B-cell-specific Moloney murine leukemia virus insertion site 1 (Bmi-1) is highly expressed in several malignant tumors and its expression level is positively correlated with tumor invasion, distant metastasis, prognosis, and recurrence. In the present study, the biological effect of small interfering RNA (siRNA) that targeted Bmi-1 expression was studied in human cervical carcinoma cell line HeLa cells. Bmi-1 siRNA inhibited the expression of Bmi-1 at the mRNA and protein levels in HeLa cells, which significantly affected proliferation, colony formation, and migration of HeLa cells in vitro and in vivo. Therefore, silencing Bmi-1 may be a potential therapeutic option for the management of some human cancers. (Cancer Sci 2009)
B-cell-specific Moloney murine leukemia virus insertion site 1 (Bmi-1) is a member of the Polycomb group gene family.(1) Bmi-1 is required for the self-renewal of normal hematopoietic(2) and nerve(3) stem cells, and determines the proliferative potential of tumor stem cells, such as leukemic,(4) neuronal,(5) and breast cancer(6) stem cells. In particular, Bmi-1 is highly expressed in several malignant tumors, such as non-small cell lung cancer,(7) breast cancer,(8) colorectal cancer,(9) nasopharyngeal carcinoma,(10) metastatic melanoma,(11) and prostate cancer,(12) and its expression level is positively correlated with tumor invasion, distant metastasis, prognosis, and recurrence. Therefore, the silencing of Bmi-1 expression has the potential for the management of some human cancers.
One method of RNA interference (RNAi) technology is to use double-stranded RNA (dsRNA), also known as small interfering RNA (siRNA), of 21–23 nt in length, to silence the sequence-specific gene. The RNA-induced silencing complex is composed of siRNA and a series of specific proteins that specifically trigger the cleavage and subsequent degradation of their target mRNA in a sequence-dependent manner. Thus, synthesis of the protein encoded by these mRNA is prevented. Therefore, RNAi technology can be used for the analysis of gene function in human cell cultures, as well as for gene-specific therapies.(13,14)
Previously, we stably transfected a plasmid that expresses antisense Bmi-1 RNA into human leukemia K562 cells and lung cancer cell line A549, and found that it inhibited proliferation of the transfected cells.(15,16) To investigate if siRNA against Bmi-1 could affect the proliferation and growth of human cervical carcinoma cell line HeLa cells, we constructed a plasmid that expressed Bmi-1-specific siRNA and transferred it into HeLa cells to observe the effects of silencing the Bmi-1 oncogene.
Materials and Methods
Construction of the siRNA expression vector. Four Bmi-1 siRNA sequences were designed according to the Bmi-1 mRNA sequence (GenBank accession no. NM_005180), and a random sequence was used as a negative control (Table 1). Basic local alignment search tool (BLAST) analysis was used to ensure these five sequences did not target other genes.
Table 1. siRNA target sequences of Bmi-1 gene used in this study
Bmi-1, B-cell-specific Moloney murine leukemia virus insertion site 1; siRNA, small interfering RNA.
5 (negative control)
DNA that corresponded to the siRNA sequence and its reverse complementary sequence were connected with a loop sequence into one chain, and a complement to this chain was synthesized. This yielded a dsDNA, with both ends especially designed for directional ligation with the BamHI and HandIII sites (Fig. 1) of the pGenesil-2 plasmid. PolIII promoter termination signal TTTTTT was added after the dsDNA. In order to identify the final plasmid, a SalI restriction site was added after the termination signal sequence. The five siRNA fragments were named small interfering sequence 1, 2, 3, 4, and 5 (sequence 5 was the negative control), respectively, and were synthesized by China Wuhan GeneSil Biotechnology (Wuhan, China).
The resulting dsDNA was inserted into pGenesil-2 (China Wuhan GeneSil Biotechnology, China) between the BamHI and HandIII sites (Fig. 2). The recombinant vector was sequenced with a DNA sequencer (ABI-377; Applied Biosystems, San Francisco, CA, USA) The final plasmids were named pGenesil-2-Bmi-11, pGenesil-2-Bmi-12, pGenesil-2-Bmi-13, pGenesil-2-Bmi-14, and pGenesil-2-HK, respectively. pGenesil-1, which harbored an enhanced green fluorescent protein (EGFP) gene that facilitated the tracking transfection efficiency, was supplied from China Wuhan GeneSil Biotechnology (China).
