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 Mucin1 (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 transfected into the CAPAN-II 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 gamma-counting.
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-II cells and PANC-1 cells showed a high level of 125I uptake after transfection with pDC316-MUC1/hNIS, and the CAPAN-II 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.