Antisense inhibition of microRNA-21 or -221 arrests cell cycle, induces apoptosis, and sensitizes the effects of gemcitabine in pancreatic adenocarcinoma.
OBJECTIVE
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 (G1 phase). Protein levels of tumor suppressor targets of the miRNAs were increased by antisense to miR-21 (PTEN and RECK) and miR-221 (p27). 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.
Park
Jong-Kook
JK
Ohio State University, Columbus, 43210, USA.
Lee
Eun Joo
EJ
Esau
Christine
C
Schmittgen
Thomas D
TD
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects* / Q000235:genetics; D002460:Cell Line; D045744:Cell Line, Tumor; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D016193:G1 Phase / Q000187:drug effects / Q000235:genetics; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D016376:Oligonucleotides, Antisense / Q000235:genetics*; D051059:PTEN Phosphohydrolase / Q000378:metabolism; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D000093542:Gemcitabine
D000964:Antimetabolites, Antineoplastic; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003841:Deoxycytidine; D035683:MicroRNAs; D016376:Oligonucleotides, Antisense; D051059:PTEN Phosphohydrolase; D000093542:Gemcitabine; C524940:MIRN21 microRNA, human; C525709:MIR221, human; C494929:PTEN protein, human