同源无启动子DNA片段抑制人胰腺癌细胞株整合基因的表达

Promoterless DNA fragments inhibiting the expression of integrated gene in human pancreatic carcinoma cells

目的观察仅转染无启动子绿色荧光蛋白(GFP)的 cDNA 全长片段能否使整合有 GFP基因的胰腺癌细胞内 GFP 表达降低。方法用 pEGFP-C1质粒转染人胰腺癌细胞株 PANC-1,G418筛选及流式细胞术分选建立 GFP 蛋白高表达的稳定细胞株。用 PCR 方法从 pEGFP-C1扩增出 GFP基因全长 cDNA 并将其克隆至无启动子的复制型质粒 PUC19,得到重组质粒即 PUC-GFP。实验分为4组:空白对照组、空质粒组(PUC 组)、GFP 重组质粒干扰组(PUC-GFP 组)、GFP 小 RNA 干扰组(siGFP 组)。分别用稳定表达 GFP 和未经处理 PANC-1细胞进行实验。用 Western 印迹、流式细胞术与倒置荧光显微镜检测重组质粒对细胞内稳定表达的 GFP 的影响及对 GFP 阴性细胞 pEGFPC1质粒瞬时转染后绿色荧光表达强度的影响。结果 PUC-GFP 可使稳定细胞株内的 GFP 表达下降,并且呈剂量依赖性。PUC-GFP 组绿色荧光强度减弱程度和空白对照及空质粒组差异有统计学意义(P<0.05)和 siGFP 组差异无统计学意义(P>0.05)。转染后第4天 GFP 表达降低并可持续至第6天,第4天后 PUC-GFP 组和 siGFP 组对 GFP 的抑制差异无统计学意义(P>0.05)。PUC-GFP 与 pEGFP-C1共转染 GFP 阴性 PANC-1细胞株可使 pEGPC1的瞬时转染效率降低。这两种情况下,荧光降低程度均和转染 PUC-GFP 的量呈正相关。结论仅转染无启动子的某个特定基因的全长 cDNA 能使哺乳细胞内相应同源性基因的表达降低。DNA 干扰可用于哺乳动物细胞。

Objective , To investigate whether introducing promoterless DNA containing the cDNA sequence of green fluorescent protein （GFP） induces gene-specific silencing in human pancreatic cancer cell line harboring genomically integrated GFP gene. Methods Using G418 selection and fluorescent separation we established a highly purified monoclonal pancreatic cancer cell line, recombinant PANC-1, which had a steady level of GFP expression. GFP cDNA was amplified by PCR from plasmid pEGFP-C1 and ligated to the promoterless plasmid PUC19. PUC-GFP was then transfected into monoclone cells along or cotransfected with pEGFP-C1 to panc-1 cells. Each had PUC plasmid transfected group as their control to eliminate possibility of plasmid toxicity and GFP small interferent RNA （siGFP） transfected group as positive control. Western blot, flow cytometry and phase contrast fluorescence microscopy were used to detect the changes of GFP expression. Results The data showed that （ 1 ） PUC-GFP inhibited the GFP expression in monoclonal cell line in a dosage dependent manner. The inhibitory effect of 3 μg PUC-GFP did not show significant difference with siGFP. （2） The significant repression appeared on the fourth day after transfecting monoclonal cells with 3 μg PUC-GFP. By the end of the sixth day, GFP expression in PUC-GFP group and siGFP group remained at a low level. （3） Cotransfecting PUC-GFP with pEGFP-C1 plasmids into PANC-1 ceils showed a decreased transfection efficiency when compared with transfecting pEGFP-C1 alone. Higher PUC-GFP vs pEGFP-C1 corresponded with lower transfection efficency. （4） When adding new pEGFP-C1 plasmid to cells after inhibition appeared, the GFP expression recovered. Conclusion Transfection of the promoterless DNA fragment containing full-length cDNA effectively induces a gene-specific silencing in mammalian cells.