Knockdown of Ezrin by RNA Interference Reverses Malignant Behavior of Human Pancreatic Cancer Cells in Vitro

Background: Pancreatic cancer is one of the most aggressive tumors with a dismal prognosis. The membranecytoskeletal crosslinker Ezrin participates in several functions including cell proliferation, adhesion, motilityand survival. There is increasing evidence that Ezrin is overexpressed in vast majority of malignant tumorsand regulates tumor progression. However, its roles in pancreatic cancer remain elusive.
Methods: Three pairsof specific Ezrin siRNAs were designed and synthetized and screened to determine the most efficient one forconstruction of a hairpin RNA plasmid targeting Ezrin. After transfection into the Panc-1 pancreatic cancer cellline, real-time quantitative PCR and Western blotting were performed to examine the expression of mRNA andprotein. The MTT method was applied to examine the proliferation and the drug sensibility to Gemcitabine.Flow cytometry was used to assess the cycle and apoptosis, while capacity for invasion was determined withtranswell chambers. Furthermore, we detected phosphorylated-Erk1/2 protein and phosphorylated-Akt proteinby Western blotting.
Results: Real-time quantitative PCR and Western blotting revealed that Ezrin expressionwas notably down-regulated at both mRNA and protein levels by RNA interference (P< 0.01). Proliferationwas inhibited and drug resistance to gemcitabine was improved (P< 0.05). Flow cytometry showed that theproportion of cells in the G1/G0 phase increased (P< 0.01), and in G2/M and S phases decreased (P< 0.05), withno apparent differences in apoptosis (P> 0.05). The capacity for invasion was markedly reduced (P< 0.01). Inaddition, down-regulating Ezrin expression had no effect on phosphorylated-Akt protein (P>0.05), but coulddecrease the level of phosphorylated-Erk1/2 protein (P< 0.05).
Conclusions: RNA interference of Ezrin couldinhibit its expression in the pancreatic cancer cells line Panc-1, leading to a potent suppression of malignantbehavior in vitro. Assessment of potential as a target for pancreatic cancer treatment is clearly warranted.