The cervical cancer C33A (HPV-negative) and HeLa (HPV-positive) cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences, and maintained in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc.) with high glucose, 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.). The cells were maintained in 6-cm plates at 37°C in an incubator with 5% CO₂. The C-33A-Wild group are untransfected C-33A cervical cancer cells.

To induce upregulation of SNAI2, the coding sequences of SNAI2 was synthesized and cloned into the GV367 overexpression plasmids by JIKAI GENE Company (https://www.genechem.com.cn/). Subsequently, lentiviruses were produced by transfecting the 293T cells using Lipofectamine 2000 (cat. no. 11668019; Thermo Fisher Scientific, Inc.) with GV367-Ctrl and GV367- SNAI2 vectors. After 48 h, the virus supernatants were harvested, filtered and concentrated. Subsequently, the lentiviruses were added to the medium, supplied with 5 µg/ml polybrene (cat. no. REVG0001; JIKAI GENE; http://www.genechem.com.cn/) to infect the C-33A cells at 37°C in an incubator with 5% CO₂ for 96 h. Multiplicity of infection was 20. Finally, the cell lines with a stable overexpression were constructed by treating the cells with 2.5 µg/ml puromycin (cat. no. HB-PU-1000; Hanbio Biotechnology Co., Ltd.) for 2 weeks.

RNA was extracted from cells with TRIzol^® reagent (Invitrogen; Thermo Fisher scientific, Inc.). The relative quantity of mRNA was determined by RT-qPCR, as previously described (8). The primer sequences were as follows: SNAI2 sense, 5′-AGGAATCTGGCTGCTGTG-3′ and antisense, 5′-GGAGAAAATGCCTTTGGAC-3′; Cyclin D1 sense, 5′-CAGATCATCCGCAAACACG-3′ and  antisense, 5′-GGGACATCACCCTCACTTAC-3′; u-PAR sense, 5′-GAGCTATCGGACTGGCTTGAA-3′ and antisense, 5′-CGGCTTCGGGAATAGGTGAC-3′; CDK1 sense, 5′-AAACTACAGGTCAAGTGGTAGCC-3′ and antisense, 5′-TCCTGCATAAGCACATCCTGA-3′ p21 sense, 5′-GGACAGCAGAGGAAGACCATGT-3′, and antisense, 5′-TGGAGTGGTAGAAATCTGTCATGC-3′; and GAPDH sense, 5′-TCATTGACCTCAACTACATGGTTT-3′ and antisense, 5′-GAAGATGGTGATGGGATTTC-3′.

The tumor cells (1.0×10⁵/well) were cultured in 6-well plates for 3 days, and were then analyzed using propidium iodide (Molecular Probes; Invitrogen; Thermo fisher scientific, Inc.) to discriminate non-viable cells. DNA synthesis or the total DNA content were measured by flow cytometry using a FACSCalibur flow cytometer (BD Accuri™ C6; BD Biosciences). FlowJo software (version 7.6.1; FlowJo LLC) was used for analysis of the results.

The tumor cells (1.0×10³/well) were cultured in 96-well plates for 2 days. The glucose consumption in cervical cancer cells, with or without upregulation of SNAI2, was detected by oxidase assay (cat. no. 09000238813; Shanghai Rongsheng Biotech Co., Ltd.), according to the manufacturer's protocol.

The cells (C-33A-Wild, C-33A-SNAI2, HeLa and HeLa-H₂O₂) were plated at a density of 3×10³ cells/well in 10% FBS-containing medium. The HeLa-H₂O₂ group cells were prepared by incubating the cells with 0.9908 mmol/l H₂O₂ at 37°C for 4 h. The senescence of tumor cells was detected by SA-beta-Gal staining at room temperature for 15 min (cat. no. C0602; Beyotime Institute of Biotechnology), according to the manufacturer's protocol.

The cells (1×10³/well) were resuspended in 100 µl 10% FBS-containing medium and were cultured on plates for 24 h. The CCK-8 assay (WST; Dojindo Laboratories, Inc.) was then used to measure cell proliferation according to the manufacturer's protocol. The cells were incubated with 10 µl CCK-8 buffer and 90 µl of DMEM at 37°C for 4 h. The OD value was then measured at 450 nm.

WB was performed as previously described (8). Total protein was extracted from the cells using RIPA buffer (Thermo Fisher Scientific, Inc.). The protein concentration was measured using the Bradford Protein Assays (Thermo Fisher Scientific, Inc.). A total of 40–70 µg proteins were then separated by SDS-PAGE on 12% gels, followed by transfer onto PVDF membranes. The PVDF membranes were blocked with 5% non-fat milk and incubated with primary antibodies [SNAI2 (1:1,000; cat. no. sc-166476; Santa Cruz Biotechnology, Inc.), ERK1/2 (1:1,000; cat. no. sc-514302; Santa Cruz Biotechnology, Inc.), phosphorylated (p-)ERK1/2 (1:2,000; cat. no. sc-7976; Santa Cruz Biotechnology, Inc.), p38 (1:1,000; cat. no. 9212; Cell Signaling Technology, Inc.), p-p38 (1:1,000; cat. no. 9211; Cell Signaling Technology, Inc.) and β-actin (1:2,000; cat. no. 4967; Cell Signaling Technology, Inc.)] at 4°C overnight. After washing with PBS-Tween (Beyotime Institute of Biotechnology), the membranes were incubated with secondary antibodies for 2 h at room temperature. All bands were washed in TBS with 0.05% Tween-20 and immunoblotted with peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies, respectively. The bands were detected using an enhanced ECL-Plus kit (cat. no. P1010-25; Applygen Technologies, Inc.) and exposed to film. ImageJ 2019 (National Institutes of Health) was used for densitometry. Actin protein was used for all western blotting as a loading control.

