MUC1启动子驱动人钠/碘共转运体基因靶向表达于胰腺癌细胞的研究

Human mucin 1 promoter drives human sodium/iodide symporter gene targeting expression in pancreatic carcinoma cells

目的克隆人 Mucin (MUC1)黏蛋白的启动子序列,并研究其驱动人钠/碘共转运体(hNIS)基因在胰腺癌细胞内的靶向性表达功能。方法利用巢式 PCR 方法从人胰腺癌细胞株CAPAN-Ⅱ细胞中扩增出 MUC1启动子。采用基因重组方法构建质粒 pDC316-MUC1/hNIS。采用脂质体转染的方法把 pDC316-MUC1/hNIS 导入到 MUCI 阳性的人胰腺癌细胞株 CAPAN一Ⅱ、PANC-1和MUC1阴性的人宫颈癌细胞株 HeLa,转染后48h 采用 RT-PCR 方法及免疫组化方法检测转染细胞内的 hNIS mRNA 水平和蛋白表达,转染48h 后检测肿瘤细胞内 NIS 对125碘吸收功能。结果扩增的MUC1启动子核心调控区序列与参考序列一致。重组 pDC316-MUC1/hNIS 转染后48h,hNIS 蛋白在CAPAN-Ⅱ、PANC-1细胞阳性表达,而在 Hela 细胞内不表达。pDC316-MUC1/hNIS 转染后仅在CAPAN-Ⅱ、PANC-1细胞内检测到高水平吸碘,分别是 pDC316-MUC1转染组的7和12倍。结论MUC1启动子能驱动 NIS 基因在 MUC1阳性的肿瘤细胞内靶向功能表达,为进一步在体内运用放射碘靶向治疗胰腺癌奠定了基础。

Objective To clone human mucin 1 （MUC1） gene promoter and apply to drive human sodium/iodide symporter （hNIS） gene targeting expression in pancreatic carcinoma cells. Methods Human Mucinl （MUC1） promoter was cloned from the 5＇ flanking region of the MUC1 gene by two-step nest PCR from human pancreatic carcinoma cells of the line CAPAN-Ⅰ , Ⅱ and then linked to pDC316 plasmid （pDC316-MUC1）. Subsequently, a recombinant plasmid containing MUC1 and hNIS was constructed （pDC316-MUC1/hNIS）. The recombinant plasmid pDC316-MUC1/hNIS, pD316-mCMV/NIS plasmid, and pDC316-mCMV/hNIS plasmid were trasfected into the CAPAN-U cells, human pancreatic carcinoma cells of the line PANC-1, and human cervical carcinoma cells of the line HeLa respectively as experimental group, positive control group, and negative control group. 48 h after the transfection RT-PCR and immunofluorescence were used to confirm the expression of hNIS mRNA and hNIS protein. Then the cells were cultured in solution with ^125I. The ^125I uptake in the cells was measured by gammacounting. Results The sequence data of regulatory element in MUC1 promoter genes was corresponded to those of reference report. The hNIS protein expression level was high in the MUC1 positive cells, as CAPAN- Ⅱ cells and PANC-1 cells,but very low in the MUC1 negative cells, such as the HeLa cells. Two days after the transfection, the CAPAN- U cells and PANC-1 cells showed a high level of ^125I uptake after transfection with pDC316-MUC1/hNIS, and the CAPAN-U cells , PANC-1 cells, and HeLa cells showed a high level of ^125I uptake after transfection with pDC316-MCMV/ hNIS. A7 - 12-fold increase in ^125I uptake was observed in the pDC316-MUC1/hNIS transfected cells compared with the pDC316-MUC1 transfected cells. Conclusion MUC1 promoter cloned from CAPAN-2 cells can be used to drive NIS genes expression in MUC1 positive pancreatic carcinoma cells . Therefore, this strategy can be used as a novel and potent gene-targeting therapy in the MUC1 positive pancreatic carcinoma in vivo.