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目的:体外合成4条靶向人组织激肽释放酶7(kallikrein-related peptidase 7,KLK7)基因的片段,并筛选最有效siRNA片段,观察沉默KLK7表达对胃癌AGS细胞增殖和凋亡的影响。方法:设计4条靶向KLK7的siRNA片段(KLK7-siRNA-416、KLK7-siRNA-596、KLK7-siRNA-474、KLK7-siRNA-795),瞬时转染AGS细胞,qRT-PCR检测各干扰组KLK7 mRNA表达的变化,Western blotting检测AGS细胞中HK7蛋白(由KLK7基因编码)的表达,MTT法检测转染后AGS细胞的增殖,流式细胞术检测AGS细胞的细胞周期及凋亡。结果:4条KLK7-siRNA片段中以KLK7-siRNA-416的干扰效率最高,KLK7-siRNA-416组KLK7 mRNA表达率显著低于NC组[(0.32±0.049)%vs(0.93±0.071)%,P<0.01],KLK7-siRNA-416组转染48 h后AGS细胞HK7蛋白的表达水平显著降低[(1.18±0.198)vs(0.52±0.096),P<0.01]。KLK7-siRNA-416转染72 h后对AGS细胞增殖的抑制率达(37.70±0.12)%(P<0.05),该转染阻滞AGS细胞于G0/G1期但不影响AGS细胞的凋亡。结论:KLK7-siRNA沉默KLK7的表达可抑制AGS细胞的增殖,可阻滞细胞于G0/G1期,对细胞凋亡的作用不明显。
OBJECTIVE: To synthesize four fragments of kallikrein-related peptidase 7 (KLK7) gene in vitro and screen the most effective siRNA fragment to observe the effect of silencing KLK7 expression on the proliferation and apoptosis of gastric cancer AGS cells. Methods: Four KLK7-targeting siRNA fragments (KLK7-siRNA-416, KLK7-siRNA-596, KLK7-siRNA-474 and KLK7-siRNA-795) were designed and transiently transfected into AGS cells. The expression of KLK7 mRNA in AGS cells was detected by Western blotting. The proliferation of AGS cells was detected by MTT assay. The cell cycle and apoptosis of AGS cells were detected by flow cytometry. Results: KLK7-siRNA-416 had the highest interference efficiency among the four KLK7-siRNA fragments. KLK7-siRNA-416 group had significantly lower KLK7 mRNA expression than that of NC group [(0.32 ± 0.049)% vs 0.93 ± 0.071% P <0.01]. After transfected with KLK7-siRNA-416 for 48 h, the expression of HK7 protein was significantly decreased in AGS cells [(1.18 ± 0.198) vs (0.52 ± 0.096, P <0.01]. The inhibition rate of AGS cells proliferation was (37.70 ± 0.12)% (P <0.05) at 72 h after transfection with KLK7-siRNA-416, which blocked the AGS cells in G0 / G1 phase but did not affect the apoptosis of AGS cells . Conclusion: KLK7-siRNA silence KLK7 expression can inhibit the proliferation of AGS cells, can block cells in G0 / G1 phase, the role of apoptosis was not obvious.