title: [Down regulation of multidrug resistance-associated protein 4 expression by RNA interference enhances radiosensitivity of colorectal carcinoma cell lines in vitro].
abstract: OBJECTIVE
  To investigate the effect of multidrug resistance-associated protein 4 (MRP4) expression on the radiosensitivity of colorectal carcinoma cell lines in vitro.
  
  
  METHODS
  The vector of shRNA for RNA interference was constructed and then transfected into HCT116 cell line to steadily down-regulate the expression of MRP4. HCT116 cells were divided into 3 groups including the CON group(non-transfected), NC group (negative control virus was added), and KD group (RNAi target was added for transfection). To test the effectiveness of RNA interference, real-time polymerase chain reaction and Western blot were used to measure the expression pattern of MRP4 at both mRNA and protein levels, respectively. For the examination of the effect of RNA interference of MRP4 on the radiosensitivity, flow cytometry was used to calculate the rate of apoptotic cells 24 h after 4 Gy radiation. Proliferation of the cells was measured via MTT assay at different time points.
  
  
  RESULTS
  ShRNA plasmid was successfully constructed. Transfection of this constructed vector into HCT116 cell line caused steady silencing of MRP4 expression (HCT116-KD). MRP4 mRNA and protein expression were significantly down-regulated following RNA interference(P<0.05). Twenty-four hours after radiation, the apoptosis rate of KD cell line was (71.7±0.8)%, significantly higher than that in the CON group [(56.1±0.9)%] and NC group[(59.8±0.8)%](P<0.05). Fourty-eight hours and 72 hours after radiation, the proliferation was significantly inhibited in KD cells compared to the control groups(P<0.05).
  
  
  CONCLUSIONS
  Expression of MRP4 is closely related to radio-tolerance of colorectal carcinoma. Down-regulation of MRP4 expression by RNA interference enhances radiosensitivity of colorectal carcinoma cell lines in vitro. MRP4 may be an effective molecular marker for predicting the radiosensitivity of colorectal carcinoma.
authors.affiliation: Department of Colorectal Surgery, The Second Military Medical University, Shanghai, China.
authors.forename: Zhi-qi
authors.initials: ZQ
authors.lastname: Yu
authors.forename: Chang
authors.initials: C
authors.lastname: Zhang
authors.forename: Rui
authors.initials: R
authors.lastname: Cai
authors.forename: Xin-yuan
authors.initials: XY
authors.lastname: Lao
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.forename: Xian-hua
authors.initials: XH
authors.lastname: Gao
authors.forename: Yi-fang
authors.initials: YF
authors.lastname: Han
authors.forename: Xiao-qing
authors.initials: XQ
authors.lastname: Zhang
authors.forename: Guang-wen
authors.initials: GW
authors.lastname: Cao
authors.forename: Chuan-gang
authors.initials: CG
authors.lastname: Fu
mesh_terms: D015179:Colorectal Neoplasms / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D045325:HCT116 Cells; D006801:Humans; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics*; D034622:RNA Interference; D011836:Radiation Tolerance / Q000235:genetics*
chemical_list: D027425:ATP-Binding Cassette, Sub-Family C Proteins; C073492:ABCC4 protein, human