The Effect Of Bmi-1-siRNA On The Proliferation Of Lung Adenocarcinoma SPC-A1Cells And Its Mechanism

Objective: The human oncogene B-cell-specific Moloney murine leukemia virusintegration site1(Bmi-1) is a member of the Polycomb group family. Bmi-1is involvedin the regulation of proliferation, self-renewal and differentiation of hematopoietic stemcells. And it regulates cell proliferation and senescence via INK4a/ARF locus. It hasbeen found Bmi-1is overexpressed in several human malignancies including breastcancer, Non-small cell lung cancer (NSCLC), gastric cancer and nasopharyngealcarcinoma. Meanwhile, overexpression of Bmi-1often correlates with poorer prognosis.Recently, it has been shown that silencing Bmi-1expression could inhibit tumor cellgrowth, proliferation and tumorigenesis. Therefore, Bmi-1may become a tumorpotential therapeutic target against human cancers.We aimed to study the effect ofBmi-1-siRNA on the proliferation of lung adenocarcinoma cell line SPC-A1cells, inwhich INK4a/ARF locus was not deleted and tried to clarify the mechanism ofBmi-1-mediated on proliferation of lung adenocarcinoma cells.Methods: In this study, we chose the most efficient siRNA chain thepGeneshl-2-Bmi-11and inserted into a pSUPER-retro-neo retroviral vector. Thepackaged Si-Bmi-1pSUPERretro-Neo retroviral vector was stably transfected into lungadenocarcinoma SPC-A1cell line. At the same time, the wild-type SPC-A1cells andSPC-A1cells infected with empty vector (Si-Bmi1-ctr pSUPERretro-Neo) were servedas controls. The transfected cells were cultured and maintained in100μg/ml G418. Aftertransfected, the levels of Bmi-1mRNA and protein expression of SPC-A1cells wereanalyzed by RT-PCR (reverse-transcriptional polymerase chain reaction) and Westernblot respectively. Trypan blue, MTT and Plate Colony Forming assay were performed to observe the proliferation capibility of SPC-A1cells and evaluate the cloning formingability in vitro. Cell cycle distribution was analyzed by FCM (Flow Cytometry) inSPC-A1cells. The ability of tumorigenesis was observed by hypodermic inoculation ofSPC-A1cells into nude mice. The levels of p16INK4a, p53, CyclinD1, PTEN, Akt andSer473p-Akt proteins were analyzed by Western blot. SPC-A1-Bmi-1-siRNA cells weretreated with various concentrationsBpv (phen) and determined whether reduction of PTEN expression inBmi-1-silenced SPC-A1cells could restore Bmi-1expression and p-Akt activity. Thefunctional correlation between PTEN and Bmi-1was detected by Co-IP (co-immunoprecipitation) assay.Results: The Si-Bmi1pSUPERretro-Neo and Si-Bmi1-ctr pSUPERretro-Neoretroviral vectors were transfected into SPC-A1cells. The expression of Neo showedthat the Bmi-1-siRNA expression vector could be successfully introduced into theSPC-A1cells. Efficiency of transfection was assessed at100%by fluorescencemicroscopy. According to the results of RT-PCR and Western blot, Bmi-1mRNA andprotein levels all significantly reduced in SPC-A1-Bmi-1-siRNA cells, compared toSPC-A1-ctr and SPC-A1-wt cells (p <0.01; p <0.01). There were no significantdifferences between the cells of SPC-A1-ctr and SPC-A1-wt cells (p>0.05). MTTassays and Trypan blue showed that knockdown of Bmi-1could inhibit the proliferationwithout the viability of SPC-A1cells influenced in vitro (p <0.05; p>0.05). Colonyforming test demonstrated that the single-cell proliferative capability ofSPC-A1-Bmi-1-siRNA cells was significantly inhibited (p <0.01), compared toSPC-A1-ctr and SPC-A1-wt cells. The transfected SPC-A1cells were arrested in G1phase (p <0.05). Knockdown of Bmi-1could weaken the ability of tumorigenesis ofSPC-A1cells in vivo (p <0.01). Western blot results showed that p16INK4a, p53and Aktexpression levels of three group cells were not affected (p>0.05; p>0.05; p>0.05).However, compared with SPC-A1-wt and SPC-A1-ctr cells, the expression of CyclinD1and Ser473p-Akt decreased (p <0.01; p <0.01), PTEN expression increased (p <0.01)in SPC-A1-siRNA-Bmi-1cells. Ablation of PTEN rescued p-Akt activity and Bmi-1expression in Bmi-1-silenced SPC-A1cells. CO-IP results showed the interactionbetween Bmi-1and PTEN in SPC-A1cells.Conclusion: Knockdown of Bmi-1gene could arrest the proliferation of SPC-A1cells in G0/G1phase of cell cycle by inhibiting CyclinD1expression indirectly and wasnot associated with p16INK4asignaling pathway. Bmi-1/PTEN/PI3K/p-Akt/CyclinD1 pathway might be crucial in the proliferation of lung aenocarcinoma cell. And theinteraction between Bmi-1and PTEN might be imporant in regulating proliferation oflung adenocarcinoma cells.