title: Silencing Bmi-1 enhances the senescence and decreases the metastasis of human gastric cancer cells.
abstract: OBJECTIVE
  To evaluate the impact of Bmi-1 on cell senescence and metastasis of human gastric cancer cell line BGC823.
  
  
  METHODS
  Two pairs of complementary small hairpin RNA (shRNA) oligonucleotides targeting the Bmi-1 gene were designed, synthesized, annealed and cloned into the pRNAT-U6.2 vector. After DNA sequencing to verify the correct insertion of the shRNA sequences, the recombinant plasmids were transfected into BGC823 cells. The expression of Bmi-1 mRNA and protein was examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The effects of Bmi-1 knockdown on cell senescence and metastasis were determined by the β-Gal activity assay and Boyden chamber assay, respectively.
  
  
  RESULTS
  The double-stranded oligonucleotide fragments of Bmi-1 short interfering RNA (siRNA) cloned into pRNAT-U6.2 vector conformed to the inserted sequence. RT-PCR and Western blotting indicated that the expression levels of Bmi-1 gene mRNA and protein were markedly decreased in transfected BGC823 cells with pRNAT-U6.2-si1104 and pRNAT-U6.2-si1356, especially in transfected BGC823 cells with pRNAT-U6.2-si1104, compared with two control groups (empty vector and blank group). In particular, Bmi-1 protein expression was almost completely abolished in cells transfected with the recombinant vector harboring shRNA targeting the sequence GGAGGAGGTGAATGATAAA (nt1104-1122). Compared with untransfected cells and cells transfected with the empty vector, the mean percentage of senescent cells increased and the number of cells passing through the Matrigel decreased in cells transfected with the recombinant vectors.
  
  
  CONCLUSIONS
  Silencing Bmi-1 by RNA interference can increase the senescent cell rate and effectively reduce the metastasis of gastric cancer cells.
authors.affiliation: Feng-Lan Gao, Wei-Shan Li, Chun-Ling Liu, Pathological Examination and Research Center, Luohe Medical College, Luohe 462002, Henan Province, China.
authors.forename: Feng-Lan
authors.initials: FL
authors.lastname: Gao
authors.affiliation: Feng-Lan Gao, Wei-Shan Li, Chun-Ling Liu, Pathological Examination and Research Center, Luohe Medical College, Luohe 462002, Henan Province, China.
authors.forename: Wei-Shan
authors.initials: WS
authors.lastname: Li
authors.affiliation: Feng-Lan Gao, Wei-Shan Li, Chun-Ling Liu, Pathological Examination and Research Center, Luohe Medical College, Luohe 462002, Henan Province, China.
authors.forename: Chun-Ling
authors.initials: CL
authors.lastname: Liu
authors.affiliation: Feng-Lan Gao, Wei-Shan Li, Chun-Ling Liu, Pathological Examination and Research Center, Luohe Medical College, Luohe 462002, Henan Province, China.
authors.forename: Guo-Qiang
authors.initials: GQ
authors.lastname: Zhao
mesh_terms: D045744:Cell Line, Tumor; D002465:Cell Movement; D016922:Cellular Senescence; D015536:Down-Regulation; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D009361:Neoplasm Invasiveness; D063150:Polycomb Repressive Complex 1 / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D013274:Stomach Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology; D014162:Transfection
chemical_list: C069077:BMI1 protein, human; D012333:RNA, Messenger; D063150:Polycomb Repressive Complex 1
keywords: Bmi-1; Gastric cancer; Metastasis; Senescence