As it involved the use of human materials, the present study was approved by the Ethics Committee of the Affiliated Hospital of Qingdao University (Qingdao, China) according to the experimental requirements of the school. HeLa, Ca Ski, SiHa and H8 cells were purchased from the National Infrastructure of Cell Line Resources of China. H8 cell line (the human cervical epithelial immortalized cell line) was used as the external control of the three cervical cancer cell lines, HeLa, Ca Ski and SiHa. Each cell line was cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) in 100 mm culture dishes (Corning Inc.) and incubated in a 5% CO₂. incubator at 37°C. The media was changed every 3 days, and the cells were passaged once they reached 80% confluence. Additionally, 293T cells (purchased from the National Infrastructure of Cell Line Resources of China) were used in co-transfection experiment and cultured with the same aforementioned treatment. To ensure cell activity, the cells from passage 2–4 were used for further experiments.

According to database sequences of the human LncRNA PVT1 (National Center for Biotechnology Information reference sequence: NR_003367.3), the pEGFP-N3 eukaryotic expression vector (Beijing Tianyi Huiyuan Bioscience & Technology Inc.) carrying the LncRNA PVT1 sequence was constructed via artificial nucleotide synthesis at Tianyi Huiyuan Biotechnology Co. Ltd. Similarly, according to the information on LncRNA PVT1 in the Ensembl genome browser 96 database (reference number, ENSMUSG00000173039). The sequences of the 3′UTR and the mutant of the 3′UTR of NF-κB gene were obtained via artificial nucleotide synthesis at Beijing Tianyi Huiyuan Bioscience & Technology Inc. and the expression plasmid constructed. The siRNA (5′-GAGCUGCGAGCAAAGAUGU-3′) and control siRNA (si-NC; 5′-GAUCGUACUAUAGCUUGUA-3′) of LncRNA PVT1 were purchased from Guangzhou RiboBio Co., Ltd.

Fugene6 transfection reagent (Roche Diagnostics) was used according to the manufacturer's protocol. The overexpression vector (final concentration 1 µg/ml) or siRNA of LncRNA PVT1 (final concentration 50 nm) were transfected into HeLa, Ca Ski and SiHa cells in 96-well plates or 100 mm dishes with 1×10⁵ cells/ml and cultured for 48 or 60 h, respectively, and the pEGFP-N3 empty vector or si-NC were also transfected with the corresponding internal controls. Additionally, HeLa cells were respectively treated with 5 µg/ml lipopolysaccharides (LPS; Beyotime Institute of Biotechnology) plus the lncRNA PVT1 siRNA, with pyrrolidinedithiocarbamate (PDTC; 50 µmol/l; Beyotime Institute of Biotechnology) alone, or with PDTC (50 µmol/l) on the basis of the lncRNA PVT1 overexpression treatment for 8 h to activate or inhibit the NF-κB pathway, and then the cells were continuously cultured for 60 h. In addition, using the Fugene6 transfection reagent, HeLa cells with 60% confluency were transiently transfected with the miR-16 mimics (final concentration 100 nm) or miR-16 inhibitor (final concentration 50 nm). All cells were collected for the experiments below following 60 h treatment.

Following culture of the cells from the four cell lines in 96-well plates with different treatments or no treatment for 60 h, cell numbers were determined using the MTT assay. The assay was conducted by adding 20 µl MTT (5 mg/ml) to each well of the plates and incubating the plates for 4 h at 37°C. Following removal of the supernatant, formazan crystals were dissolved in 200 µl dimethyl sulfoxide, and the absorbance was measured at 490 nm by microplate reader (Thermo Fisher Scientific, Inc.). There were eight replicate wells for each group to ensure the accuracy of the experiments.

Following treatment with or without the LncRNA-PVT1 siRNA for 48 h, HeLa cells were collected to use for cell cycle or cell apoptosis analyses using a FACSCanto II flow cytometer (Becton, Dickinson and Company). For the cell cycle analysis, cells were centrifuged at 300 × g for 5 min at 4°C and washed with ice-cold PBS following digestion with trypsin. The cells were then mixed with 0.25% Triton X-100 and 5 µl propidium iodide (PI) and incubated for 30 min at room temperature in the dark. Cells were resuspended in 0.5 ml PBS and analyzed immediately.

For cellular apoptosis analysis, cells were digested with trypsin, centrifuged at 300 × g for 5 min at 4°C, and washed with ice-cold PBS. The cell pellets were resuspended in 100 µl of Annexin V binding buffer, transferred to a 5 ml culture tube containing 5 µl Annexin V-FITC, and mixed with 10 µl PI by using the Annexin V-FITC Apoptosis Detection kit (Beyotime Institute of Biotechnology). The tubes were gently vortexed and incubated for 15 min at room temperature in the dark. Subsequently, 300 µl binding buffer was added and the cells were analyzed immediately.

