The Molecular Mechanisms Of BMI-1 In Tumorigenesis

PartⅠThe detection of BMI-1 mRNA in leukemic cells with real-time fluorescent quantitative reverse transcription-polymerase chain reactionObjectiveTo establish a SYBR GreenⅠreal-time reverse transcription-polymerase chain reaction (RT-PCR) for quantitating human BMI-1 mRNA and study its expression in leukemic cells.MethodsTotal RNA was extracted with TRIzol and mRNA was transcribed reversely into cDNA. SYBR GreenⅠreal-time PCR was used to quantitate mRNA expression of BMI-1 and reference gene ABL in leukemic cell lines (K562, HL-60, Daudi, Jurkat, Molt-4, U937 and THP-1), 10 samples of bone marrow cells from acute myeloid leukemia and 8 samples of normal peripheral blood cells. The specific amplicons were measured by gelelectrophoresis with ethidium bromide staining, and the characteristic of their melting temperatures(Tm) were analysed by melting curves.Results⑴BMI-1 mRNA was detected in all the leukemic cell lines in the study. U937 showed a high expression level and HL-60 showed a low expression level.⑵The relative expression level of BMI-1 mRNA in leukemic cells and normal controls was 0.793±0.204 and 0.084±0.026, respectively (P<0.01).Conclusions ⑴SYBR GreenⅠreal-time quantitative RT-PCR is a rapid, sensitive and good specific method to detect BMI-1 mRNA.⑵BMI-1 is involved in leukemogenesis and may be a new tumor marker.PartⅡConstruction of recombinant eukaryotic vector BMI-1-pEGFP and its over-expression in Hela cellsObjectiveTo construct the mammalian expression vector of BMI-1-pEGFP, and observe whether it could express in human cervix cancer cell line Hela, and detect the effect on cell cycle and its possible downstream target genes expression of BMI-1 over-expression.MethodsThe cDNA fragment of BMI-1 obtained by RT-PCR from human breast cancer cell line MCF-7 was inserted into pEGFP-N1. The recombinant plasmid BMI-1-pEGFP was confirmed by restriction enzyme digestion, PCR and DNA sequencing. BMI-1-pEGFP was transfected into Hela cells with Lipofectamine 2000. The expression of BMI-1-pEGFP was determined by EGFP fluorescence and Western Blot analysis. SYBR GreenⅠreal-time RT-PCR was used to quantitate BMI-1, P16INK4a, hTERT, HOXA9, HOXB4 and HOXC13 mRNA. FACS analysis was used to detect cell cycle change.ResultsThe correct construction of the recombinant plasmid BMI-1-pEGFP has been shown by restriction enzyme digestion, PCR and DNA sequencing. BMI-1-pEGFP could express both EGFP reporter gene and exogenous BMI-1-EGFP fusion protein in Hela cells. FACS analysis showed a decrease from 65.68 % to 50.53% in G1 population and a significant increase from 27.17 % to 39.59 % in S population 48 hours after transfection. In Hela cells transfected with BMI-1-pEGFP, real-time RT-PCR showed that BMI-1 mRNA expression was increased by 1259.8-fold; P16INK4a, HOXA9 and HOXC13 mRNA expression were reduced to 9.2%, 10.9% and 69.7%, respectively; but hTERT and HOXB4 mRNA expression did not changed significantly.ConclusionsThe vector of BMI-1-pEGFP has been successfully constructed and it could be expressed. BMI-1 over-expression in Hela cells has reduced P16INK4a, HOXA9 and HOXC13 mRNA expression, and decreased in G1 population and increased in S population. BMI-1 may be involved in tumorigenesis of cervix carcinoma.PartⅢConstruction of eukaryotic vector BMI-1 shRNA and its inhibition in Hela cellsObjectiveTo construct eukaryotic vector expressing short hairpin RNA (shRNA) of BMI-1, to knock-down BMI-1 expression in human cervix cancer cell line Hela, and to detect the effect on cell cycle and its possible downstream target genes expression.MethodsDesigning four different shRNA targeting the coding sequence of the BMI-1, the BMI-1 shRNA 1-4 were constructed by inserting the designed shRNA to the eukaryotic expression vector psiRNA-hH1neo, and transfected into Hela cells with Lipofectamine 2000. In Hela cells transfected with BMI-1 shRNA 1-4 and selected by G418, SYBR GreenⅠreal-time RT-PCR was used to quantitate BMI-1, P16INK4a, hTERT, HOXA9, HOXB4 and HOXC13 mRNA Western Blot analysis was used to BMI-1 protein expression. FACS analysis was used to detect cell cycle change. ResultsThe constructed eukaryotic vectors BMI-1 shRNA 1-4 were identified by partial nucleotide sequencing. In BMI-1 shRNA 2 and 4 transfected Hela cells as compared with untransfected Hela cells, the BMI-1 mRNA expression was significantly suppressed by 59.9% and 67.4%, respectively, and Western Blot analysis showed BMI-1 protein level was decreased significantly. In Hela cells transfected with BMI-1 shRNA 2 and 4 and selected by G418, real-time RT-PCR showed that P16INK4a, HOXA9 and HOXC13 mRNA expression were increased by 