Results were expressed as the mean ± standard deviation of three independent experiments with a total of six samples and interpreted by one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference. SPSS software (version 17.0; SPSS, Inc.) was used for statistical analyses.

SNAI2 expression was detected using RT-qPCR and WB in cervical cancer cell lines. A high level of SNAI2 expression was detected in the HPV-positive cervical carcinoma HeLa cell line, but almost no SNAI2 expression was detected in the HPV-negative cervical carcinoma C-33A cell line (Fig. 1A and B).

To further investigate the function of SNAI2 in HPV-negative cervical cancer cells, exogenous SNAI2 was stably overexpressed in C-33A cells (C-33A-SNAI2) using GV367-SNAI2 lentiviral particles, and expression was determined using RT-qPCR, WB and fluorescence microscopy (Fig. 1C). The results showed that the mRNA and protein expression levels of SNAI2 were high in C-33A-SNAI2 cells (Fig. 1).

To investigate whether SNAI2 expression promoted the dormancy of HPV-negative cervical cancer cells, C-33A-Wild, C-33A-SNAI2 and HeLa cells were first cultured for 24 h, and cell proliferation was tested using the CCK-8 method. The results revealed that the viability of C-33A-SNAI2 cells was significantly lower than that of their respective control cells (C-33A-Wild and HeLa), indicating that upregulation of the SNAI2 gene suppressed the proliferation of HPV-negative cervical cancer C-33A cells (Fig. 2).

It was further investigated whether the upregulation of SNAI2 affected the cell cycle regulation in HPV-negative cervical cancer C-33A cells. The present results revealed that a high percentage of HPV-negative cervical cancer C-33A cells remained in the G0/G1 phase, with a smaller S and G2/M cell population, when SNAI2 expression was upregulated, suggesting that the cells remained arrested at G0 phase (Fig. 3). In addition, HPV-positive cervical cancer HeLa cells maintained the highest viability, with the smallest population in the G0/G1 phase. In conclusion, SNAI2 may administrate difference function in HPV-positive and HPV-negative cervical cancer cells.

All of these results demonstrated that overexpression of the SNAI2 gene inhibited the proliferation of HPV-negative cervical cancer cells by inducing cell cycle arrest during the G0/G1 phase of the cell cycle.

Senescence plays a fundamental role in some age-related diseases, such as osteoarthritis, glaucoma, diabetes and cancer (14). The critical characteristic for senescence is cell cycle arrest; and senescent cells exhibit permanent proliferation arrest with increased expression of cell cycle inhibitors (15), meaning that senescent cells cannot restart proliferation after release from cell cycle arrest (14,15).

To further examine whether SNAI2-mediated C-33A cell cycle arrest was due to the promotion of cell entry into dormancy or senescence, tumor cells were stained for expression of the senescence marker SA-beta-Gal. It was observed that C-33A-SNAI2 cells lacked the typical morphological signs of senescence (including cell enlargement, flattening and long cytoplasmic projections), and the percentage of cells positive for the senescence marker SA-beta-Gal was significantly decreased in C-33A-SNAI2 cells, compared with that in C-33A-Wild cells, indicating that the upregulation of SNAI2 was required for the proliferating-to-dormant switch of HPV-negative cervical cancer C-33A cells via the induction of G0/G1 arrest, but not the enhancement of cell senescence (Fig. 4A and B).

Glucose plays an important role in the survival and proliferation of tumor cells, and it was previously demonstrated that the dependence of cancer cells on glucose promotes survival and cellular proliferation (16). To further identify whether the upregulation of the SNAI2 gene was involved in regulating glucose consumption in C-33A cells, glucose consumption of tumor cells was assessed using oxidase assays. The present results revealed that C-33A-SNAI2 cells had lower glucose consumption than C-33A-Wild and HeLa tumor cells (Fig. 4C).

SNAI2 is involved in mediating the proliferation, metastasis and angiogenesis of tumors (17). SNAI2 enhances the kinase activity of cyclin D1/CDK4/CDK6, which is a central mediator in the transition from the G1 to the S phase, to further promote the switch to tumor cell proliferation (18). Therefore, SNAI2 may participate in the proliferating-to-dormant switch of tumor cells.

The u-PAR is identified in tumor cells with a high potential for invasion, metastasis, dormancy and proliferation, upon downregulation of which, the p38SAPK pathway is induced, and a G0/G1 arrest representing tumor cell dormancy is initiated (19).

To confirm the role of SNAI2 in the proliferation-to-dormancy switch of HPV-negative cervical cancer cells, the dormant tumor cells were analyzed using RT-qPCR and WB. As demonstrated in Fig. 5A, the upregulation of SNAI2 could enhance the increase in p21 expression, and the decrease in CDK1, u-PAR and cyclin D1 expression in C-33A cells, compared with that of C-33A-Wild cells.

A previous study reported that the ERK activity level and ERK/p38 activity balance are valid general predictors of tumorigenicity and dormancy, and a high ERK/p38 ratio allows for cell cycle progression and tumor growth (20). Disruption of this complex caused an inversion in the ERK/p38 ratio toward p38 signaling; a change that results in cell cycle arrest and dormancy in breast cancer, prostate cancer, melanoma and fibrosarcoma cell lines (20).

To further clarify the mechanisms underlying the determination of HPV-negative cervical cancer C-33A cell dormancy, the activation of ERK and p38MAPK was detected using WB. The results verified that the sustained activation of p-ERK and p-p38MAPK was significantly decreased when SNAI2 expression was upregulated in C-33A cells (Fig. 5B and C). The p-ERK/p-p38 ratio in C-33A-SNAI2 cells also revealed a downward trend compared with that of C-33A cells (Fig. 5D).