The SKOV3 and CAOV3 human ovarian cancer cell lines were obtained from Qilu Medical College of Shandong University. The cells were maintained in DMEM (Hyclone; Cytiva) supplemented with 10% fetal bovine serum (Hyclone; Cytiva), 100 U/ml penicillin and 100 mg/ml streptomycin (Biosharp Life Sciences) in a humidified incubator at 37°C with 5% CO₂. Recombinant human CTGF (rhCTGF; PeproTech, Inc.; 500 mg/l) was added to the culture medium to examine its role in the cell phenotype.

For transfection, SKOV-3 and CAOV3 cells were seeded in 24-well plates at a density of 2×10⁵ cells/well. Then, 50 nM small interfering RNA (siRNA) targeting PVT1 (si-PVT1) or negative control siRNA (si-NC) was respectively transfected into cells at ambient temperature using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Cells were subjected to further experiments 48 h after transfection. The sequence of the PVT1-specific siRNA was 5′-GGCACCTTCCAGTGGATTT-3′. The si-NC was scrambled siRNA with the following sequences: Sense, UGCUGACUCCAAAGCUCUGdTdT and anti-sense, CAGAGCUUUGGAGUCAGCAdTdT. The si-PVT1 and si-NC were purchased from Guangzhou Ruibo Biotechnology Co., Ltd.

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). RT was performed to generate cDNA using a ReverTra Ace™ qPCR RT kit (Toyobo Life Science) at 42°C for 1 h on a PCR machine. The relative expression level of each gene was measured using Thunderbird SYBR qPCR Mix (Toyobo Life Science) on a 7500 Real-Time PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following thermocycling conditions were applied: 40 cycles of denaturation at 95°C for 15 sec, primer annealing at 58°C for 20 sec and extension at 72°C for 20 sec. The relative expression levels of the target genes were normalized to GAPDH using the 2^−∆∆Cq method (12). The primer sequences used are shown in Table I.

The transfected cells (5×10³ cells/well) were seeded in 96-well plates. The cells were cultured for 0, 24, 48, 72, 96 and 120 h, respectively. After culture, 10 µl CCK-8 solution (Dojindo Laboratories, Inc.) was added to each well and the cells were further incubated for 1 h. The absorbance at 450 nm was measured using a microplate reader (Thermo Fisher Scientific, Inc.).

The transfected cells (5×10² cells/well) were seeded in 6-well plates and co-cultured with or without rhCTGF for 14 days. The cell medium was refreshed every 3 days. On day 14, the cells were fixed with 4% paraformaldehyde at room temperature for 10 min and then stained with 0.5% crystal violet (Beyotime Institute of Biotechnology) for 20 min at ambient temperature. Subsequently, the number of colonies (with >50 cells considered to be a colony) was counted manually under a Leica AM6000 microscope (Leica Microsystems GmbH).

Transfected SKOV-3 and CAOV3 cells with or without rhCTGF were seeded into 6-well plates at a density of 5.0×10⁵ cells/well. Cells were serum-starved for 18 h. When the degree of confluence reached ~90%, a scratch wound was created in the cell layer using a sterile 200-µl pipette tip in the central region of each well. The wounded cells were incubated at 37°C for 48 h. Cell images were then captured using an inverted light microscope (Leica AM6000). The distance that the cells migrated was analyzed using ImageJ software (version 1.8.0) (National Institutes of Health). The migration rate was calculated as ratio of the wound distance at 48 h to the wound distance at 0 h.

The migration and invasion ability of the ovarian cancer cells was detected using a 24-well Transwell chamber (Costar; Corning, Inc.) with an 8.0-µm pore size. The transfected cells were suspended in serum-free DMEM and inoculated in the upper chamber at a density of 5.0×10⁵ cells/well. Matrigel-coated chambers (BD Biosciences) were used for the invasion assay, while chambers without Matrigel coating were used for the migration assay. The chamber coating was performed at 37°C for 30 min. Medium containing 10% FBS was added to the lower chamber with or without rhCTGF. After 48 h of incubation at 37°C, the migratory or invading cells on the membrane were fixed with methanol and stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) at ambient temperature for 20 min. The number of migrating and invading cells was counted in 5 random fields using a Leica AM6000 microscope.

