ABSTRACT
OBJECTIVE: To study the effect of shRNA expressing vector targeting to id1 gene on the biological behavior of the esophageal cancer cell. METHODS: The specific shRNA sequence was designed, synthesized and cloned into pGFU6/neo vector. Then, the resulting recombinants were transfected into Eca-109 cells using Lipofetamine 2000 following the instruction of the manufactures. The expression levels of id1 mRNA were analyzed by RT-PCR and the levels of Id1, p16 and PCNA protein were detected by Western blot assay. The growth of Eca-109 cells was analyzed using clone formation assay and trypan blue exclusion assay. Distribution of cell cycle and proliferation of Eca-109 cells were assessed by flow cytometry. RESULTS: The sequence of recombinant plasmid was confirmed by sequence analysis. The expression levels of id1 mRNA and Id1 protein significantly decreased in Eca-109 cells transfected with pGFU6/neo-Id1-1 vector compared to the negative control group (P < 0.05). The recombinant plasmid expressing id1 shRNA could effectively inhibit cell proliferation and induce cell cycle arrest in G0/G1 phase with a remarkably decrease of cells in S phase (P < 0.05). CONCLUSION: All the above data suggested that the expression of id1 gene was significantly inhibited in Eca-109 cells transfected with pGFU6/neo-id1-1 recombinant. Furthermore, the proliferation of id1 knockdown cells was depressed.