Cell culture. The HeLa cells were cultured in RPMI-1640 medium (Hyclone, Logan, UT, USA) supplemented with 10% bovine calf serum (Hyclone, USA) in a humidified chamber at 37°C with 5% CO2.
Plasmid transfection. The HeLa cells were seeded in six-well plates at a density of 3 × 105 cells per well on the day before transfection. After 24 h culture, the cells were transfected with purified pGenesil-2-Bmi-11, pGenesil-2-Bmi-12, pGenesil-2-Bmi-13, pGenesil-2-Bmi-14, pGenesil-2-HK, and pGenesil-1 using FuGENE HD Transfection Reagent (Roche, Indianapolis, IN, USA), according to the manufacturer’s instructions. The transfectants with pGenesil-2-Bmi-11, pGenesil-2-Bmi-12, pGenesil-2-Bmi-13, pGenesil-2-Bmi-14, and pGenesil-2-HK were named HeLa-S1, HeLa-S2, HeLa-S3, HeLa-S4, and HeLa-V cells, respectively. The parental HeLa cells (wild type) were named HeLa-W. Forty-eight hours after transfection, the HeLa cells were harvested for further analysis. pGenesil-1, which harbored an EGFP gene, was transfected into cells to facilitate visualization of transfection under fluorescence microscopy (BX-51; Olympus, Omachi, Japan). Transfection efficiency was evaluated by the visual counting of fluorescent cells in random fields.
Reverse transcription–polymerase chain reaction. Total RNA was isolated from HeLa-S1, HeLa-S2, HeLa-S3, HeLa-S4, HeLa-V, and HeLa-W cells using Trizol (Invitrogen, Carlsbad, CA, USA). cDNA was first synthesized using a reverse transcription–polymerase chain reaction (RT-PCR) kit (TaKaRa, Japan), according to the manufacturer’s instruction, and PCR was performed under the following conditions: denaturation at 94°C for 1 min, and then 30 cycles of denaturation for 30 s at 94°C, annealing for 30 s at 60°C, and extension for 2 min at 72°C. The neo gene was amplified by the forward 5′-CAAGATGGATT GCACGCAGG-3′ and reverse primers 5′-ATGCTCTTCGTCCAG ATCAT-3′. The Bmi-1 gene was amplified by the forward 5′-TCATCCTTCTGCTGATGCT G-3′ and reverse primers 5′-GCATCACAGTCATTGCTGCT-3′. GAPDH was used as an internal marker and amplified by the forward 5′-GAAGGTGAAGGTC GGAGTC-3′ and reverse primers 5′-GAAGATGGTGATGGGATTTC-3′. The amplified products were separated on 1% agarose gel that contained 0.5% ethidium bromide.
Western blotting. Forty-eight hours after the transfection, HeLa cells were harvested. Protein extracts were obtained using the KEYGEN total protein extraction kit (Nanjing, China). Protein concentrations of the supernatant fractions were determined using the Bio-Rad protein assay (Richmond, CA, USA). Sixty micrograms of protein per sample was loaded and separated by 10% sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE), followed by electrotransfer onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). After blocking with 5% fat-free milk in Tris-buffered saline containing 0.05% Tween-20, the membrane was first incubated with the following primary antibodies: mouse monoclonal anti-Bmi-1 (ab54897; Abcam, Cambridge, UK) and mouse monoclonal anti-β-actin (AC-15; Sigma, St Louis, MO, USA). The protein–antibody complexes were detected using horseradish peroxidase-conjugated secondary antibodies and the SuperECL Plus chemiluminescence system (Applygen, Beijing, China). The relative amount of protein on the blots was determined by densitometry using LabWorks software (UVP, Upland, CA, USA). β-Actin was used as a loading control.
