| BMI-1(B-cell-specific Moloney murine leukemia virus integration site 1) , a Polycomb group repressor, is always present as a PcG complex combined with RING1, HPC2, Mph2, et al. BMI-1 prolongs cells'capacity to proliferate and represses their senescence by repressing the transcription of CDKN2A through acetylhydrolasing or deacetylhydrolasing the Polycomb response elements in chromosome. There have been many experimental studies proving that BMI-1 plays a critical role in the formation and proliferation of tumor. BMI-1 has been found in many malignant tumors such as Hodgkin's and non-Hodgkin's lymphoma, AML, breast cancer, colorectal cancer, medulloblastoma, et al. In 2003, Lessard reported that BMI-1 regulates the proliferation of leukaemic stem cells. Nowdays, BMI-1 has been considered as an oncogene.In 2004, Soek Ying Neo, et al from National University of Singapore found the expression level of BMI-1 of HCC tumor specimens associated with HBV is obviously higher than nontumor liver tissue specimens. But the function of BMI-1 in the formation and progression of HCC is still unknown. For the survival period of normal human hepatic epithelial cells is only in two weeks, it is difficult to establish the hepatic epithelial cell line expressing BMI-1 stably to study the function of BMI-1 in the formation of HCC. HaCaT is a spontaneously immortalized keratinocyte cell line derived from normal adult human skin. It can persist the stable genotype and phenotype and sustain the non-tumorigenic phenotype up to passages 283. So HaCaT cell line is a good model to study the tumorigenic alterations. In our research, we introduced BMI-1 cloned from the human HCC tissues into HaCaT cells to study the function and molecular mechanisms in the malignant transformation of HaCaT. Furthermore, we can presume BMI-1 may play a certain role in the formation of HCC.First, we detected the expression level of BMI-1 in HCC tissues and four hepatic tumor cell lines:Huh7, HepG2, 7420, SMMC using RT-PCR. We found BMI-1 is expressed highly in all HCC tissues and hepatic tumor cell lines except the normal human adult hepatic tissues. Then BMI-1 cDNA was cloned from HCC by RT-PCR. The expression vector, pcDNA3.0-BMI-1, was constructed and identified through digestion and nucleotide sequencing. HaCaT cells were transfected with pcDNA3.0-BMI-1 by the electroporation method. The monoclone HaCaT cell line expressing BMI-1 stably was obtained by using G418 and identified by RT-PCR and Western Blot. The proliferation and clonal formation capacities of HaCaT cells were promoted prominently after expressing BMI-1 highly. The results of soft agar experiments and injected subcutaneously into SCID mice showed that BMI-1 induced HaCaT cells malignant transformation. Molecular markers such as p14ARF, p16,CCND1, CDKN1A, CCNE1, CCNA2,CDKN2C, CDKN1C were assayzed with RT-PCR and Real-time PCR when the immortal cells transformed. The transcription level of INK4α-ARF was downregulated. The downstream molecules of INK4α-ARF were upregulated. The results proposed a probable molecular mechanism of the malignant transformation induced by BMI-1: BMI-1 inhibits the transcriptive activity of INK4α-ARF.To our knowledge, it is the first time to find that the high expression of BMI-1 in HCC tissues and human hepatic tumor cell lines, it is the first time to clone BMI-1 from the HCC tissue and it is the first time to introduce BMI-1 into HaCaT cells resulting their malignant transformation. However, the precise relation between BMI-1 and human primary hepatic cancer is still unclear. It will be invaluable for the research of hepatic cancer biologic behavior and the treatment of hepatic cancer.
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