title: Reversal of multi-drug resistance by pSUPER-shRNA-mdr1 in vivo and in vitro.
abstract: OBJECTIVE
  To explore the possibility of reversing multi-drug resistance (MDR) to HepG2/mdr1 in vitro and in vivo with RNA interference (RNAi).
  
  
  METHODS
  HepG2/mdr1 was obtained by cloning the whole gene mdr1 into HepG2 cells. shRNA targeting sequence was designed to be homologous to the P-gp encoding MDR1 mRNA consensus sequence. pSUPER-shRNA/mdr1 was constructed using the enzyme-digested technique. HepG2/mdr1 cells were transfected with vectors of pSUPER-shRNA/mdr1 to measure their efficacy by real-time PCR for mdr1 mRNA, flow cytometry (FCM) for P-gp expression, and Rhodamine efflux, MTT method for HepG2/mdr1 function, respectively. In vivo, mice tumors were treated by injecting pSUPER-shRNA/mdr1 in situ and into intra-abdominal cavity. Tumors were collected to create cell suspension and cryosections after chemotherapy with adriamycin and mytomycin. The cell suspension was incubated in RPMI-1640 supplemented with G418 to screen stable cells for appreciating the reversal of MDR. Cryosections were treated with immunohistochemistry technique to show the effectiveness of transfection and the expression of P-gp.
  
  
  RESULTS
  pSUPER-shRNA/mdr1 was successfully constructed, which was confirmed by sequencing. The MDR phenotype of HepG2/mdr1 was decreased significantly in vitro transfection. HepG2/mdr1 showing its MDR was reversed notably in P-gp expression (11.0% vs 98.2%, P<0.01). Real-time PCR showed that mRNA/mdr1 was lower in test groups than in control groups (18.73+/-1.33 vs 68.03+/-2.21, P<0.001). Compared with HepG2, the sensitivity of HepG2/mdr1 and HepG2/mdr1-dsRNA cells to ADM was decreased by 1.64 times and 15.6 times, respectively. The accumulation of DNR in positive groups was decreased evidently. In vivo, the p-gp expression in positive groups was significantly lower than that in control groups (65.1% vs 94.1%, P<0.05). The tumor suppressing rate in test groups was 57.8%. After chemotherapy, the growth rate in test groups was lower than that in control groups (700.14+/-35.61 vs 1659.70+/-152.54, P<0.05). Similar results were also observed under fluorescence microscope, and confirmed by Image-Pro Plus 4.5 analysis.
  
  
  CONCLUSIONS
  pSUPER-shRNA/mdr1 vector system allows simple, stable and durable nonviral knockdown of P-gp by RNAi in malignant cells and animals to restore their sensitivity to adriamycin.
authors.affiliation: Department of General Surgery, People's Hospital of Liuzhou, Medical University of Guangxi, Liuzhou 545001, Guangxi Zhuang Autonomous Region, China. pgdhx@126.com
authors.forename: Guang-Dong
authors.initials: GD
authors.lastname: Pan
authors.forename: Jian-Qing
authors.initials: JQ
authors.lastname: Yang
authors.forename: Lv-Nan
authors.initials: LN
authors.lastname: Yan
authors.forename: Guang-Ping
authors.initials: GP
authors.lastname: Chu
authors.forename: Qiang
authors.initials: Q
authors.lastname: Liu
authors.forename: Yi
authors.initials: Y
authors.lastname: Xiao
authors.forename: Lin
authors.initials: L
authors.lastname: Yuan
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors* / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D045744:Cell Line, Tumor; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D019450:Genes, MDR; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009374:Neoplasms, Experimental / Q000188:drug therapy; D010957:Plasmids / Q000235:genetics; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D004317:Doxorubicin