Cell growth curve and viability assay. Cell growth curves were measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, HeLa-S1, HeLa-S3, HeLa-V, and HeLa-W cells were plated in triplicate at a density of 3 × 103 cells per well in 96-well plates. On each of the five consecutive days, 10 μL MTT stock solution (5 mg/mL in phosphate-buffered saline [PBS]) was added to each microtiter well, and the plates were incubated at 37°C for 4 h. After aspiration of the supernatant, 150 μL dimethylsulfoxide was added and mixed, and absorbance was measured with a microELISA reader (Thermo Scientific, Waltman, MA, USA) at a wavelength of 492 nm. Growth curves were plotted as optical density (492 nm) against days after seeding. Cell viability was determined by trypan blue exclusion assay, as described previously.(17)
Colony-forming assay. Four-hundred HeLa-S1, HeLa-S3, HeLa-V, and HeLa-W cells in 2 mL growth medium were plated in triplicate in the six-well plates. After 10 days, the cells were rinsed with PBS twice, fixed with 10% formaldehyde, and stained with 0.1% crystal violet in 10% ethanol. The numbers of colonies were counted.
In vitro migration assays. The inhibitory effect of RNAi on Hca-F cell migration in vitro(18) was demonstrated using 24-well transwell units (Corning, Rochester, NY, USA) with 8-μm pore size polycarbonate filters. HeLa-S1, HeLa-S3, HeLa-V, and HeLa-W cells were harvested in serum-free medium (3 × 105/mL) and added in a volume of 100 μL to the upper chamber. The lower chamber contained 600 μL RPMI-1640. The cells were incubated for 24 h at 37°C in a 5% CO2 incubator. After incubation, the cells on the upper surface of the filter were removed completely by wiping with a cotton swab. The filters were fixed with 10% formaldehyde and stained with 0.1% crystal violet in 10% ethanol. The cells that had migrated to the lower surface of the filter were counted under a light microscope at a magnification of ×100. The number of migratory cells was calculated.
Cell cycle assay. The cell cycle was analyzed by using flow cytometry with propidium iodide (PI; Sigma, USA) staining. In each group, the cells were harvested and washed with PBS and fixed overnight in ice-cold 70% ethanol. The cells were washed twice with PBS and treated with 1 mg/L RNase A (TaKaRa, Japan) for 15 min. Finally, the cells were stained with 50 mg/L PI in the dark for 1 h. The cell cycle analysis was performed with a fluorescence-activated cell sorter (BD Biosciences, Franklin Lakes, NJ, USA), and PI fluorescence was measured at 488 nm. Each group was analyzed in triplicate and 10 000 cells were analyzed in each experiment.
In vivo tumorigenesis assay. Nude mice (Balb/c nu/nu) were obtained from the Animal Facility of Dalian Medical University, Dalian China. For the measurement of tumor growth in vivo, HeLa-S1, HeLa-S3, HeLa-V, and HeLa-W cells (1 × 107 cells/mouse) were injected subcutaneously into the armpit of nude mice (n =6). After 3 weeks, tumors were isolated from nude mice, weighed, and fixed in formalin.
In vivo tumor metastasis assay. In vivo tumor metastasis assay was performed as described previously.(19) Thirty nude mice were divided equally into five groups. HeLa-S1, HeLa-S3, HeLa-V, and HeLa-W cells (2 × 106 each) were injected into nude mice via the tail vein. After 3 weeks, nude mice were killed and their armpit lymph nodes were isolated, fixed, sectioned, and stained with hematoxylin–eosin (HE), and blood-borne metastasis rates were determined.
Statistical analysis. Data were expressed as mean ± SD, and analyzed by one-way ANOVA with SPSS version 13.0 (SPSS, Chicago, IL, USA). P <0.05 was considered statistically significant.
