Antisense Inhibition of microRNA-21 or-221 Arrests Cell Cycle, Induces Apoptosis, and Sensitizes the Effects of Gemcitabine in Pancreatic Adenocarcinoma

Objectives: The contribution of overexpressed microRNA-21 and -221 (miR-21 and miR-221) to the malignant phenotype was determined by inhibiting these miRNAs using antisense oligonucleotides. Methods: The effects of antisense to miR-21 and miR-221 on cell proliferation, cell cycle arrest, induction of apoptosis, combinatorial effects with gemcitabine, and effects on target protein levels were studied. Results: Low nanomolar concentrations of both antisense oligonucleotides reduced proliferation of pancreatic cancer cell lines. Reduced proliferation was less pronounced in the normal ductal epithelial cell line human pancreatic Nestin-expressing cell or in pancreatic cancer cell lines exposed to an irrelevant control oligonucleotide. Inhibition of miR-21 and miR-221 increased the amount of apoptosis in HS766T cells by 3- to 6-fold compared with the control oligonucleotide. HS766T cells exposed to miR-21 antisense resulted in cell cycle arrest (G(1) phase). Protein levels of tumor suppressor targets of the miRNAs were increased by antisense to miR-21 (PTEN and RECK) and miR-221 (p27(kip1)). Antisense to miR-21 and miR-221 sensitized the effects of gemcitabine, and the antisense-gemcitabine combinations were synergistic at high fraction affected. Conclusions: We demonstrate that antisense to miR-21 and miR-221 results in significant cell killing under various conditions and that antisense oligonucleotides targeted to miRNA represents a potential new therapy for pancreatic cancer.