摘要
目的研究靶向鸟苷酸环化酶C(GC-C)的短发夹RNA(shRNA)对人胃癌SGC-7901细胞增殖、凋亡和细胞周期等生物学功能的影响。方法构建针对人GC-C基因的shRNA真核表达载体,将其转染入人胃癌SGC-7901细胞,采用半定量RT-PCR和Western blotting法检测细胞中GC-C在mRNA和蛋白水平的变化。以CCK-8法、流式细胞术和原位末端标记法检测转染GC-CshRNA真核表达载体的SGC-7901细胞增殖、凋亡及细胞周期等生物学指标的变化。结果经DNA测序鉴定,所得到的表达载体插入片段序列与GenBank中的序列一致。shRNA-GC-C真核表达载体转染能有效抑制GC-C基因的表达,以shRNA-GC-CⅢ序列的抑制效果最佳。转染GC-CshRNA真核表达载体后SGC-7901细胞的增殖能力受到抑制(P<0.05);细胞形态发生改变,细胞体积缩小,核固缩,染色质浓缩聚集于核膜;流式细胞术检测显示G1峰前出现亚二倍体峰,转染48、72h后凋亡率分别为5.47%和5.63%(P<0.05)。结论靶向GC-C基因的shRNA可有效抑制人胃癌SGC-7901细胞生长,并促进细胞凋亡,该作用与下调靶基因GC-C的表达有关;GC-C可能是胃癌治疗的一个潜在靶点。
Objective To investigate the effects of synthetic guanylyl cyclase-C（GC-C） short hairpin RNA（shRNA） on the biological function of human gastric cancer,SGC-7901 cell line,including cell proliferation,cell cycle,cell apoptosis,etc.Methods SGC-7901 cell line was cultured.shRNA eukaryotic expression vector that can express shRNA of GC-C was constructed,and transfected into SGC-7901 in vitro.GC-C expression was measured by RT-PCR and Western blotting.Cell viability was analyzed by CCK-8.Cell cycle was determined with flow cytometry（FCM）.Apoptosis analysis was performed with TUNEL assay.Results The sequencing results confirmed that the sequences of the three shRNAs were correct,and shRNA-GC-C Ⅲ was found to have the best inhibitory effect on GC-C expression.The inhibitory rate was about 75%.The proliferation of SGC-7901 cell line was markedly inhibited by shRNA-GC-C Ⅲ（P〈0.05）.There was changes in cell morphology,such as cell volume reduction,karyopyknosis and chromatin conglomeration.FCM showed that GC-C inhibition caused the appearance of sub-G0/G1 peak which increased from 5.47%（48h after transfection） to 5.63%（72h after transfection）.Conclusions The shRNA of GC-C gene can effectively induce growth inhibition of SGC-7901 cells and cell apoptosis,which is possibly induced by down regulating GC-C gene.GC-C might be a potential target for the therapy of gastric cancer.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2009年第10期1164-1167,共4页
Medical Journal of Chinese People's Liberation Army