Transient transfection confirmation through RT-PCR and fluorescence. The HeLa cells were transfected with pGenesil-2-Bmi-1siRNA or pGenesil-2. After 48 h, a 500-bp band of antineomycin gene products was amplified with RT-PCR from transiently-transfected HeLa-S1, HeLa-S2, HeLa-S3, HeLa-S4, and HeLa-V, but not HeLa-W cells (Fig. 3). HeLa cells were transfected with pGenesil-1, and after 48 h, the ratio of transfection was observed by fluorescence microscopy (Fig. 4). According to the strength of fluorescence, transfection efficiency was estimated at approximately 90% (transfection efficacy = number of fluorescence-emitting cells under fluorescent microscopy/total cell number under light microscopy). These results indicated that transiently-transfected HeLa cells contained the neo gene and that the siRNA was introduced successfully into the HeLa cells.
Bmi-1siRNA decreased the Bmi-1 expression at the mRNA and protein levels. Real-time quantitative RT-PCR and western blotting were performed to examine the effect of siRNA transfection on Bmi-1 expression levels in HeLa cells. As shown in Figure 5, the Bmi-1 mRNA and protein levels were significantly decreased in HeLa-S1, HeLa-S2, HeLa-S3, and HeLa-S4 cells compared to HeLa-V and HeLa-W cells (P <0.05). There were no significant differences between HeLa-V and HeLa-W cells (P >0.05). Thus, Bmi-1 siRNA could effectively downregulate the in vitro Bmi-1 mRNA expression and protein translation in HeLa cells.
Bmi-1 siRNA inhibited HeLa cell growth without inducing cell death in vitro. The growth rates of HeLa-S1, HeLa-S3, HeLa-V, and HeLa-W cells were determined by MTT assay (Fig. 6A). HeLa-S1 and HeLa-S3 cells grew significantly slower than HeLa-W or HeLa-V cells (P <0.05) from the second day after seeding, and there was no significant difference between HeLa-V and HeLa-W cells (P >0.05). In order to determine if the slower growth of HeLa-S1 and HeLa-S3 cells was caused by cell death, a trypan blue exclusion test was performed to assess cell viability. As shown in Figure 6(B), there were no significant differences in cell viability between HeLa-S1, HeLa-S3, HeLa-V, and HeLa-W cells (P >0.05). Therefore, the slower growth rate of HeLa-S1 and HeLa-S3 cells was not attributed to increased cell death.
Bmi-1 siRNA blocked cell cycle distribution at the G1/G0 phase of HeLa cells. Cell cycle distribution was measured by flow cytometry to determine whether the inhibition of cell growth was related to cell cycle arrest. DNA distribution histograms and the analyses of cell cycles are shown in Figure 7. G0/G1 phase accumulation was observed in HeLa-S1 and HeLa-S3 cells compared to HeLa-V or HeLa-W cells (Fig. 7A,B), and was accompanied by a decrease in the number of cells at the S phase (P <0.05) (Fig. 7C).
Bmi-1 siRNA inhibited the clonogenicity of HeLa cells in vitro. In order to determine the effect of Bmi-1 siRNA on the clonogenicity of HeLa cells, we performed colony-forming assays. Our results demonstrated that compared with HeLa-W and HeLa-V cells, colony numbers of HeLa-S1 and HeLa-S3 cells were slightly decreased, but there was no significant difference (P <0.01, Fig. 8). There were also no significant differences between HeLa-V and HeLa-W cells (P >0.05). Thus, Bmi-1 siRNA significantly inhibited the clonogenicity of HeLa cells.
Bmi-1 siRNA diminished the migration of HeLa cells in vitro. To examine whether the targeted downregulation of Bmi-1 in HeLa cells affected their migration, we performed an in vitro transwell migration assay. HeLa-W and HeLa-V cells had a similar ability to migrate through the filter, because the numbers of migrating cells were roughly equal (P >0.05). The number of HeLa-S1 and HeLa-S3 cells migrating through the filter was markedly lower than the number of HeLa-W and HeLa-V cells (P <0.01, Fig. 9). Thus, Bmi-1 siRNA could diminish HeLa cell migration in vitro.
Bmi-1 siRNA suppressed the tumorigenesis of HeLa cells in vivo. The in vitro assay described earlier suggests that tumorigenicity in vivo might also be inhibited by Bmi-1 siRNA. To evaluate whether the downregulation of Bmi-1 expression could inhibit the tumorigenic capability of HeLa cells in vivo, HeLa-S1, HeLa-S3, HeLa-V, or HeLa-W cells were injected subcutaneously into the armpit of nude mice. After 21 days, we observed a significant reduction in the mean tumor weight in the Bmi-1 siRNA groups when compared with the control groups (P <0.05, Fig. 10). Thus, the downregulation of Bmi-1 could attenuate the ability of HeLa cells to form tumors in vivo.
