A total of 30 normal cervical and 17 cervical cancer samples without chemotherapy, immunotherapy or radiotherapy were obtained via surgery from The Second Affiliated Hospital of the Medical College of Zhengzhou University (Zhengzhou, China) between January 2012 and December 2013. The present study was approved by the Ethics Committee of the Medical College of Zhengzhou University and informed consent was obtained from the patients prior to sample collection.

Human cervical cancer cell lines (SiHa, C33A and CaSki) and the normal colonic epithelial cell line (CRL-1831) were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA). All cell lines were cultured in RPMI-1640 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (HyClone), 100 U/ml penicillin and 100 µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA) and grown at 37°C in an incubator with 5% CO₂. The medium was replaced once every 2–3 days and cells were digested with 0.25% trypsin and passaged when they reached 80% confluence.

The full-length coding sequence of the human SASH1 gene was amplified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and ligated into the pGEM-T vector (Clontech Laboratories, Inc., Cambridge, UK) at the EcoRI and KpnI restriction sites. Following sequence confirmation, positive clones were subcloned into the pcDNA3.1 expression vector to construct the recombinant expression vector pcDNA3.1-SASH1.

For in vitro transfection, the cells were seeded in six-well plates at a density of 1.0×10⁵ cells/well and cultured overnight. When the cells grew to 70–80% confluence, pcDNA3.1-SASH1 and the empty vector were transfected using Lipofectamine 2000™ (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions.

RNA was extracted from macrodissected primary tumor tissues or cultured cells homogenized in TRIzol reagent according to the manufacturer's instructions (Invitrogen; Thermo Fisher Scientific, Inc.). The Superscript First-Strand kit was used to synthesize first strand cDNA (Invitrogen; Thermo Fisher Scientific, Inc.). RT-qPCR was performed using an ABI Prism 7500 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and the following primers were used: SASH1, sense 5′-CGG GAA AGC GTC AAG TCG GA-3′ and antisense 5′-ATC TCC TTT CTT GAG CTT GAG-3′; matrix metalloproteinase (MMP)-2, sense 5′-CCC CAG ACA GGT GAT CTT GAC-3′ and antisense 5′-GCT TGC GAG GGA AGA AGT TG-3′; MMP-9, sense 5′-CGCTGGGCTTAGATCATTCC-3′ and antisense 5′-AGG TTG GAT ACA TCA CTG CAT TAG G-3′. The levels of individual gene mRNA transcripts were initially normalized to the control β-actin. Subsequently, the differential expression of these genes was analyzed using the 2^-ΔΔCq method.

Equal quantities (30 µg) of protein were solubilized in Laemmli buffer (62.5 mM Tris/HCl pH 6.8, 10% glycerol, 2% SDS, 5% mercaptoethanol and 0.00625% bromophenol blue), boiled for 5 min, separated by 12% polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes (EMD Millipore, Bedford, MA, USA). The membranes were probed with rabbit anti-SASH1 (cat. no. LS-C285219; LifeSpan Biosciences, Seattle, WA, USA), rabbit anti-MMP-2 (cat. no. 4022S), rabbit anti-MMP-9 (cat. no. 3852S), rabbit anti-FAK (cat. no. 3285S) or mouse anti-β-actin (cat. no. 4967S) (all from Cell Signaling Technology Inc., Beverly, MA, USA) in Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% BSA (Sigma-Aldrich) at 4°C overnight, followed by three washes in TBST for 5 min per wash. The membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG polyclonal antibody (cat. no. MBS560261; Mybiosource, San Diego, CA, USA) for 1 h at room temperature. The blotted protein bands were exposed to and visualized using enhanced chemiluminescence (ECL) reagent. Developed films were digitized by scanning, and the optical densities were analyzed using Image J software 1.61 (National Institutes of Health, Bethesda, MD, USA).

Cell viability was determined using an MTT assay as described previously (8). In brief, cells were plated in 96-well culture plates at 5×10³ cells per well and incubated overnight. Cells were treated with pcDNA3.1-SASH1 vector or empty vector for 24, 48, 72 and 96 h. Following this, MTT was added to each well. The resulting formazan was then dissolved in 100 µl of dimethyl sulfoxide and the absorbance was recorded at 540 nm using a Bio-Rad 3350 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).

