title: Inhibition of multi-drug resistance of ovarian carcinoma by small interfering RNA targeting to MRP2 gene.
abstract: OBJECTIVE
  To investigate the inhibiting mechanism of multi-drug resistance (MDR) using expression vectors of short hairpin RNA (shRNA) in a MDR human ovarian cancer cell line (A2780/cp70).
  
  
  METHODS
  Two shRNA expression vectors were constructed and introduced into A2780/cp70 cells. Expression of MRP2 mRNA was assessed by RT-PCR, and Mrp2 expression was determined by Western blot and immunocytochemistry. Apoptosis and sensitization of the cells to cisplatinum were quantified by flow cytometry and methyl-thiazol-tetrazolium (MTT) assays. Cellular cisplatinum accumulation was assayed by laser scanning confocal microscopy (LSCM).
  
  
  RESULTS
  In A2780/cp70 cells transfected with MRP2-A and MRP2-B shRNA expression vectors, RT-PCR showed that MRP2 mRNA expression was reduced by 41.8% (P < 0.05), 30.9% (P < 0.01) (transient transfection) and 39.6% (P < 0.05), 29.4% (P < 0.01) (stable transfection), respectively. Western blot and immunocytochemistry showed that Mrp2 expression was significantly and specifically inhibited. Resistance against cisplatinum was decreased from 173- to 119-fold (P < 0.05), 64-fold (P < 0.01) (transient transfection) and to 117-fold (P < 0.05), 60-fold (P < 0.01) (stable transfection). Furthermore, shRNA vectors significantly enhanced the cellular cisplatinum accumulation. The combination of shRNA vectors and cisplatinum significantly induced the apoptosis of cells.
  
  
  CONCLUSIONS
  shRNA expression vectors effectively reduce MRP2 expression and can restore the sensitivity of drug-resistant cancer cells to conventional chemotherapeutic agents.
authors.affiliation: Department of Obstetric and Gynecology, Xijing Hospital, Fourth Military Medical University, Xi'an, 710033, China, majiajia@fmmu.edu.cn.
authors.forename: Jia-jia
authors.initials: JJ
authors.lastname: Ma
authors.forename: Bi-liang
authors.initials: BL
authors.lastname: Chen
authors.forename: Xiao-yan
authors.initials: XY
authors.lastname: Xin
mesh_terms: D017209:Apoptosis; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002945:Cisplatin / Q000378:metabolism / Q000494:pharmacology; D018432:Drug Resistance, Multiple / Q000235:genetics*; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D010051:Ovarian Neoplasms / Q000188:drug therapy* / Q000235:genetics; D016133:Polymerase Chain Reaction; D034741:RNA, Small Interfering / Q000235:genetics*; D014162:Transfection
chemical_list: D002945:Cisplatin; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D034741:RNA, Small Interfering; C000715087:ABCC2 protein, human