title: Knockdown of Ezrin by RNA interference reverses malignant behavior of human pancreatic cancer cells in vitro.
abstract: BACKGROUND
  Pancreatic cancer is one of the most aggressive tumors with a dismal prognosis. The membrane cytoskeletal crosslinker Ezrin participates in several functions including cell proliferation, adhesion, motility and survival. There is increasing evidence that Ezrin is overexpressed in vast majority of malignant tumors and regulates tumor progression. However, its roles in pancreatic cancer remain elusive.
  
  
  METHODS
  Three pairs of specific Ezrin siRNAs were designed and synthetized and screened to determine the most efficient one for construction of a hairpin RNA plasmid targeting Ezrin. After transfection into the Panc-1 pancreatic cancer cell line, real-time quantitative PCR and Western blotting were performed to examine the expression of mRNA and protein. 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 with transwell chambers. Furthermore, we detected phosphorylated-Erk1/2 protein and phosphorylated-Akt protein by Western blotting.
  
  
  RESULTS
  Real-time quantitative PCR and Western blotting revealed that Ezrin expression was notably down-regulated at both mRNA and protein levels by RNA interference (P< 0.01). Proliferation was inhibited and drug resistance to gemcitabine was improved (P< 0.05). Flow cytometry showed that the proportion of cells in the G1/G0 phase increased (P< 0.01), and in G2/M and S phases decreased (P< 0.05), with no apparent differences in apoptosis (P> 0.05). The capacity for invasion was markedly reduced (P< 0.01). In addition, down-regulating Ezrin expression had no effect on phosphorylated-Akt protein (P>0.05), but could decrease the level of phosphorylated-Erk1/2 protein (P< 0.05).
  
  
  CONCLUSIONS
  RNA interference of Ezrin could inhibit its expression in the pancreatic cancer cells line Panc-1, leading to a potent suppression of malignant behavior in vitro. Assessment of potential as a target for pancreatic cancer treatment is clearly warranted.
authors.affiliation: Department of General Surgery, Beijing Tiantan Hospital, Capital Medical University, Beijing, China.
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Zhong
authors.forename: Mao-Min
authors.initials: MM
authors.lastname: Song
authors.forename: Ying
authors.initials: Y
authors.lastname: He
authors.forename: Shi
authors.initials: S
authors.lastname: Cheng
authors.forename: Hui-Sheng
authors.initials: HS
authors.lastname: Yuan
mesh_terms: D000964:Antimetabolites, Antineoplastic / Q000494:pharmacology; D017209:Apoptosis; D015153:Blotting, Western; D002448:Cell Adhesion; D002453:Cell Cycle; D002465:Cell Movement; D049109:Cell Proliferation; D003598:Cytoskeletal Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D015536:Down-Regulation; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D005434:Flow Cytometry; D006801:Humans; D009361:Neoplasm Invasiveness; D010190:Pancreatic Neoplasms / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D000093542:Gemcitabine; D000099149:Ezrin
chemical_list: D000964:Antimetabolites, Antineoplastic; D003598:Cytoskeletal Proteins; D003841:Deoxycytidine; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000093542:Gemcitabine; D000099149:Ezrin