Bmi-1 siRNA inhibited the metastatic capability of HeLa cells in vivo. Previous in vitro experiments have shown that Bmi-1 siRNA can inhibit the migration of HeLa cells. Based on this, tumor cells were injected into nude mice via the tail vein to observe whether the downregulation of Bmi-1 expression could inhibit the metastatic capability of HeLa cells in vivo. After 3 weeks, the nude mice were killed, and the lymph nodes were isolated, fixed, stained with HE, and observed by light microscopy (Olympus, Japan). There were no or only occasional tumor cells in the lymph nodes of the nude mice injected with HeLa-S1 and HeLa-S3 cells, while large numbers of tumor cells were noted in the lymph nodes of nude mice injected with HeLa-W and HeLa-V cells (Fig. 11).
It has been proposed that cancer stem cells (CSC) are responsible for tumorigenesis and contribute to cancer resistance to chemotherapeutic agents. CSC are biologically distinct from other cancer cell types. Furthermore, certain natural proprieties of CSC are likely to increase their resistance to standard chemotherapy agents. Thus, it is necessary that chemotherapy agents are targeted against CSCs, otherwise the latter drive tumor expansion.(20) Designing agents that target CSC may lead to more efficient cancer therapy.
Bmi-1 has been identified as an oncogene that cooperates with c-myc in generating murine lymphoma.(21,22) Recently, Bmi-1 has been shown to be necessary for the proliferative potential of tumor stem cells and is overexpressed in a variety of human cancers. Lessard and Sauvageau(4) found that Bmi-1+/+ and Bmi-1−/− fetal liver cells were infected with a retrovirus that contained the Hoxa9 and Meis1a genes, and transduced cells were transplanted into sublethally-irradiated syngeneic mice. All recipients of Hoxa9/Meis1-transduced fetal liver cells developed acute myeloid leukemia (AML) within a similar latency period. Leukemic cells derived from the bone marrow of the different primary recipients from groups A (Bmi-1+/+ Hoxa9/Meis1) and B (Bmi-1−/− Hoxa9/Meis1) were transplanted into syngeneic mice. Mice with (Bmi-1+/+ Hoxa9/Meis1)-transplanted tumors developed AML within 30 ± 25 days after transplantation; none of the secondary recipients of (Bmi-1−/− Hoxa9/Meis1) leukemic bone marrow cells developed AML, and no leukemic cells were detected in long-term recipients analyzed at 4 and 11 months after transplantation. This indicates that silencing Bmi-1 gene expression inhibits the proliferation of leukemic stem cells. Therefore, the downregulation of Bmi-1 expression might be a potential therapeutic strategy against human CSC.
RNAi can knockdown specific gene expression in mammals, which may provide a new approach for gene therapy of tumors.(23,24) In the present study, we used RNAi to directly downregulate the expression of Bmi-1 in HeLa cells. We designed four pairs of siRNA against the Bmi-1 gene to ensure that there was at least one Bmi-1 siRNA that could inhibit gene expression effectively. Transfection with the four Bmi-1 siRNA-expressing vectors all decreased the expression of Bmi-1 at the mRNA and protein levels in HeLa cells. This suggests that Bmi-1 expression was knocked down successfully by Bmi-1 siRNA. Therefore, pGenesil-2-Bmi-11 and pGenesil-2-Bmi-13 were chosen for the subsequent in vitro and in vivo experiments.
A cell growth assay was used to study whether the downregulation of Bmi-1 by RNAi influenced the proliferation of HeLa cells in vitro. Transfection with the Bmi-1 siRNA vectors had a significant inhibitory effect on HeLa cell growth, as compared with the controls transfected with empty vectors and their parental cells. Previous studies have shown that the Bmi-1 gene can regulate normal cell proliferation,(10,25,26) and Bmi-1−/− leukemic cells show proliferation arrest.(4) Our previous work has also demonstrated that reducing Bmi-1 levels by antisense RNA expression suppresses the proliferation of A549 cells.(16) Thus, consistent with previous results, the silencing of Bmi-1 may impede HeLa cell proliferation and growth in vitro. Our finding indicates that Bmi-1 plays important roles in the proliferation of cervical cancer cells.
