Human cervical cancer cell lines, including HeLa, SiHa and C33A, used in the present study were generously provided by the Scientific Research Center in Zhongnan Hospital of Wuhan University. The cells were maintained in DMEM (Hyclone, China) containing 10% fetal bovine serum, 1% penicillin and streptomycin in a humid 5% CO₂ atmosphere at 37°C.

Human FoxM1-specific RNAi plasmid vectors that express shRNAs or empty vectors were purchased from Shanghai Genechem Co. Ltd. (Shanghai, China). All of the vectors were expressed under the control of a CMV promoter. In brief, 2.0×10⁵ HeLa cells/lane were seeded in a 6-well culture plate and transfected with the appropriate plasmids by using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. The cells were incubated at 37°C for 6 h. Afterwards, lipid and plasmid complexes were removed and a fresh medium was added. At 48 h after transfection, stable transfectants were selected from 700 μg/ml G418 for four weeks. Individual clones were isolated using pipette tips and maintained in G418 (350 μg/ml).

We applied MTT assay to determine cell viability. The number of cells was counted every 24 h for 7 days. A TUNEL apoptosis detection kit (Promega) was used for DNA fragmentation fluorescence staining according to the manufacturer’s protocol. Positively stained, fluorescein-labelled cells were visualised and counted using a fluorescence microscope (Nikon Eclipse 80i, China).

In immunofluorescent staining, the cells were fixed with 4% formaldehyde, blocked for 30 min in 1% BSA prepared in PBS and incubated overnight in primary antibody at a concentration of 1:100 at 4°C. The cells were subsequently incubated in appropriate fluorescence-labelled secondary antibody for 1 h at room temperature. The slides were then mounted with DAPI (Beyotime, China) to visualize the nucleus. Fluorescent photomicrographs were obtained using a fluorescence microscope.

The assays were assessed using uPA, MMP-2, MMP-9 and VEGF ELISA kits according to the manufacturer’s protocol. The cultivated cells were incubated in 6-well plates for 24 h. UPA, MMP-2, MMP-9 and VEGF concentrations were measured using the corresponding ELISA kits (Elabscience, China), afterwards, the culture medium was collected and centrifuged to remove cell debris.

Cell migration and invasion assays were obtained using 24-well chambers (8-μm pore size) with or without Matrigel according to the manufacturer’s protocol. The cells that penetrated the membrane were determined by counting the mean cell number of five ×20 magnification fields randomly and photographed under an inverted phase-contrast microscope (Nikon Eclipse 80i). These experiments were repeated in triplicate.

Nude mice (BALB/c nu/nu, females; 4–5-week-old) were purchased from the Laboratory Animal Center of Wuhan University and housed under SPF conditions. The experimental protocols were approved by the Animal Research Committee of Zhongnan Hospital of Wuhan University. The nude mice were randomly assigned to three groups. FoxM1-shRNA, empty vector and parental HeLa cells suspended in PBS were inoculated subcutaneously on the right oxter with 1×10⁷ cells. Tumor growth was measured at an interval of 6 days after injection by using a calliper, and tumor volume was calculated according to the following formula: length × width² × 0.5 (22). All the mice were euthanized at day 35 post-inoculation. Harvested tumor tissues were removed from each mouse, weighed and cut into two parts. One part was placed in liquid nitrogen and then frozen at −80°C; the remaining part was fixed in 10% buffered formalin, embedded in paraffin, sectioned and stained.

Total RNA was extracted from the cultured cells or tumor xenografts by using TRIzol. The purity of the extracted RNA was then determined by spectrophotometry. The designed premier sequences of uPA, MMP-2, MMP-9, VEGF and β-actin genes are shown in Table I. The first-strand cDNA was synthesized from 4.823 μg of total purified mRNA in 96-well plates with these primers in a total volume of 20 μl. The targeted cDNAs were amplified using SYBR Green Master Mix. The PCR conditions of the genes included the following: one cycle of 50°C for 2 min and 95°C for 10 min; 40 cycles of 95°C for 30 sec and 60°C for 30 sec.

Tumor protein was extracted from cells and tumor xenografts by using RIPA buffer. Equal amounts of protein (50 μg/lane) were electrophoresed on SDS-PAGE gels and blotted on a PVDF membrane (Millipore). Rabbit anti-FoxM1 (1:500, Santa, China), anti-uPA (1:500, Santa, China), anti-MMP2 (1:600, Bioworld, China), anti-MMP9 (1:600, Bioworld), anti-VEGF (1:500, Abcam, China) and β-actin (1:1,000, Boster, China) were used as primary antibodies. HRP-conjugated goat anti-rabbit (1:50,000, Boster) was used as a secondary antibody. The protein bands were detected on X-ray film by using an enhanced chemiluminescence detection system.

