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AIM: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-1gene 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 theβ-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 pRNATU6.2-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.CONCLUSION:Silencing Bmi-1 by RNA interference can increase the senescent cell rate and effectively reduce the metastasis of gastric cancer cells.
AIM: 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-1gene 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 theβ-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 proteins were markedly decreased in transfected BGC823 ce lls with pRNAT-U6.2-si1104 and pRNATU6.2-si1356, especially in transfected BGC823 cells with pRNAT-U6.2-si1 104, compared with two control groups (empty vector and blank group) expression was almost completely abolished in cells transfected with the recombinant vector harboring shRNA targeting the sequence GGAGGAGGTGAATGATAAA (nt 1104-1122). 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. CONCLUSION: Silencing Bmi-1 by RNA interference can increase the senescent cell rate and effectively reduce the metastasis of gastric cancer cells.