngeal carcinoma cell (NPC) CNE-2Z.Methods The NPC tissues and adjacent normal tissues were collected from 10 NPC patients, and the content of Derlin-1 mRNA in the tissues was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The CNE-2Z cells were divided into blank control group (control group), negative control group (NC group), knockdown group (si-Derlin-1 group). RT-qPCR method was adopted to detect the expression of Derlin-1 mRNA in NPC, the tetramethylazazole blue (MTT) method to detect the proliferation of cells of the three groups at different cisplatin concentrations, Annexin V-FITC/PI double staining method to detect the apoptotic rate of cells of the three groups at cisplatin concentration of 5 μmol/L, Transwell to detect cell invasion and migration ability of the three groups at the cisplatin concentration of 5 μmol/L. RT-qPCR, Western blot were used to detect relative mRNA and protein expressions of Bcl-2, Bax, matrix metalloproteinase (MMP)2, and MMP9.Results RT-qPCR results showed that the relative expression of Derlin-1 mRNA in NPC tissues was significantly lower than that in adjacent normal tissues, and the difference was statistically significant (P<0.05). Compared with the control group and NC group, Derlin-1 mRNA in the si-Derlin-1 group was significantly decreased, and the difference was statistically significant (P <0.05). The results of MTT method revealed that the cell proliferation rate in the si-Derlin-1 group was significantly lower than those of the control group and NC group, and the difference was statistically significant (P<0.05). Annexin V-FITC/PI double staining showed that the apoptosis rate of si-Derlin-1 group was (29.65±2.35)% when the cisplatin concentration was 5 μmol/L, which was elevated compared with the control group (13.24±1.43)% and the NC group (15.09±1.32)%, the differences were statistically significant (P<0.05). The cell migration rate and invasion rate in the Derlin-1 group were (20.15±2.20)% and (22.33±3.5)% respectively, which were significantly decreased compared with those of the control group [(100±1.3)%, (99±2.43)%] and the NC group [(96.72 ±3.22) %, (94.44 ±1.21)%], and the differences were statistically significant (F migration rate=1080.610，P=0.000),(F invasion rate=848.590, P=0.000). RT-qPCR results showed that compared with the control group and NC group, the expression of mRNA of the apoptosis inhibitor Bcl-2 in the si-Derlin-1 group decreased, the expression of mRNA of apoptosis-inducing factor Bax was up-regulated, and the relative expressions of mRNA of migration-related factors MMP2 and MMP9 were significantly decreased, and the differences were statistically significant (all P<0.05); Western blot results showed that compared with the control group and NC group, the expression of apoptosis-inhibiting protein Bcl-2 in si-Derlin-1 group was decreased, and the expression of apoptosis-inducing protein Bax was up-regulated, the relative expression of MMP9 protein was significantly decreased, and the differences were statistically significant (all P<0.05).Conclusion Nasopharyngeal carcinoma; Derlin-1; Chemosensitivity; Apoptosis; Invasion and migration