沉默NF-κB基因对缺氧胃癌细胞增殖的影响及其机制

The influence of silencing of NF-κB gene on the proliferation of gastric cancer cells exposed to hypoxia and its mechanism

目的:探讨通过RNA干扰技术沉默核因子-κB(nuclear factor-κB,NF-κB)基因对缺氧胃癌SGC-7901细胞增殖的影响及其可能的机制。方法:将沉默NF-κB基因的重组载体pcDNA^(TM)6.2-GW/EmGFPmiR-NF-κB转染SGC-7901细胞,并筛选稳定转染的细胞株。实验分为对照组(SGC-7901细胞在常氧状态下培养)、缺氧组(SGC-7901细胞在缺氧状态下培养)、干扰组(稳定转染pcDNA^(TM)6.2-GW/EmGFPmiR-NF-κB后的SGC-7901细胞在常氧状态下培养)和联合组(稳定转染pcDNA^(TM)6.2-GW/EmGFPmiR-NF-κB后的SGC-7901细胞在缺氧状态下培养)。应用锥虫蓝染色法检测各组SGC-7901细胞的存活率,平板克隆形成实验和细胞倍增时间法检测各组SGC-7901细胞的增殖变化,蛋白质印迹法检测各组SGC-7901细胞中NF-κB、缺氧诱导因子-1α(hypoxia inducible factor-1α,HIF-1α)和磷脂酶D-1(phospholipase D-1,PLD-1)蛋白的表达水平。结果:重组载体pcDNA^(TM)6.2-GW/EmGFPmiR-NF-κB成功转染至SGC-7901细胞,并筛选获得稳定转染的细胞株。与对照组比较,缺氧组SGC-7901细胞的存活率变化不明显,干扰组和联合组SGC-7901细胞的存活率降低(P<0.05);缺氧组SGC-7901细胞的克隆形成数增加,细胞倍增时间缩短(P<0.05);干扰组和联合组SGC-7901细胞的克隆形成数减少,细胞倍增时间延长(P<0.05);干扰组SGC-7901细胞中NF-κB、HIF-1α和PLD-1蛋白的表达水平明显下调,缺氧组SGC-7901细胞中NF-κB、HIF-1α和PLD-1蛋白的表达水平明显上调(P<0.05)。与缺氧组比较,联合组SGC-7901细胞中NF-κB、HIF-1α和PLD-1蛋白的表达水平明显下调(P<0.05)。结论:缺氧条件下,SGC-7901细胞中HIF-1α与NF-κB可能存在相互关系,沉默NF-κB基因可降低HIF-1α和PLD-1的表达,从而抑制缺氧条件下胃癌SGC-7901细胞的增殖能力。

Objective： To investigate the influence of nuclear factor-κB （NF-κB） gene silence on the proliferation of gastric cancer SGC-7901 cells exposed to hypoxia and its possible mechanism. Methods： SGC-7901 cells were transfected with recombinant vector pcDNATM 6.2-GW/EmGFPmiR-NF-κB in which the NF-κB was silenced, and the stably transfected cell line SGC-7901 was screened out. There are four groups were designed in this study： control group （SGC-7901 cells were cultured under normoxic conditions）, hypoxic group （SGC-7901 cells were cultured under hypoxic conditions）, transfection group （SGC-7901 cells were transfected with recombinant vector pcDNATM 6.2-GW/EmGFPmiR-NF-κB and cultured under normoxic conditions） and combination group （SGC-7901 cells were transfected with recombinant vector pcDNATM6.2-GW/EmGFPmiR-NF-κB and cultured under hypoxic conditions）. The viability of SGC-7901 cells in each group was detected byTrypan blue staining. The colony-formation assay and cell doubling time assay were used to examine the proliferation of SGC-7901 cells. The expression levels of NF-κB, hypoxia-inducible factor-l~ （HIF-I~） and phospholipase D1 （PLD1） proteins in SGC-7901 cells were examined by Western blotting. Results： The SGC-7901 cells were successfully transfected with recombinant vector pcDNATM6.2-GW/EmGFPmiR-NF-KB, and the stably transfected cell line was established. As compared with the control group, the viabilities of SGC-7901 cells in the transfection group and the combination group were obviously decreased （P 〈 0.05）, but there was no significant change in the hypoxic group. As compared with the control group, the colony-forming number of SGC-7901 cells in the hypoxic group was increased and the cell doubling time was shortened （P 〈 0.05）, while the numbers of colony-forming in the transfection group and combination group were decreased and the cell doubling time was prolonged （P 〈 0.05）. The expression levels of NF-κB, HIF-lα and PLD1 proteins in SGC-7901 cells in the transfection group were obviously suppressed, while these expressions in the hypoxic group were obviously up-regulated （P 〈 0.05）. As compared with the hypoxic group, the expression levels of NF-KB, HIF-Iα and PLD1 proteins in SGC-7901 cells in the combination group were significanly down-regulated （P 〈 0.05）. Conclusion： In SGC-7901 cells exposed to hypoxic conditions, there may be an interaction between HIF-lcL and NF-κB. The suppression of NF-KB expression can inhibit the proliferative ability of gastric cancer SGC-7901 cells under hypoxic conditions via down-regulation of HIF-lα and PLD-1 expressions