Notch配体DLL3在胃癌细胞中的表达及对胃癌细胞增殖、转移的影响

EXPRESSION,PROLIFERATION AND METASTASIS OF NOTCH LIGAND DLL3 IN GASTRIC CANCER CELLS

目的研究分析Notch配体DLL3在胃癌细胞中的表达以及对胃癌细胞增殖、转移的影响。方法通过选用PCR扩增人全长DLL3基因并克隆至真核表达载体pCMV-Tgh4中,根据转染pCMV-Tag4质粒和无转染HEK293T细胞分为阴性对照和空白对照,进一步检测人全长DLL3基因在人正常胃上皮细胞GES-1和AGS等3株胃癌细胞中的表达差异,检测DLL3过表达后胃癌细胞的增殖以及DLL3下调后胃癌细胞的增殖。结果全长人DLL3/pCMV-Tag4为模板,PCR产物通过琼脂糖凝胶电泳后结果表示特异性目的条带可于1 800 bp左右可见,重组质粒能够得到4 300 bp与1 800 bp的片段,将其进一步进行DNA测序,测序结果比对后将该阳性克隆命为DLL3/pCMV-Tag4。无转染质粒HEK293T细胞与对照组相比,DLL3的mRNA水平明显升高,且于78 000 bp处存在一条特异性目的条带,对照组尚未检测出。人DLL3mRNA于AGS等胃癌细胞中表达明显高于正常胃上皮细胞GES-1,人DLL3在胃癌细胞中的表达量明显高于正常胃上皮细胞GES-1。转染DLL3/pCMV-Tag4后于68 000 bp以及78 000 bp处存在两条特异性目的条带,而对照组只有1条目的条带,DLL3在胃癌细胞中过表达成功,且DLL3的过表达能够促进胃癌细胞的增殖。转染24 h,DLL3-siRNA转染组中DLL3表达明显少于Control siRNA组,说明特异性人DLL3-siRNA在胃癌细胞中成功转移DLL3表达,且转移DLL3抑制了胃癌细胞的增殖。结论人全长DLL3/pCMV-Tgh4真核表达质粒能够成功建立,Notch配体中的DLL3与胃癌细胞增殖具有相关性,下调DLL3的表达水平能够有效抑制胃癌细胞的增殖。

Objective To investigate the expression,proliferation and metastasis of Notch ligand DLL3 in gastric cancer cells.Methods Human full-length DLL3 gene was amplified by PCR and cloned into eukaryotic expression vector pCMV-Tgh4.The transfected pCMV-Tag4 plasmid and non-transfected HEK293 T cells were divided into negative control and blank control to further detect the differential expression of human full-length DLL3 gene in human gastric epithelial cells GES-1 and AGS of three gastric cancer cells.The proliferation of cancer cell after DLL3 over-expression and the proliferation of gastric cancer cells after DLL3 down-regulation were detected.Results The full-length human DLL3/pCMV-Tag4 was used as a template,and the results of PCR product electrophoresis on agarose gel showed that the specific target band could be seen at around 1 800 bp,and that the recombinant plasmid could obtain 4 300 bp and 1 800 bp fragments,which were further subjected to DNA sequencing,then the positive clone named as DLL3/pCMV-Tag4.Compared to control group,the mRNA level of DLL3 was significantly increased in non-transfected plasmid HEK293 T cells,and a specific target band was present at 78 000 bp,which was not detected in the control group.The expression of human DLL3 mRNA in gastric cancer cells such as AGS was significantly higher than that in normal gastric epithelial cells;the expression of human DLL3 in gastric cancer cells was significantly higher than that innormal gastric epithelial cells.After transfection with DLL3/pCMV-Tag4,there were two specific target bands at 68 000 bp and 78 000 bp,while the control group had only one band.DLL3 was successfully overexpressed in gastric cancer cells,and overexpression of DLL3 promoted gastric cancer cells proliferation.At 24 h after transfection,the expression of DLL3 in the DLL3-siRNA transfection group was significantly lower than that in the Control si RNA group,indicating that the specific human DLL3-siRNA successfully transferred DLL3 expression in gastric cancer cells,and the transfer of DLL3 inhibited the proliferation of gastric cancer cells.Conclusion The full-length DLL3/pCMV-Tgh4 eukaryotic expression plasmid can be successfully constructed.The DLL3 in the Notch ligand was correlated with the proliferation of gastric cancer cells,which could be inhibited by the down-regulation of DLL3 expression.