The cell cycle and cell proliferation are closely related. The length of the cell cycle depends on the G0/G1 phase, and the cell cycle can be extended by G0/G1 phase arrest. In order to determine the suppressive mechanism of Bmi-1 siRNA on HeLa cell growth, the cell cycle was analyzed by flow cytometry. The effect of Bmi-1 gene silencing on the cell cycle varies between different cell types. Bmi-1−/− leukemic cells displayed accumulation at the G0/G1 phase and fewer cells at the S phase.(4) Our previous study found that A549 cells transfected with antisense Bmi-1 displayed accumulation at the G0/G1 phase and a reduction at the S phase.(16) However, there are also conflicting reports. For example, Cui et al. demonstrated that Bmi-1 knockdown by siRNA has no significant effect on cell cycle status in human neuroblastoma I BE(2)-C cells.(27) We showed that HeLa cells transfected with Bmi-1 siRNA displayed accumulation at the G1 phase and demonstrated G0/G1 phase arrest, a reduction at the S phase, prevention of mitosis, and ultimately the inhibition of cell proliferation; Bmi-1 maintains the proliferation potential of HeLa cells through a mechanism that is largely dependent on cell cycle regulation. CSC should be rare, quiescent, and capable of self-renewal and maintaining tumor growth and heterogeneity.(28) Bmi-1 has been shown to have an essential role in regulating the proliferative activity of normal and leukemic stem cells.(4) Subsequently, Liu et al. demonstrated that Bmi-1 plays an important role in regulating self-renewal of normal and tumorigenic human mammary stem cells.(6) To determine whether Bmi-1 could determine the proliferative potential of HeLa cells, we analyzed the effect of Bmi-1 silencing on the transformation ability of HeLa cells in vitro and in vivo. We showed that the inhibition of Bmi-1 expression by siRNA downregulation inhibited the cell colony-forming ability. To confirm the in vitro results, an in vivo tumorigenesis assay was carried out. This showed that HeLa-S1 and HeLa-S3 cells became less tumorigenic, as demonstrated by the reduced tumor weight, than HeLa-W or HeLa-V cells. Therefore, these results suggest that Bmi-1 siRNA inhibited the transformation of HeLa cells in vitro and had a strong antitumor effect in vivo.
Tumor cell invasion via the destruction of the extracellular matrix and migration into the vascular system is an important part of the process of tumor metastasis. CSC may migrate to distal sites from the primary tumor and cause metastasis.(29) The migratory ability of tumor cells reflects the metastatic potential of the tumor. Our previous work also demonstrated that reducing Bmi-1 levels by antisense RNA expression suppressed the migratory capacity of A549 cells in vitro.(16) To further determine whether the downregulation of Bmi-1 by RNAi influenced the migration of HeLa cells in vitro, we performed a transwell migration assay. This showed that the silencing of Bmi-1 diminished the migratory capacity of HeLa cells in vitro. Subsequently, we found that the silencing of Bmi-1 reduced the blood-borne metastatic ability of HeLa cells in nude mice. These results provide compelling evidence that the knock down of Bmi-1 expression contributes to tumor metastasis and suggest that Bmi-1 has a significant correlation with the metastatic ability of cervical cancer cells.
In conclusion, Bmi-1 siRNA effectively inhibits HeLa cell proliferation and migration in vitro and in vivo by downregulating Bmi-1 expression. Our results suggest that the Bmi-1 gene is a good target for the gene therapy of cervical cancer and deserves further investigation as a novel approach to cancer therapy.
This research was supported by a grant from Liaoning Province Technology Department of China (grant no. 20072169). We thank Mrs Daping Nie and Mr. Jie Zhu for their technical assistance with the fluorescence-activated cell sorter analysis.