9.35-fold and 12.57-fold; 13.25-fold and 14.36-fold; 7.36-fold and 7.89-fold, respectively; but hTERT and HOXB4 mRNA expression did not changed significantly. FACS analysis showed a significant increase from 65.68 % to 73.00% and 76.41% in G1 population and a decrease from 27.17 % to 19.21% and 17.66% in S population.ConclusionsBMI-1shRNA 2 and 4 can efficiently knock-down BMI-1 expression in Hela cells. BMI-1 under-expression in BMI-1 shRNA 2 and 4 -transfected Hela cells have up-regulated P16INK4a, HOXA9 and HOXC13 mRNA expression, and increased in G1 population and decreased in S population. Therefore, BMI-1 may be a good target for cervical cancer gene therapy.PartⅣBMI-1 over-expression in MDA-MB-231, MCF-7, K562, Jurkat cells and its effect on possible downstream target genes expressionObjectiveTo observe whether the mammalian expression vector BMI-1-pEGFP constructed successfully could express in cell line MDA-MB-231, MCF-7, K562 and Jurkat, and to detect the effect on its possible downstream target genes expression of BMI-1 overexpression.MethodsBMI-1-pEGFP was transfected into MDA-MB-231, MCF-7, K562 and Jurkat cells with Lipofectamine 2000. The expression of BMI-1-pEGFP was determined by EGFP fluorescence and Western Blot analysis. SYBR GreenⅠreal-time RT-PCR was used to quantitate BMI-1, P16INK4a, hTERT, HOXA9, HOXB4 and HOXC13 mRNA.Results⑴BMI-1-pEGFP could express both EGFP reporter gene and exogenous BMI-1-EGFP fusion protein in MDA-MB-231, MCF-7, K562 and Jurkat cells.⑵In MDA-MB-231, MCF-7, K562 and Jurkat cells transfected with BMI-1-pEGFP and selected by G418, real-time RT-PCR showed that BMI-1 mRNA expression was increased by 78.63-fold, 65.72-fold, 43.25-fold and 36.78-fold, respectively; Western Blot analysis showed exogenous BMI-1-EGFP fusion protein.⑶For MDA-MB-231 and MCF-7 cells of BMI-1 over-expression, hTERT mRNA expression increased by 36.53-fold and 27.33-fold, respectively; P16INK4a, HOXA9 and HOXC13 mRNA expression were reduced to 62.8% and 57.4%, 65.7% and 63.5%,73.5 % and 69.3 %, respectively. MCF-7 cells did not express HOXB4 mRNA and the mRNA expression level of HOXB4 in MDA-MB-231 cells transfected with BMI-1-pEGFP did not changed significantly.⑷For K562 and Jurkat cells of BMI-1 over-expression, they did not express P16INK4a and HOXA9 mRNA; HOXC13 mRNA expression was reduced to 64.8% and 67.2%; and the mRNA expression level of hTERT and HOXB4 did not changed significantly.ConclusionsThe vector of BMI-1-pEGFP could be expressed in MDA-MB-231, MCF-7, K562 and Jurkat cells. BMI-1 over-expression in MDA-MB-231 and MCF-7 cells has increased hTERT mRNA expression and has reduced P16INK4a, HOXA9 and HOXC13 mRNA expression. BMI-1 over-expression in K562 and Jurkat cells has reduced HOXC13 mRNA expression. BMI-1 may be involved in tumorigenesis. PartⅤBMI-1 gene silence via small interfering RNA in MDA-MB-231, MCF-7, K562, Jurkat cells and its effect on possible downstream target genes expressionObjectiveTo observe whether the mammalian expression vector BMI-1 shRNA 2 and 4 could express in cell line MDA-MB-231, MCF-7, K562 and Jurkat and knock-down BMI-1 expression, and detect the effect on its possible downstream target gene expression of BMI-1 under-expression.MethodsBMI-1 shRNA 2 and 4 were transfected into MDA-MB-231, MCF-7, K562 and Jurkat cells with Lipofectamine 2000. SYBR GreenⅠreal-time RT-PCR was used to quantitate BMI-1, P16INK4a, hTERT, HOXA9, HOXB4 and HOXC13 mRNA. BMI-1 protein expression was detected by Western Blot analysis.Results⑴BMI-1 shRNA 2 and 4 could knock-down BMI-1 mRNA and protein expression in MDA-MB-231, MCF-7, K562 and Jurkat cells.⑵In MDA-MB-231 and MCF-7 cells transfected with BMI-1 shRNA 2 and 4 and selected by G418, real-time RT-PCR showed that P16INK4a, HOXA9 and HOXC13 mRNA expression were increased by 7~9-fold, 10~12-fold and 6~8-fold, respectively. MCF-7 cells did not express HOXB4 mRNA and the mRNA expression level of HOXB4 in MDA-MB-231 cells transfected with BMI-1 shRNA 2 and 4 did not changed significantly.⑶In K562 and Jurkat cells transfected with BMI-1 shRNA 2 and 4 and selected by G418, real-time RT-PCR showed that HOXC13 mRNA expression was increased by 8~10-fold and 9~11-fold, respectively. The mRNA expression level of hTERT and HOXB4 did not changed significantly.ConclusionsBMI-1 shRNA 2 and 4 can efficiently knock-down BMI-1 expression in MDA-MB-231, MCF-7, K562 and Jurkat cells. BMI-1 under-expression in MDA-MB-231 and MCF-7 cells has reduced hTERT mRNA expression and has increased P16INK4a, HOXA9 and HOXC13 mRNA expression. BMI-1 under-expression in K562 and Jurkat cells has increased HOXC13 mRNA expression. Therefore, BMI-1 may be a good target for cancer gene therapy.