The protein levels of CTGF, E-cadherin and vimentin were examined by western blotting. Total protein was extracted ovarian cancer cells using RIPA lysis buffer containing protease inhibitor cocktail (Thermo Fisher Scientific, Inc.). Cells suspended in the RIPA buffer were lysed on ice for 10 min and then centrifuged at 13,200 × g for 10 min. The supernatant containing total protein lysate was quantified using a BCA Protein assay kit (Beyotime Institute of Biotechnology,). Then, 10 µg protein sample per lane was loaded for separation on 12% gels by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins on the SDS-PAGE gel were transferred onto polyvinylidene fluoride membranes (Bio-Rad Laboratories, Inc.). After blocking with 5% skimmed milk for 1 h at ambient temperature, the membrane was incubated with the following primary antibodies at 4°C overnight: Anti-CTGF (ab5097; dilution 1:1,000; Abcam,), E-cadherin (ab231303; dilution 1:1,500; Abcam), vimentin (ab92547; dilution 1:1,000; Abcam) and anti-GAPDH (BM1985; dilution 1:2,500; Boster Biological Technology) The membranes were washed three times with TBS with 2.5% Tween 20 (TBST) buffer and then incubated with HRP-conjugated secondary antibody (#7074; dilution 1:3,000; Cell Signaling Technology, Inc.) at room temperature for 1 h. After further washes with TBST buffer, the protein signals were developed using Super ECL Plus Detection Reagent (Tanon Science and Technology Co., Ltd.) and images captured using a gel imager system (Bio-Rad Laboratories, Inc.). The western blot experiment was performed once for each condition.

SPSS 25.0 (IBM Corp.), ImageJ and GraphPad Prism 8 (GraphPad Software, Inc.) were used for data analysis. Data are presented as the mean ± standard deviation of three independent experiments. Unpaired Student's t-test was used for comparisons between two groups. Comparisons among multiple groups were analyzed using one-way analysis of variance (ANOVA) with Tukey's post hoc test for subsequent pairwise comparisons. Comparisons of data at multiple time points were examined using two-way ANOVA, with Bonferroni's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To investigate the biological roles of lncRNA PVT1 in the phenotype of ovarian cancer cells, si-PVT1 and si-NC were transfected into SKOV3 and CAOV3 cells. The transfection of si-PVT1 significantly reduced the expression of PVT1 in SKOV3 and CAOV3 cells compared with that in cells transfected with si-NC (Fig. 1A). Comparison of the transfected cells in CCK-8, colony formation, wound healing and Transwell assays revealed that the proliferation, migration and invasion abilities of both cell lines were significantly impaired following PVT1 silencing (Fig. 1B-E). These results suggest that lncRNA PVT1 contributes to the malignant phenotype of ovarian cancer cells.

To investigate whether PVT1 modulate the EMT process, the level of CTGF and EMT-associated genes, namely E-cadherin and vimentin, were examined using RT-qPCR and western blotting. In SKOV3 and CAOV3 cells, PVT1 silencing suppressed the expression of CTGF and vimentin but increased the expression of E-cadherin at the mRNA and protein levels (Fig. 2). These results indicate that the downregulation of PVT1 inhibits the expression of CTGF and suppresses the EMT process.

As CTGF expression was found to be decreased by PVT1 knockdown, whether CTGF is a downstream mediator of PVT1 function was investigated via the addition of rhCTGF to the cell culture. Consistent with the previous results, the proliferation, migration and invasion abilities of SKOV3 and CAOV3 cells were repressed by PVT1 siRNA. However, the inhibitory effect was partially attenuated by treatment with rhCTGF (Fig. 3). In addition, the rhCTGF treatment also attenuated the reduction in the expression levels of CTGF and vimentin induced by PVT1 knockdown, and reduced the PVT1 knockdown-induced increase in the expression level of E-cadherin (Fig. 4). Together, these results suggest that CTGF mediates the biological effects of lncRNA PVT1 in ovarian cancer cells.