Total RNA was extracted from three cervical cancer cells and H8 cells with different treatments using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. First-strand cDNA was synthesized using a PrimeScript RT Master kit (Takara Biotechnology Co., Ltd.) according to the manufacturer's protocol (37°C for 15 min, 85°C for 5 sec and then stored at 4°C). The qPCR was performed using SYBR Green reagents (Takara Biotechnology Co., Ltd.) and a 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). Following initial denaturation for 30 sec at 95°C, amplification was performed for 40 cycles (95°C for 5 sec and 60°C for 32 sec). The specific primer sequences were as follows: miR-16, forward 5′-TAGCAGCACGTAAATATTGGCG-3′; LncRNA PVT1, forward 5′-TGCTCTAGAATCTGATGCACGTTCCACC-3′, reverse 5′-CCGGAATTCCTTAATTCTCCAATCTCAAAATAC-3′; NF-κB, forward 5′-GCATCCAGACCAACAACAACC-3′, reverse 5′-AGAGTTTCGGTTCACTCGGC-3′; and cyclin D1 (CCND1), forward 5′-CAGATCATCCGCAAACACGC-3′, reverse 5′-AAGTTGTTGGGGCTCCTCAG-3′. Expression was normalized using β-actin or U6 using the 2^−ΔΔCq method (40). Primer sequences were as follows: β-actin, forward 5′-GCTCGTCGTCGACAACGGCTC-3′, reverse 5′-CAAACATGATCTGGGTCATCTTCTC-3′; U6, forward 5′-CTCGCTTCGGCAGCACA-3′, reverse 5′-GCGAGCACAGAATTAATACGAC-3′. A melting curve was constructed to verify the amplification of a single PCR product. Experiments were performed in triplicate with standard deviations of the Cq values not exceeding 0.5 on a within-run basis.

Following the sequence alignment analysis of NF-κB-3′UTR and miR-16, the pGL3-NF-κB-3′UTR reporter plasmid (binding sites, UUAUAA), the corresponding mutant plasmid (AAGGCC) and the empty plasmid (Beijing Tianyi Huiyuan Bioscience & Technology Inc.) were constructed. Using the Fugene6 transfection reagent, 293T cells with 60% confluency were transiently transfected with the pGL3-NF-κB-3′UTR reporter plasmid (final concentration 1 µg/ml) and miR-16 mimics (final concentration 100 nm) in the dual-luciferase reporter system, the corresponding mutant plasmid and the empty plasmid were transfected as the internal controls. After incubation for 36 h, firefly and Renilla luciferase activities were measured and compared using the dual-luciferase reporter assay kit (Promega Corporation) by Thermo Scientific™ Fluoroskan Ascent™ FL microplate reader (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Total cell lysates were extracted from HeLa cells with the different treatments using a radioimmunoprecipitation assay (RIPA) buffer (Beijing Solarbio Science & Technology Co., Ltd.) to detect the pNF-κB (p65), NF-κB (p65), Iκ-Bα, Bcl-2 and CCND1 protein levels, and total protein levels in all samples were assessed using a BCA Protein Assay kit (Beijing Solarbio Science & Technology Co., Ltd.). After denaturation, equal amounts of protein were loaded into each well of a 10–12.5% polyacrylamide gel and subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were then transferred onto polyvinylidene difluoride (PVDF) membranes, which were blocked with 5% skimmed milk (Sigma-Aldrich, Merck KGaA) at room temperature for 1 h on a shaker. Next, the membranes were incubated with primary antibodies overnight at 4°C and subsequently washed three times for 30 min before being incubated with goat anti-rabbit IgG-HRP (dilution 1:5,000, cat. no. ab205718; Abcam) at room temperature for 1 h. The bound antibodies were visualized with the ECL Plus Western Blotting Detection System (GE Healthcare Life Sciences). The following monoclonal antibodies were used: Rabbit anti-pNF-κB (p65) (65 kDa, dilution 1:10,000, cat. no. ab76302), rabbit anti-NF-κB (p65) (65 kDa, dilution 1:1,000, cat. no. ab32536), rabbi anti-Iκ-Bα (17 kDa, dilution 1:1,000, cat. no. ab178847), rabbi anti-Bcl-2 (26 kDa, dilution 1:1,000, cat. no. Ab32124), rabbi anti-CCND1 (34 kDa, dilution 1:10,000, cat. no. ab134175) and rabbit anti-Tubulin antibody (55 kDa, dilution 1:5,000, cat. no. ab59680) served as an internal protein loading control (Abcam).

All statistical analyses were performed using one-way ANOVA followed by a Student-Newman-Keuls test (the treatments with siRNA, siRNA plus LPS, overexpression, PDTC, or PDTC plus overexpression, respectively) with the Statistical Analysis Systems software (version 8.2; SAS Institute, Inc.) to determine significance. The data are expressed as the mean ± SD P<0.05 was considered to indicate a statistically significant difference.

