In total, 100 women who underwent surgery between 2012 and 2017 at Yonsei Severance Hospital, Yonsei University (Seoul, Korea) were included in this study. Specimens from patients with newly diagnosed invasive stage IA to IVB cervical cancer (International Federation of Gynecology and Obstetrics) who had not received prior treatment were evaluated. Additionally, 22 normal cervical tissues from patients undergoing simple hysterectomy because of uterine leiomyomata were obtained as controls. This study was approved by the Ethics Committee of Yonsei Severance Hospital, and informed consent was obtained from all patients. All specimens were immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction.

The human cervical squamous carcinoma SiHa cells was obtained from the Korean Cell Line Bank (Seoul, Korea) and provided by the Korea Gynecologic Cancer Bank through the Bio and Medical Technology Development Program of the Minister of Science, Information and Communication Technology and Future Planning, Korea. A total of 293 cells were purchased from American Type Culture Collection (Manassas, VA, USA). SiHa and 293 cells were cultured in Dulbecco's modified Eagle's medium. All culture media were supplemented with 10% (v/v) fetal bovine serum and 1% penicillin/streptomycin, and cell lines were maintained at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. Culture medium was replaced with fresh medium every 2–3 days, and cells that had been passaged less than 20 times were used in the experiments.

Total RNA was extracted using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. One microgram of total RNA was reverse transcribed into first-strand cDNA using a reverse transcription reagent kit (Bioline, London, UK). The cDNA template was amplified by qRT-PCR using SensiFAST SYBR Hi-ROX Mix (Bioline). qRT-PCR was performed on an ABI StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). All quantifications were performed with U6 as the internal standard. Relative gene expression was analyzed using the 2^−ΔΔCT method, and the results were expressed as extent of change with respect to control values. qRT-PCR experiments were replicated at least three times. Primers used for PCR are shown in Table I.

Full-length human SRA cDNA was amplified by PCR and inserted into the pLenti6/V5-D-TOPO vector using the ViraPower Lentiviral Expression System (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The plasmid was transfected into 293FT cells for packaging, and the resulting lentivirus was used to infect the desired cell lines. The selection of SRA stably transfected cells was performed in medium containing blasticidin (Invitrogen; Thermo Fisher Scientific, Inc.).

Cell proliferation was evaluated using Cell Counting kit-8 (CCK-8) assays (Dojindo Laboratories, Kumamoto, Japan). Cells were seeded into 6-well flat-bottomed plates (1×10⁵ cells/well) in 2 ml complete medium. The cells were incubated overnight to allow for cell attachment and recovery and were subsequently subjected to SRA1 overexpression for 24, 48, 72, or 96 h. Next, 10 µl CCK-8 solution was added to each well, and cells were incubated for 1 h. The absorbance was measured at 450 nm using an auto-microplate reader to calculate the number of viable cells in each well. The cell survival rate was expressed as the absorbance relative to that of the SiHa, veoctor cells. Three independent experiments were performed in triplicate.

Matrigel invasion assays were performed using BD Biocoat Matrigel Invasion Chambers (pore size: 8 µm, 24-well; BD Biosciences, Bedford, MA, USA), according to the manufacturer's protocol. Briefly, 1×10⁵ cells were plated in the upper chambers in serum-free medium, and complete medium was added to the bottom chamber. The chambers were then incubated for 48 h at 37°C in an atmosphere containing 5% CO₂. Noninvading cells were removed from the upper chambers using cotton-tipped swabs. Cells that had invaded through the pores into the lower side of the filter were stained (Diff Quik; Sysmes, Kobe, Japan), and these cells were then counted using a hemocytometer. Invasion cell was measured using ImageJ software (NIH, Bethesda, MD, USA) and the percentage of the invasion cell was calculated. Results were standardized to SiHa cells. The number of cells that invaded the membrane was counted in 10 fields under the ×20 objective lens. Original magnification, ×200. The assay was replicated at least three times.

Cell migration was assessed using wound healing assays. Briefly, 1×10⁶ cells were seeded into 6-well culture plates with serum-containing medium and allowed to grow to 90% confluence in complete medium. The serum-containing medium was removed, and cells were serum starved for 24 h. When the cell density reached ~100% confluence, an artificial homogenous wound was created by scratching the monolayer with a sterile 200-µl pipette tip. After scratching, the 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. Scratch width was measured using NIH ImageJ software, and the percentage of the scratch area closed was calculated as (width at 0 h-width at 48 h)/width at 0 h. Results were standardized to SiHa cells. The migration cells was counted in 10 fields under the ×20 objective lens. Original magnification, ×200. The assay was performed in triplicate.

Proteins were extracted with RIPA buffer (Thermo Fisher Scientific, Inc.). Protein concentrations were measured using a Pierce BCA Protein assay kit (Thermo Fisher Scientific, Inc.). After boiling with 5X sample buffer, proteins were resolved on 10% sodium dodecyl sulfate-polyacrylamide gels and transferred electrophoretically to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 5% nonfat dried milk in 1X Tris-buffered saline containing 0.1% Tween-20 (pH 7.6) at room temperature for 1 h and were then incubated with primary antibodies at 4°C overnight under constant agitation. The primary antibodies included rabbit anti-human E-cadherin (1:1,000 dilution), rabbit anti-human β-catenin (1:1,000 dilution), and mouse anti-human Snail (1:1,000 dilution; all Cell Signaling Technology, Danvers, MA, USA). Proteins were visualized 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).

