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目的探讨慢病毒介导的核转录因子-κB诱导激酶和IκB激酶连接蛋白(NIBP)基因沉默后对人结肠癌HCT116细胞上皮-间质转化(EMT)的影响。方法通过DNA重组技术,进行设计并构建人NIBP基因的慢病毒载体以及无关序列的Lenti-EGFP慢病毒载体,感染HCT116细胞后分别为转染组(NIBP-RNAi组)和阴性对照组(NC组),而未予转染处理的HCT116细胞则为空白对照组(CON组)。将实验分为非肿瘤坏死因子-α(TNF-α)组和TNF-α组:非TNF-α组包括NIBP-RNAi组、NC组、CON组,TNF-α组包括NIBP-RNAi+TNF-α组(NIBP-RNAi+组)、NC+TNF-α组(NC+组)、CON+TNF-α组(CON+组),其中TNF-α组均予TNF-α进行干预。检测非TNF-α组NIBP mRNA及蛋白的表达情况,以及各组上皮性钙黏附分子(E-cadherin)和神经性钙黏附分子(N-cadherin)mRNA及蛋白的表达;采用倒置相差显微镜观察各组细胞形态的变化。结果 NIBP-RNAi组NIBP的mRNA及蛋白表达均显著低于NC组及CON组(P<0.05);与CON组比较,CON+组、NC+组的细胞发生明显的EMT,E-cadherin的mRNA和蛋白的表达均明显降低、N-cadherin的mRNA和蛋白的表达均明显上升(P<0.05),NIBP-RNAi组及NIBP-RNAi+组的细胞由间质细胞形态转变为上皮细胞形态,E-cadherin的mRNA和蛋白的表达均明显上升、N-cadherin的mRNA和蛋白的表达均明显降低(P<0.05);而CON组和NC组比较,以及CON+组和NC+组E-cadherin及N-cadherin的mRNA和蛋白表达比较,差异均无统计意义(P>0.05)。结论靶向下调NIBP基因表达可抑制人结肠癌HCT116细胞EMT的发生。
Objective To investigate the effect of lentivirus-mediated nuclear factor-kappa B-induced kinase and IκB kinase-linked protein (NIBP) gene silencing on the epithelial-mesenchymal transition (EMT) of human colon cancer HCT116 cells. Methods The lentiviral vector of human NIBP gene and Lenti-EGFP lentiviral vector of unrelated sequence were designed and constructed by DNA recombination technology. After being infected with HCT116 cells, the transfected group (NIBP-RNAi group) and the negative control group (NC group) ), While untransfected HCT116 cells were blank control group (CON group). The experiment was divided into non-tumor necrosis factor-α (TNF-α) group and TNF-α group: NIBP-RNAi group, NC group, CON group and TNF- TNF-αgroup (NIBP-RNAi + group), NC + TNF-αgroup (NC + group) and CON + TNF-αgroup (CON + group). The expression of NIBP mRNA and protein in non-TNF-α group and the expression of E-cadherin and N-cadherin mRNA and protein were detected by inverted phase contrast microscope Group cell morphology changes. Results The mRNA and protein expressions of NIBP in NIBP-RNAi group were significantly lower than those in NC group and CON group (P <0.05). Compared with CON group, EMT, E-cadherin mRNA and protein (P <0.05). The expression of N-cadherin mRNA and protein in NIBP-RNAi group and NIBP-RNAi + group was changed from mesenchymal cell morphology to epithelial cell morphology. The expression of E-cadherin (P <0.05). Compared with CON group and NC group, the mRNA expressions of E-cadherin and N-cadherin in CON + group and NC + group were significantly increased Compared with the protein expression, there was no significant difference (P> 0.05). Conclusion Targeted down-regulation of NIBP gene expression can inhibit the occurrence of EMT in human colon cancer HCT116 cells.