Antisense transfection of gene ANK-B inhibits proliferation, invasion and tumorigenicity of Panc1 cells

Introduction: Cytoskeletal protein ankyrin-B (encoded by ANK-B) gene was cloned by RACE method in previous work. It exhibited an overexpression in pancreatic cancer cells and tissues. The antisense ANK-B was introduced into Panc1 cells, resulting in a reduced proliferation, invasion and tumorigenicity of transfected cells.

Methods: Rapid amplification of cDNA end (RACE) was used to clone the full-length cDNA of ANK-B. The gene expression was inhibited by transfecting antisense ANK-B into Panc1 cells. The effect on cell proliferation and metastasis of ANK-B silencing was evaluated by WST-1 assay, invasion and migration assay, zymography, in vivo assay.

Results: Downregulation of gene ANK-B was confirmed by RT-PCR, real time RT-PCR, and Western blot. By WST-1 assay, a reduction of 30% proliferation of transfected cells was observed. Silencing of ANK-B decreased the invasive (45.9%) and migratory (64.6%) capability of transfected cells in comparison to parental Panc1 cells. Moreover, RT-PCR and zymography exhibited inhibition of MMP2 expression both in RNA level and protein level. Most importantly, the tumor forming efficiency of antisense ANK-B transfected cells was attenuated both in vitro: analyzed by soft agar assay (59.5%), and in vivo: detected by tumorigenicity assay (45%).

Conclusion: This study shows that suppression of ANK-B not only attenuates proliferation, invasion in vitro, but also attenuates tumorigenicity of Panc1 pancreatic carcinoma cells in vivo. Our findings suggest that ANK-B is implicated in the development of pancreatic cancer. The identification of pancreatic cancer related ‘novel’ gene-ANK-B may contribute to diagnosis and treatment of pancreatic disease.