The results of the statistical analyses (SPSS, version 24.0; IBM, Chicago, IL, USA) were expressed as means ± standard deviations (SDs). Pearson's χ² tests, Student's t-tests, and Fisher's exact tests were used to evaluate the associations between SRA expression and clinicopathological characteristics. To evaluate the performance of the models with respect to their discrimination ability, statistics were used chi-square values from the logrank test from the receiver operating characteristic (ROC) analysis. The median value (1.64) was set as the cut-off value. The groups were classification into high and low SRA expression groups at values above and below the cut-off value (1.64), respectively. The Kaplan-Meier method was used to analyze overall survival times. The log-rank test was used to estimate between group differences. Stepwise Cox regression model analysis was used for multivariate survival analysis of the parameters that were significant in the univariate analysis. The statistical tests were two-sided; differences with P<0.05 was considered to indicate a statistically significant difference.

To determine whether SRA expression in tissues was linked to the clinicopathological features of cervical cancer, we evaluated the expression of SRA in cervical cancer tissues (n=100) and corresponding normal tissues (n=22). SRA expression in cervical cancer tissues was more than 3.15-fold that of noncancerous tissues (P=0.00007; Fig. 1). Additionally, we examined the relationships between SRA expression and clinicopathological information in 100 patients with cervical cancer (Table II). The mean follow-up period was 60 months. Additionally, patients with high SRA expression exhibited higher rates of lymphatic invasion and invasive cell type relative to patients with low SRA expression; this relationship was statistically significant.

The median 5-year survival duration was significantly higher in the low SRA expression group than in the high SRA expression group (19.7 and 25.1 months, respectively; log-rank test: P=0.041; Fig. 2A). The risk model in SRA data had an area under curve (AUC) of 0.737 in predicting (P=0.001; Fig. 2B). Moreover, Cox univariate proportional hazards analysis showed that stage [hazard ratio (HR) =2.809; P=0.004], tumor size (HR=2.260; P=0.011), and recurrence (HR=2.479; P=0.05) were independent prognostic factors for overall survival (Table II). Cox multivariate proportional hazards analysis showed that SRA expression (HR=3.714; P=0.031), stage (HR=2.809; P=0.004), tumor size (HR=2.143; P=0.047), and lymphatic invasion (HR=3.44, P=0.03) were independent prognostic factors for overall survival (Table III). Both univariate and multivariate proportional hazards analyses showed that stage and tumor size were independent prognostic factors of overall survival.

We previously examined SRA expression levels in several cervical cancer cell lines by qRT-PCR (15). Here, lentiviral-mediated overexpression of SRA was performed to determine the functional role of this lncRNA in SiHa cells. SRA-expressing plasmid was prepared using as template a PCR product that contained the SRA sequences (Fig. 3A). RT-PCR analysis showed that SRA was successfully overexpressed in SiHa cells compared with that in control cells (P=0.01; Fig. 3B). We next examined the impact of SRA overexpression on cell proliferation. The results of CCK-8 assays showed that overexpression SRA in SiHa cells increased cell proliferation (Fig. 3C), suggesting that SRA was involved in the proliferation of cervical cancer cells.

Next, the effects of SRA on the invasive and migratory behaviors of cells were assessed by Matrigel invasion and wound healing assays. Overexpression of SRA resulted in increased migration of SiHa cells relative to empty vector-expressing controls (P=0.001; Fig. 4A). There was a significant difference between the scratch width percentages of each cell line 24 and 48 h after in the SiHa, empty vector and SRA overexpression cells (P=0.001; Fig. 4B). Furthermore, SRA overexpression in SiHa cells significantly increased invasion relative to that in empty vector-expressing cells (P=0.001; Fig. 4C). The invasion relative percentages of each cell line 48 h after in SiHa, empty vector and SRA overexpression groups was significantly different (P=0.001; Fig. 4D). The SiHa cells percentages were 100±4.58. The empty cell line percentages were 72±13.52. The SRA overexpression cell line percentages were 173±5.38. Altogether, these results indicated that SRA promoted SiHa cell invasion and migration in vitro.

Because the EMT is important in cell migration and invasion, the present study examined whether SRA was required for EMT using RT-qPCR and western blotting. The overexpression of SRA decreased E-cadherin expression and increased N-cadherin, Snail, Wnt5β, Vimentin and β-catenin expression (Fig. 5A). Levels of protein in cells with overexpressed SRA had decreased E-cadherin expression and increased N-cadherin, β-catenin, Vimentin, Wnt5β, Twist and Snail expression (Fig. 5B). The western blot density percentages of each cell line 48 h after in the SiHa, empty vector and SRA overexpression cells are presented in Fig. 5C. In addition, the expression of Snail, a transcription factor that mediates the EMT, was upregulated in SRA-overexpressing SiHa cells compared with that in cells transfected with the empty vector (Fig. 5A-C). These data suggested that upregulation of EMT-associated genes could partly explain the involvement of SRA in cervical cancer cell migration and invasion.