NF-κB p65的敲减加重结肠上皮HCT116细胞氧化损伤

Knockdown of NF-κB p65 Sensitizes Cellular Oxidative Damage in HCT116 Human Colonic Epithelial Cells

炎症细胞诱导的活性氧类生成和肠道氧化应激与慢性炎症性肠疾病以及结直肠肿瘤的发病密切相关.目前,核因子κB(NF-κB)信号通路在氧化应激反应、结直肠炎症和肿瘤发生中的作用还未研究清楚.本研究将化学合成的一对编码小干扰RNA序列、靶向人NF-κB基因长60 bp寡核苷酸链定向克隆至pSUPER小干扰RNA表达载体中,经单酶切、双酶切及测序证实重组RNA干扰载体构建成功.将构建成功的质粒转染至结肠上皮细胞HCT116中敲减p65,分别采用免疫印迹方法检测NF-κB p65蛋白表达水平,(3-(4,5-二甲基噻唑-2)-3,5-二苯基四氮唑溴盐(MTT)方法检测细胞存活情况.结果显示,pSUPER-NF-κB p65载体可特异性下调NF-κB p65蛋白表达;下调p65表达可导致过氧化氢诱导的HCT116内活性氧类物质生成增高,存活细胞数目显著减少,氧化损伤加重.研究表明,在人结肠上皮细胞内NF-κB p65通路的抑制显著加重了结肠上皮细胞氧化损伤情况.

Inflammation induces the generation reactive oxygen species （ROS） and tissue oxidative stress during the cause of chronic inflammatory disease and cancer in the digestive tract. The nuclear faetor-kappaB （NF-κB） signal pathway plays important roles in intestinal oxidative stress, colorectal inflammation and cancers. In this study, a small interference RNA （siRNA） of 60 base pairs targeting the NF-κB p65 gene was chemically synthesized, as well as cloned into a pSUPER plasmid. The plasmid was transfected into HCT116 cells. Western blot and MTT assays were conducted to determine the expression of NF-κB p65 and cell viability. The results demonstrated that down-regulation of p65 increased the production of ROS and sensitized the oxidative damage induced by H2O2. It suggested that inhibition of NF-κB signal pathway might sensitize cancer ceils to oxidative stresses.