Background and purpose: The human oncogene B-cell-specific moloney murine leukemia virus integration site 1 (Bmi-1) is an important member of the polycomb group family

and it regulates cell proliferation and senescence via INK4a/ARF locus. This study investigated the effects of Bm

i-1-siRNA on the proliferation of lung adenocarcinoma cell line SPC-A1 cells with INK4a/ARF locus and clarify the mechanism of Bmi-1-mediated effect on proliferation of lung adenocarcinoma cells. Methods: In this study

we chose the most efficient siRNA chain the pGeneshl-2-Bmi-1 sense-1 and inserted into a pSUPER-retro-neo retroviral vector. The packaged si-Bmi-1 pSUPERretro-neo retroviral vector was stably transfected into lung adenocarcinoma SPC-A1 cell line. The stably transfected cells were cultured and passed. After transfection

the levels of Bmi-1 mRNA and protein expression of SPC-A1 cells were analyzed by RT-PCR and Western blot respectively. Trypan blue

MTT and plate colony forming assay were performed to observe the proliferation capibility of SPC-A1 cells and evaluate the cloning forming ability in vitro. The potency of tumorigenesis was observed in nude mouse through hypodermic inoculation of SPC-A1 cells. Cell cycle distribution was analyzed by flow cytometry (FCM) in SPC-A1 cells. The expression levels of proliferation proteins including p16

INK4a

p53

Cyclin D1

PTEN

Akt and Ser473p-Akt were analyzed by Western blot. Results: The mRNA and protein expression levels of Bmi-1 were significantly reduced in SPC-A1-Bmi-1-siRNA cells transfected with pSUPER-retroneo retroviral vector. Knockdown of Bmi-1 could inhibit the growth

colony formation in vitro and tumorigenesis invivo of SPC-A1 cells (P0.01). The transfected SPC-A1 cells were arrested in G

1

phase ［(64.6±1.2)%

P0.05］.Compared with two control groups

p16

INK4a

p53 and Akt were not affected (P0.05)

while Cyclin D1 and Ser473p-Akt were downregulated (P0.01) and PTEN was up-regulated (P0.01) in the SPC-A1-Bmi-1-siRNA cells. SPC-A1-Bmi-1-siRNA cells were treated with various concentrations of PTEN inhibitor to determine expression levels of PTEN

Bmi-1 and Ser473p-Akt protein. Ablation of PTEN rescued Bmi-1 and Ser473p-Akt expression in SPC-A1-Bmi-1-siRNA cells. Conclusion: Knockdown of Bmi-1 gene can arrest the

proliferation of SPC-A1 cells through G

0

/G

1

phasearrest by inhibiting Cyclin D1 expression indirectly

which may be not associated with p16

INK4a

signaling pathway.