To compare the number of capillaries that formed, we immunohistochemically analyzed the serial sections from xenograft tumors of each group. The sections were blocked with 3% H₂O₂ at room temperature for 15 min and the slides were incubated with primary antibody CD31, which is considered as an endothelial cell-specific marker (1:200, Bioworld), overnight at 4°C. Secondary antibodies biotin-labelled anti-rabbit IgG (Bioworld) were used to visualize the specific markers by avidin-HRP/DAB reaction. Negative controls were obtained by replacing the primary antibody with PBS. Microvessel density (MVD) was quantified by observing the number of vessels and immunoreactive endometrial cells per field at ×100 high-power magnification in four vascular ‘hot spots’, and other details were conducted as described previously (23).

Data were expressed as mean ± SD from at least three separate experiments. Statistical analysis was performed using GraphPad Prism 5 software and SPSS 13.0 software. The statistical significance of differences was determined by Student’s two-tailed t-test in two groups and one-way ANOVA in multiple groups. P<0.05 was considered statistically significant.

In advance, the baseline expression of FoxM1 in a panel of cervical cells in our laboratory was determined by western blot analysis. The highest expression of FoxM1 was observed in HeLa cells, which were then used in our study (Fig. 1A). After FoxM1-shRNA transfection was performed, FoxM1 expression was remarkably decreased as revealed by qPCR and western blot analysis compared with empty vector-transfected cells (Fig. 1B and C). These results indicated that FoxM1 expression was effectively suppressed by the specific shRNA of FoxM1 in HeLa cells.

To clarify whether or not FoxM1 downregulation can be a functional alteration, we examined cell viability by conducting MTT assay. We found that FoxM1 knockdown significantly inhibited cell growth from the second day (Fig. 1D).

To evaluate the effect of FoxM1 knockdown on cell apoptosis, we investigated nuclear morphology by TUNEL and DAPI staining. In TUNEL-positive cells, nuclear condensation and fragmentation representing apoptosis was observed. By contrast, the normal cells only showed blue DAPI-stained nucleus (Fig. 2A). The number of TUNEL-positive FoxM1 shRNA-transfected HeLa cells significantly increased compared with the control cells (Fig. 2B). Thus, FoxM1 knockdown markedly induced apoptosis of HeLa cells.

Several proteins that perform primary functions in the invasion, migration and metastasis of cervical cancer include uPA, MMP-2, MMP-9 and VEGF. Immunofluorescence analysis results demonstrated that FoxM1 downregulation decreased the expressions of uPA, MMP-2, MMP-9 and VEGF (Fig. 3A). This result is consistent with the signal differences in the fluorescence-labelled cells. We conducted qPCR and western blot analysis to determine whether or not the expression levels of uPA, MMP-2, MMP-9 and VEGF are influenced by suppressed FoxM1 in HeLa cells. The mRNA and protein levels of uPA, MMP-2, MMP-9 and VEGF were markedly decreased in FoxM1 shRNA-transfected cells (Fig. 3B and C). We also conducted ELISA assays and observed similar patterns in the activities of uPA, MMP-2, MMP-9 and VEGF in stable-transfected cells (Fig. 3D–G). The primary FoxM1-shRNA xenografts were associated with the downregulation of these four factors at mRNA and protein levels (Fig. 4). These results suggested that the downregulation of FoxM1 inhibited uPA, MMP-2, MMP-9 and VEGF expressions in vivo and in vitro and prevented aggressive tumor invasion.

FoxM1 downregulation inhibited the expression and impaired the activity of several important factors involved in tumor cell migration and invasion. We further examined whether or not FoxM1 downregulation affects cell invasion and migration ability by using a transwell system. The FoxM1 shRNA-transfected cells showed a low level of penetration into the membrane with (invasion) or without (migration) Matrigel compared with the control cells (Fig. 5). These results showed that FoxM1 downregulation notably suppressed cell migration and invasion.

As expected, all of the three groups of cells developed tumors (Fig. 6A). The tumor growth curves showed that the growth pattern in the shRNA-FoxM1 group was significantly slower than that in the two control groups (Fig. 6B). The mice were sacrificed at day 35 after injection. Tumor weight in the shRNA-FoxM1 group were significantly smaller than those in the parental HeLa group and the empty vector group (Fig. 6C). Tumor inhibitory rate in shRNA-FoxM1 group reached 72.76% compared with the empty vector group. No differences were observed between the two control groups in terms of growth pattern, tumor size and weight. These data indicated that the suppressed FoxM1 expression of HeLa cells could inhibit tumorigenicity in nude mice.

Tumor induces angiogenesis to maintain the flow of nutrients for the increasing number of cells. To gain insights into tumor angiogenesis, we performed the MVD test. These results indicated that the number of capillary was significantly lower in the FoxM1-shRNA group compared with control group (Fig. 7). This result indicated a decrease in the angiogenic potential compared with the control groups.