Effects Of SiRNA-mediated Silencing Of Bmi-1 Gene Expression On The Biological Behaviour Of HeLa Cells

Objective: Bmi-1 is member of the Polycomb-group (PcG) family, is required for regulation of the proliferation and self-renewal of normal and tumor stem cells. In recent years, it has been found that Bmi-1 is overexpressed in varieties of human cancers, and is closely related to the prognosis of tumors. Therefore silencing of Bmi-1 expression might be a potential therapeutic strategy in management of human cancers. In this study, sequences for small interfering RNAs (siRNAs) against Bmi-1 were chosen according to its gene sequence, the chosen siRNAs were connected to the eukaryotic expression plasmid pGensil-2, and the recombinants were transfected into human cervical carcinoma cell line Hela cells. The effects of silencing the oncogene Bmi-1 on cytobiological behavior and its mechanism in Hela cells with these siRNAs were explored.Methods: Four Bmi-1 siRNA sequences and a random sequence of negative control were chosen according to GENEBANK of Bmi-1 mRNA, Sequence NM₀05180, the uniqueness of siRNA sequences with the BLAST was confirmed. The siRNA sequences were connected to the pGenesil-2 plasmid in reverse complementary sequences at both ends of Stem-loop trails through manipulation of the restriction site. The resultant Bmi-1 siRNA expression vectors were amplified in E coli and purified, and transfected into the Hela cells with lipofectamine method. Neo gene was detected by RT-PCR for confirming the success of transfection, and the efficiency of transfection was observed by measuring expression of EGFP in Hela cells. The level of Bmi-1 mRNA and its protein in Hela cells transfected with Bmi-1 siRNA were analyzed by RT-PCR, and western blotting respectively. The proliferation of Hela cells transfected with Bmi-1 siRNA were analyzed with MTT and typan blue exclusion methods. Flow cytometry was used for cell cycle study. Plate colony forming assay was used to analyze single-cell proliferative capacity. Trans-well assay was used to examine cell migration. Finally, the abilities of tumorigenesis and metastasis of Hela cells transfected with Bmi-1 siRNA was observed in nude mouse through hypodermic inoculation and direct tail vein injection of the Bmi-1 siRNA plasmid transfected HeLa cells respectively.Results: The recombinant plasmids that could express siRNAs were successfully constructed (four Bmi-1 siRNA expression plasmids and a negative control plasmid), and could be successfully transfected to and expressed in HeLa cells, the efficiency of transfection was estimated to be about 90%. These siRNAs were proved to silence the endogenous exp- ression of Bmi-1 both in mRNA and the protein levels. The proliferation of Bmi-1 siRNA tranfected Hela cells was inhibited without inducing cells death in vitro. The transfected Hela cells were arrested in G0-G1 phase, and the cell proliferation index decreased significantly. The single-cell prolix- ferative capacity of Bmi-1 siRNA transfected Hela cells was inhibited. Trans-well assay showed that migration ablitity of the transfected Hela cells was inhibited. The tumorigenesis and metastasis abilites of the transfected Hela cells were significantly attenuated in nude mice compared to the negative control plasmid tranfected HeLa cells.Conclusion: Silencing Bmi-1 gene through siRNA inhibits the prolix- feration and migration properties of Hela cells in intro, and attenuates the tumorigenesis and metastasis of Hela cells in vivo. Detection of Bmi-1 might be a biological marker of tumor maligancy and Bmi-1 silencing through siRNA technique might be used as a biological approche in the management of cervical cancer.