title: RNAi silencing targeting RNF8 enhances radiosensitivity of a non-small cell lung cancer cell line A549.
abstract: OBJECTIVE
  The E3 ubiquitin ligase RNF8 regulates the accumulation or removal of a number of proteins at DNA lesions, thereby playing a critically important role in DNA damage response. The present study investigated the possibility of using RNF8 as a new target in the radiation treatment of human non-small cell lung cancer.
  
  
  METHODS
  We used RNA interference technology to silence the expression of RNF8 in A549 cells, and then detected the radiation response by colony forming assays. DNA repair was monitored by γ-H2AX foci formation after RNF8 depletion. Expression of Ku70 and Rad51 were assessed by immunofluorescent staining and Western blotting. Cell cycle and apoptosis were measured by flow cytometry assays.
  
  
  RESULTS
  After lentivirus-mediated siRNA transfection, expression of RNF8 in A549 cells downregulated which led to an increased radiosensitivity and impaired DNA repair. RNF8 knockdown did not affect Ku70 expression, however, Rad51, a key player in homologous recombination (HR) repair, was abrogated at sites of DNA damage. Furthermore, we observed an extended G2/M arrest and an increased induction of apoptosis after ionizing radiation in the absence of RNF8.
  
  
  CONCLUSIONS
  RNF8 silencing effectively downregulates Rad51 therefore maybe impairing HR repair, and prolongs the G2/M accumulation as well as cell apoptosis upon radiation, which all suggest an enhanced radiosensitivity on A549 cells.
authors.affiliation: Cancer Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
authors.forename: Hongxia
authors.initials: H
authors.lastname: Zhou
authors.forename: Xiaoqian
authors.initials: X
authors.lastname: Mu
authors.forename: Jing
authors.initials: J
authors.lastname: Chen
authors.forename: Hongli
authors.initials: H
authors.lastname: Liu
authors.forename: Wei
authors.initials: W
authors.lastname: Shi
authors.forename: Enming
authors.initials: E
authors.lastname: Xing
authors.forename: Kunyu
authors.initials: K
authors.lastname: Yang
authors.forename: Gang
authors.initials: G
authors.lastname: Wu
mesh_terms: D017209:Apoptosis / Q000528:radiation effects; D002289:Carcinoma, Non-Small-Cell Lung / Q000532:radiotherapy*; D002453:Cell Cycle / Q000528:radiation effects; D045744:Cell Line, Tumor; D053903:DNA Breaks, Double-Stranded; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D015536:Down-Regulation; D006657:Histones / Q000032:analysis; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008175:Lung Neoplasms / Q000532:radiotherapy*; D034622:RNA Interference; D051135:Rad51 Recombinase / Q000235:genetics; D011836:Radiation Tolerance; D044767:Ubiquitin-Protein Ligases
chemical_list: D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; C486521:RNF8 protein, human; D044767:Ubiquitin-Protein Ligases; D051135:Rad51 Recombinase