Squamous cell cervical carcinoma (SiHa) cells and epidermoid cell cervical carcinoma (CaSki; originally derived from a small-bowel mesentery metastasis) cells and human cervical cancer cell lines [American Type Culture Collection (ATCC) Manassas, VA, USA], were maintained in Dulbecco's modified Eagle's medium (Gibco-BRL, Gaithersburg, MD, USA) containing 10% (v/v) fetal bovine serum and penicillin/streptomycin. Cells were maintained at 37°C in an atmosphere of 95% air and 5% CO₂. Only cells that had been passaged <20 times were utilized in the experiments.

We extracted total RNA using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturers instructions. Total RNA (2 µg) was reverse transcribed into first-strand cDNA using a reverse transcription reagent kit (Invitrogen). The cDNA template was amplified by qRT-PCR using the SYBR-Green Real-Time PCR kit (Toyobo Co., Ltd., Osaka, Japan) on an ABI StepOnePlus Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). We used U6 as the internal standard for all quantifications. We analyzed the relative gene expression using the 2^−ΔΔCt method, and the results are expressed as the extent of change relative to control values. All qRT-PCR experiments were repeated at least three times. Primers used for the PCR reactions are listed in Table I.

We obtained siRNA for SRA lncRNA and negative-control siRNA (siNC) from Genolution Pharmaceuticals (Seoul, Korea). Cells (5×10⁴ cells/well) were seeded into 6-well plates and transfected with 10 nM siRNA in phosphate-buffered saline using the G-Fectin kit (Genolution Pharmaceuticals) according to the manufacturers instructions. The siRNA-transfected cells were used in the in vitro assays 48 h after transfection. The target sequence for the siSRA was, 5′-CTCCCTTCTTACCACCACCA-3′. Experiments were repeated at least three times.

Full-length human SRA-transcript cDNA was amplified by PCR and inserted into the pLenti6/V5-D-TOPO vector using the ViraPower lentiviral expression system (Invitrogen) according to the manufacturers instructions. We then transfected the plasmid into 293FT cells for packaging, with the resultant lentivirus used to infect the desired cell lines. Stably SRA-transfected cells were selected in medium containing blasticidin (Invitrogen).

Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) was utilized to evaluate cell proliferation. Cells (2×10³ cells/well) were seeded into 96-well flat-bottomed plates containing 100 µl of complete medium per well. The cells were incubated overnight to allow for cell attachment and recovery, and subsequently transfected with siNC or siSRA for 24, 48, 72 or 96 h. An aliquot of 10 µl of CCK-8 solution was supplemented into each well, followed by incubation for 2 h. Absorbance was measured at 450 nm to estimate the number of viable cells in each well. The assay was performed in triplicate.

We performed a Matrigel invasion assay using the BD Biocoat Matrigel invasion chamber (pore size, 8-µm; 24-wells; BD Biosciences, Bedford, MA, USA) according to the manufacturers protocol. Cells (5×10⁴) were seeded in the upper chamber, which contained serum-free medium, and complete medium was added to the lower chamber. The Matrigel-invasion chamber was incubated at 37°C under 5% CO₂ for 48 h. Non-invading cells were removed from the upper chamber using cotton-tipped swabs. Cells that had invaded through the pores onto the lower side of the filter were stained (Diff-Quik; Sysmex, Kobe, Japan) and counted using a hemocytometer. The assay was repeated at least three times.

We evaluated cell migration using a wound-healing assay. Approximately 5×10⁵ cells were seeded into 6-well culture plates containing serum-enriched medium and allowed to grow to 90% confluence in complete medium. The serum-containing medium was eliminated, and cells were serum-starved for 24 h. When the cells reached 100% confluence, an artificial homogenous wound was made by scratching the monolayer using a sterile 200-µl pipette tip. Following the scratching, cells were washed with serum-free medium. Images of cells migrating into the wound were captured at 0, 24 and 48 h using a microscope. Three independent experiments were performed in triplicate.