Cells were seeded into 10-mm dishes at a density of 2×10² cells/dish and then cultured at 37°C for 10–14 days until the visible cell clones were formed. Following this, the cells were fixed with methanol. Fixative buffer was discarded and the cells were stained with Giemsa dye for 10 min. After the staining solution had been discarded, the number of cell clones was counted.

Cell invasion was measured using a modified Boyden chamber (BD Biosciences, Bedford, MA, USA). Cells were treated with overexpression-SASH1 or the mock for 4 h, and were then seeded in the upper chamber. DMEM medium with 10% fetal bovine serum was added into the lower chamber. After 24 h of incubation, cells that passed through the lower side of the membrane were stained with hematoxylin and eosin (Sigma-Aldrich) and quantified using a BX51 fluorescence microscope (Olympus Optical Co., Tokyo, Japan) by counting six high-powered fields in the center of each well.

Statistical analysis was performed using SPSS software version 16.0 (SPSS, Inc., Chicago, IL, USA). For comparison among groups, a χ² test or a one-way analysis of variance followed by a post hoc Tukey's test was performed. P<0.05 was considered to indicate a statistically significant difference. The data are presented as the mean ± standard deviation.

To examine the role of SASH1 in cervical cancer tumorigenesis, RT-qPCR analyses of cervical cancer tissues and normal cervical tissues was performed. The results demonstrated that the mRNA expression of SASH1 was markedly lower in cervical cancer tissues compared with the normal cervical tissues (Fig. 1A). Similar results were obtained from the analysis of cervical cancer cell lines. As shown in Fig. 1B and C, the mRNA and protein expression levels of SASH1 were also significantly lower in cervical cancer cell lines compared with the CRL-1831 group. Collectively, these data suggest that SASH1 is downregulated in cervical cancer.

To further investigate the effect of SASH1 on cervical cancer tumorigenesis, the recombinant expression vector pcDNA3.1-SASH1 was constructed, and then RT-qPCR and western blot analysis were employed to determine the efficiency of SASH1 transfection. As shown in Fig. 2A, the mRNA expression of SASH1 was significantly higher in the pcDNA3.1-SASH1-transfected group relative to the control group. No significant difference in the mRNA expression level of SASH1 between the control group and the empty vector group was identified. Consistent with the results of RT-qPCR, western blot analysis demonstrated that the expression of SASH1 protein was also significantly increased in the pcDNA3.1-SASH1-transfected group (Fig. 2B). Collectively, these data show that the expression of SASH1 was successfully elevated in the CaSki cell line.

Subsequently, the effect of SASH1 on cervical cancer cell proliferation was investigated. As shown in Fig. 3A, SASH1 significantly inhibited the proliferation of cervical cancer cells, in a time-independent manner. In addition, the effect of SASH1 on colony formation in soft agar was investigated. SASH1 also reduced the colony formation in soft agar relative to the control (lentiviral vector)-transduced cells (Fig. 3B). These data indicate that SASH1 inhibits cervical cancer cell proliferation.

The invasive behavior of cervical cancer cells was determined using a Boyden chamber assay. Cervical cancer cells demonstrated a reduced capacity for invasion when transfected with pcDNA3.1-SASH1 (Fig. 4A). Furthermore, since the upregulation of MMP-2 and MMP-9 expression contributes to the invasiveness of cancer cells (9,10), the effects of SASH1 on MMP-2 and MMP-9 was investigated in cervical cancer cells. As shown in Fig. 4B and C, SASH1 significantly decreased the expression levels of MMP-2 and MMP-9.

The FAK pathway is involved in cell proliferation and invasion. The present study thus examined whether SASH1 affects the level of FAK. As shown in Fig. 5, SASH1 overexpression significantly inhibited the expression level of FAK. This indicated that SASH1 may be involved in the regulation of the FAK pathway as an upstream molecule, suggesting that SASH1 regulates cell proliferation and invasion via the FAK pathway.