Compared with H8 cells, LncRNA PVT1 expression levels were significantly increased in the three cervical cancer cells (Fig. 1A), and the cell numbers had also significantly increased (Fig. 1B). To further identify the effect of LncRNA PVT1 on cervical cancer, HeLa, Ca Ski and SiHa cells were transfected with either the LncRNA PVT1 overexpression vector or siRNA for 60 h, and then cells were counted. Following transfection with the overexpression vector, expression of the LncRNA PVT1 was significantly increased in the three cervical cancer cells (9–12-fold changes), and the knockdown efficiency of the LncRNA PVT1 siRNA was also apparent (75–90%; Fig. 1C). The results also revealed that cell numbers were significantly (P<0.05 or P<0.01) higher in the three cervical cancer cell lines treated with the LncRNA PVT1 overexpression vector and significantly lower (P<0.01) in those treated with siRNA compared with that of the corresponding internal control cells (empty vector or si-NC; Fig. 1D). Given that LncRNA PVT1 had similar effects on the cell numbers of the three cervical cancer cell types, HeLa cells were used for subsequent further analysis in the present study.

Furthermore, the FCM results revealed that cell percentages in S phase were significantly lower (P<0.05) and in the G₁ phase were significantly higher (P<0.05; Fig. 2A), and the apoptosis rate was significantly higher (P<0.05; Fig. 2B) in HeLa cells with the interference vector compared with that of untreated HeLa cells. CCND1 mRNA and Bcl-2 protein expression was decreased in HeLa cells treated with siRNA of LncRNA PVT1 compared with untreated HeLa cells (P<0.01). However, CCND1 mRNA and Bcl-2 protein expression was increased in HeLa cells with LncRNA PVT1 overexpression compared with those of untreated HeLa cells (Fig. 2C and D).

As the NF-κB pathway has a role in cell apoptosis and proliferation, whether LncRNA PVT1 could activate the NF-κB pathway was investigated. The results revealed that both NF-κB expression and activity levels were lower in HeLa cells with the siRNA targeting LncRNA PVT1, and higher in HeLa cells with the overexpression vector compared with that of untreated HeLa cells. Similarly, Iκ-Bα levels changed in a manner opposite to that of NF-κB activity in HeLa cells given the different treatments. Furthermore, HeLa cells were co-treated with 5 µg/ml LPS (as an activator of NF-κB) following transfection with siRNA LncRNA PVT1. After 8 h of co-treatment, NF-κB activity recovered in the untreated HeLa cell levels, but Iκ-Bα levels decreased again, demonstrating an opposite effect to that NF-κB (Fig. 3A), which was accompanied by corresponding changes in CCND1 and Bcl-2 expression (Fig. 3B). Furthermore, PDTC was used as a specific inhibitor of NF-κB to investigate the specific mediating action of NF-κB in the present study. When HeLa cells were treated with PDTC for 8 h, the CCND1 and Bcl-2 expression levels exhibited opposite effects to those observed following LPS treatment (Fig. 3C). In addition, the cell number of HeLa cells treated with both siRNA and LPS was significantly increased (P<0.05) compared with that of the HeLa cells treated with siRNA targeting LncRNA PVT1 only (Fig. 3D). However, the cell number of HeLa cells was significantly decreased by treatment with PDTC (P<0.05) compared with that in HeLa cells without PDTC, and a significant decrease (P<0.05) was also observed in HeLa cells treated with both PDTC and transfected with the LncRNA PVT1 overexpression vector compared with that in HeLa cells transfected with the overexpression of LncRNA PVT1 only (Fig. 3D).

According to the target regulation of miR-16 to NF-κB, miR-16 expression levels were assessed in the present study. Firstly, compared with H8 cells, miR-16 expression levels were significantly lower in the three cervical cancer cell lines (Fig. 4A). Subsequently, miR-16 expression levels were significantly reduced (P<0.01) in HeLa cells treated with the LncRNA PVT1 overexpression vector compared with HeLa cells transfected with the vector control (Fig. 4B). On the contrary, miR-16 expression levels were significantly upregulated (P<0.01) in HeLa cells transfected with LncRNA PVT1 siRNA compared with the control siRNA (Fig. 4B). Subsequently, when the HeLa cells were transfected with miR-16 mimics and an miR-16 inhibitor (Fig. 4C), NF-κB expression levels were reduced by miR-16 mimics and increased by miR-16 inhibitor (Fig. 4D). Considering the target regulation of miR-16 to NF-κB as previous reported (41), additional experiments were performed in order to investigate the regulatory association between miR-16 and NF-κB in the present study. The results of the dual-luciferase reporter assay revealed that luciferase activity significantly increased in 293T cells (P<0.01) following co-transfection with the miR-16 mimics and the pEGFP-N3-3′UTR vector of the NF-κB gene (Fig. 4E), providing direct evidence that miR-16 targets NF-κB mRNA.