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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。
AIM: To investigate the effect of silenced nuclear factor-κB (NF-κB) gene on the proliferation of human gastric cancer cell line SGC-7901 by RNA interference and its possible mechanism. Methods: SGC-7901 cells were transfected with pcDNA (TM) 6.2-GW / EmGFPmiR-NF-κB silenced NF-κB gene and the stable transfected cells were screened. The experiment was divided into control group (SGC-7901 cells cultured in normoxia state), hypoxia group (SGC-7901 cells cultured in hypoxia), interference group (pcDNA6.2-GW / EmGFPmiR SGC-7901 cells cultured in normoxia with NF-κB) and in combination group (SGC-7901 cells stably transfected with pcDNA ™ 6.2-GW / EmGFPmiR-NF-κB were cultured in hypoxia ). The survival rate of SGC-7901 cells in each group was detected by trypan blue staining. The proliferation of SGC-7901 cells was detected by plate clone formation assay and cell doubling time method. The expressions of NF- κB, Hypoxia inducible factor-1α (HIF-1α) and phospholipase D-1 (PLD-1) protein levels were detected. Results: The recombinant plasmid pcDNA (TM) 6.2-GW / EmGFPmiR-NF-κB was successfully transfected into SGC-7901 cells and the stable transfected cells were screened. Compared with the control group, the survival rate of SGC-7901 cells in hypoxia group did not change significantly, while the survival rate of SGC-7901 cells in interference group and combination group decreased (P <0.05). The number of clone formation in SGC-7901 cells increased (P <0.05). The number of clone formation and cell doubling time of SGC-7901 cells in the interference group and the combination group were prolonged (P <0.05). The expressions of NF-κB, HIF-1α And the expression of PLD-1 protein in SGC-7901 cells were significantly down-regulated. The expression of NF-κB, HIF-1α and PLD-1 in SGC-7901 cells was significantly up-regulated in hypoxia group (P <0.05). Compared with hypoxia group, the expression of NF-κB, HIF-1α and PLD-1 in SGC-7901 cells was significantly down-regulated (P <0.05). CONCLUSIONS: Hypoxia can affect the expression of HIF-1α and NF-κB in SGC-7901 cells. Silencing NF-κB may decrease the expression of HIF-1α and PLD-1, 7901 cells proliferation ability.