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目的构建重组慢病毒介导的Nup88-sh RNA载体,由RNA干扰(RNAi)技术沉默Nup88,研究其对结肠癌HT-29细胞的增殖、黏附、侵袭和转移的影响,为结肠癌的临床基因治疗寻找新的靶点。方法 Nup88重组慢病毒载体的构建后包装检验效价,以最佳感染复数转染结肠癌HT-29细胞,实验分为沉默组、阴性对照组和空白组3组,其中沉默组分为Nup88-sh RNA1、Nup88-sh RNA2、Nup88-sh RNA3、Nup88-sh RNA4 4个亚组。通过逆转录-聚合酶链反应(RT-PCR)和Western blot印迹检测各组表达效率,主要是HT-29细胞的m RNA和蛋白;四甲基偶氮唑盐(MTT)法和流式细胞术检测Nup88基因被干扰后对HT-29细胞增殖和凋亡的影响;细胞侵袭实验检测Nup88基因被干扰后对HT-29细胞侵袭力的影响。结果沉默4组病毒及1组阴性对照均构建成功,滴度均为4E+8 TU/m L。沉默组Nup88 m RNA与阴性对照组和空白组相比,蛋白表达差异均有显著统计学意义(P<0.01)。Nup88-sh RNA1转染后,沉默率可达到86%。沉默组细胞增殖程度显著减少,与空白组和阴性对照组相比,差异有统计学意义(P<0.05)。沉默组细胞凋亡率(30.28%±0.19%)显著增加,与阴性对照组(4.15%±0.24%)和空白组(2.98%±0.27%)相比,差异有统计学意义(P<0.05)。培养肿瘤细胞常规24 h后,沉默组穿膜细胞数(120.5±8.7)和抑制率(49.22%)与阴性对照组(232.2±13.4,-2.14%)和空白组(276.4±10.2,0)相比,穿膜细胞数明显减少,抑制率明显增加,差异具有统计学意义(P<0.05)。结论 Nup88重组慢病毒可以通过RNAi成功抑制HT-29细胞中Nup88基因的表达,并能显著抑制其增殖及远处的侵袭能力。
Objective To construct a recombinant lentivirus-mediated Nup88-sh RNA vector and silence the expression of Nup88 by RNA interference (RNAi) technology to investigate the effect of Nup88 on the proliferation, adhesion, invasion and metastasis of colon cancer HT-29 cells. Treatment looking for new targets. Methods Nup88 recombinant lentiviral vector was used to construct recombinant retroviral vector. The recombinant plasmid was transfected into HT-29 cells with the best multiplicity of infection. The experiment was divided into three groups: silencing group, negative control group and blank control group. Nup88- sh RNA1, Nup88-sh RNA2, Nup88-sh RNA3, Nup88-sh RNA4 four subgroups. The expression efficiency of each group was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, mainly m RNA and protein of HT-29 cells; MTT assay and flow cytometry The effect of Nup88 gene on the proliferation and apoptosis of HT-29 cells was detected by the interference assay. The cell invasion assay was used to detect the influence of Nup88 gene on the invasiveness of HT-29 cells. Results Silencing 4 viruses and 1 negative control group were successfully constructed with 4E + 8 TU / m L titer. Compared with the negative control group and the blank group, Nup88 m RNA expression in the silence group was significantly different (P <0.01). Nup88-sh RNA1 transfection, the silence rate can reach 86%. Silencing cells significantly reduced the degree of cell proliferation, compared with the blank group and the negative control group, the difference was statistically significant (P <0.05). Compared with the blank control group (4.15% ± 0.24%) and the blank control group (2.98% ± 0.27%), the apoptosis rate of the silencing group was significantly increased (30.28% ± 0.19%, P <0.05) . After cultured for 24 h, the number of transmembrane cells (120.5 ± 8.7) and the inhibition rate (49.22%) in the silencing group were significantly lower than those in the negative control group (232.2 ± 13.4 and -2.14%) and the blank group (276.4 ± 10.2 and 0) Compared with the control group, the number of transmembrane cells was significantly decreased and the inhibition rate was significantly increased. The difference was statistically significant (P <0.05). Conclusion Nup88 recombinant lentivirus can successfully inhibit the expression of Nup88 gene in HT-29 cells through RNAi, and significantly inhibit its proliferation and distant invasion ability.