We used radioimmunoprecipitation assay buffer to extract proteins, and a Pierce BCA protein assay kit (both from Thermo Fisher Scientific, Waltham, MA, USA) was used to measure protein concentration. Proteins were boiled with 2X sample buffer, subsequently resolved on 10% sodium dodecyl sulfate-polyacrylamide gels, and transferred electrophoretically to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat dried milk in 1X Tris-buffered saline containing 0.1% Tween-20 (pH 7.6) at room temperature for 1 h, the membranes were incubated with primary antibodies at 4°C overnight under continual agitation. The following primary antibodies were used: rabbit anti-human vascular endothelial growth factor (VEGF) (1:500), rabbit anti-human matrix metalloproteinase (MMP)-2 (1:500) (both from Abcam, Cambridge, UK, USA), rabbit anti-human MMP-9 (1:1,000), rabbit anti-human E-cadherin (1:1,000), rabbit anti-human β-catenin (1:1,000) (all from Cell Signaling Technology, Danvers, MA, USA), mouse anti-human vimentin (1:1,000; Sigma-Aldrich, St. Louis, MO, USA), mouse anti-human Snail (1:1,000), rabbit anti-human SOX-2 (1:1,000), rabbit anti-human Nanog (1:1,000), rabbit anti-human Oct4 (1:1,000) (all from Cell Signaling Technology), and mouse anti-human β-actin (1:5,000; Sigma-Aldrich). We visualized the proteins using an enhanced chemiluminescence system (Amersham, Little Chalfont, UK), and band intensities were quantified using a luminescent image analyzer (LAS-4000 Mini; Fujifilm, Uppsala, Sweden).

IBM SPSS version 23 for Windows (SPSS, Inc., Chicago, IL, USA) was used for statistical analysis. The Kolmogorov-Smirnov test was used to validate standard normal-distributional assumptions. Statistical significance was determined using the Fisher's exact-test, Pearson's Chi-square test and Student's t-test. Mean differences were considered significant at P<0.05. Results are presented as the means ± standard deviation.

We performed qRT-PCR to estimate the expression levels of SRA in five different cell lines, one of which was derived from human embryonic kidney cells (HEK293), whereas the other four were derived from human cervical cancers. We observed that SRA expression levels were significantly higher in epithelioid cervical carcinoma (HeLa), CaSki and SiHa cells as compared with levels observed in epidermoid cervical carcinoma cells (ME-180) (Fig. 1).

To examine the functional role of SRA in cervical cancer cells, siRNA was utilized to downregulate SRA expression in the CaSki and SiHa cells, which showed significantly higher levels of SRA expression. We evaluated the knockdown efficiency of siSRAs, and verified that siSRA transfection was more efficient in silencing SRA as compared with siNC (Fig. 2A and C). We then performed a CCK-8 assay to determine the effect of SRA knockdown on cell proliferation. Our results showed that siRNA-mediated knockdown of SRA in CaSki and SiHa cells substantially decreased cell proliferation (Fig. 2B and D). These findings indicated that SRA is involved in the proliferation of cervical cancer cells.

Wound-healing and Matrigel-invasion assays were performed to determine whether SRA plays a role in cervical cancer cell migration and invasion. In the wound-healing assay, siRNA-mediated knockdown of SRA inhibited cell migration in the CaSki and SiHa cells (Fig. 3A and D). Moreover, SRA-knockdown inhibited cell invasion in the CaSki and SiHa cells according to the results of the Matrigel-invasion assay (Fig. 3B and E). The extent of inhibition upon SRA knockdown was quantified by qRT-PCR (Fig. 3C and F).

To elucidate the molecular mechanisms underlying the inhibitory effect of SRA knockdown on cell migration and invasion, we explored the relationship between SRA expression and MMP-2, MMP-9 and VEGF expression. We observed that siSRA treatment decreased levels of MMP-2, MMP-9 and VEGF mRNA in the CaSki cells (Fig. 4A) as well as MMP-2, MMP-9 and VEGF protein levels (Fig. 4B). These findings indicated that SRA promoted cervical cancer cell migration and invasion via upregulation of MMP-9, MMP-2 and VEGF expression.

Since EMT is crucial to cell migration and invasion, we examined whether SRA is required for EMT. EMT was monitored using qRT-PCR and western blotting following siRNA-mediated knockdown of SRA in CaSki cells. SRA knockdown resulted in increased E-cadherin expression and decreased β-catenin and vimentin expression (Fig. 5A and B). Additionally, the EMT-mediating transcription factor Snail and Twist were downregulated in the siSRA-transfected cells relative to levels observed in the siNC-transfected cells (Fig. 5A and B). These results suggested that SRA may be involved in the dysregulation of EMT-related genes in cervical cancer cells, thereby promoting migration and invasion.

To elucidate the mechanism by which SRA promotes a malignant phenotype in cervical cancer cells, we assessed the status of important signaling cascades controlled by NOTCH in the SRA-knockdown cells. SRA knockdown in the CaSki cells resulted in downregulation of NOTCH1, HES1 and p300 expression [both mRNA (Fig. 6A) and protein (Fig. 6B)]. These data indicated that SRA promoted tumor growth in vitro via the EMT and NOTCH signaling pathways.