Functional implications of PABPC1 in the development of ovarian cancer

2.1 Data acquisition

Initially, we analyzed the expression of PABPC1 using the data set GSE54388 which was downloaded from Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) database. The RNA sequencing data sets were acquired from The Cancer Genome Atlas (TCGA, https://cancergenome.nih.gov) and normal samples were downloaded from Genotype-Tissue Expression (GTEx, https://gtexportal.org/home/) database. Different data sets including Bonome Ovarian data set and Yoshihara Ovarian data set from Oncomine (https://www.oncomine.org) database were also used to analyze the PABPC1 expression pattern. A total of 374 cases of OC in TCGA cohort were divided into high and low PABPC1 expression groups according to the median expression of PABPC1 for evaluating the correlation between PABPC1 and the overall survival rate.

2.2 Cell lines

Human OC cell lines SK-OV-3 and A2780 were obtained from the Shanghai Cell Bank, Chinese Academy of Medical Sciences (Shanghai, China), and OVCAR-3 was acquired from the American Type Culture Collection (Rockville, VA, USA). From Procell Technology (Wuhan, Hubei, China), normal OC cell line IOSE80 was purchased. All cells were incubated at 37°C under the 5% CO₂ atmosphere in Roswell Park Memorial Institute-1640 medium (Procell) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution.

2.3 RNA interference and transfection

Small interfering RNAs (siRNAs) targeting PABPC1 (si-PABPC1#1: 5′-CATCGACAATAAAGCACTAT-3′; si-PABPC1#2: 5′-CTAGCCAAATTGCTCAACTA-3′) and their control siRNA (si-con: 5′-CGAACTCACTGGTCTGACC-3′) were designed to downregulate PABPC1 expression. Plasmid pcDNA3.1-PABPC1 and empty vector pcDNA3.1 were conducted to overexpress PABPC1. All siRNA sequences and plasmid vectors were constructed by the GenePharma Corporation (Shanghai, China).

Lipofectamine™ 2000 transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA) served to carry out transfection according to the manufacturer’s protocols. Upon transfection for 24 h, the transfection efficacy was detected.

2.4 RNA extraction and real-time quantitative polymerase chain reaction (RT-qPCR)

Using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA), total RNAs from cells were extracted and then reverse transcribed by PrimeScript RT Reagent kit (TaKaRa, Tokyo, Japan). The mRNA level of PABPC1 was tested by RT-qPCR with the SYBR Premix Ex Taq (TaKaRa) on the Applied Biosystems 7500 Thermocycler (Thermo Fisher Scientific) according to the manufacturer’s protocols. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as the control. Primer sequences for RT-qPCR were:

PABPC1 forward: 5′-CAGAGAATGGCAAGTGTACGAGC-3′,

PABPC1 reverse: 5′-GCTAGGAGGATAGTATGCAGCAC-3′;

GAPDH forward: 5′-TGTGTCCGTCGTGGATCTGA-3′,

GAPDH reverse: 5′-CCTGCTTCACCACCTTCTTGA-3′.

2.5 Western blot

After transfection for 24 h, proteins were extracted from the cells using the radio immunoprecipitation assay lysis buffer (Beyotime, Nantong, China) on ice. Protein concentration was measured via the bicinchoninic acid protein assay kit (Beyotime). Equal amount of protein (20 μg) was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes, which were then blocked for an hour with 5% skimmed milk powder. Next the membranes were incubated with the primary antibodies including anti-PABPC1 (ab233280), anti-E-cadherin (ab1416), anti-N-cadherin (ab202030), anti-snail (ab229701) and anti-vimentin (ab193555) overnight at 4°C. Additionally, anti-PABPC1 and all EMT-related protein primary antibodies were purchased from Abcam Trading Company Ltd (Shanghai, China). GAPDH rabbit monoclonal antibody (AF1186; Beyotime) was regarded as an internal control. Subsequently, these membranes were washed with Tris-buffered saline Tween thrice for 5 min and were incubated with the horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The protein luminescence effect was evaluated via an enhanced chemiluminescence plus with a bioimaging system. Image J software served to scan the gray values.

2.6 Cell proliferation assay

After transfection for 24 h, cells at a density of 1,000 cells/well were routinely incubated in 96-well plates. After incubation for 0, 24, 48 and 72 h, 10 μL of cell counting kit 8 (CCK-8; Dojindo, Tabaru, Japan) reagent was supplied to each well. Next the optical density (OD) values were recorded 2 h later using a microplate reader at 450 nm wavelength.

2.7 Plate clone formation assay

To prepare cell suspension, trypsin was first used to digest the cells of logarithmic growth. Then at 400 cells/dish, the cell suspension was plated into 60 mm dishes containing 5 mL preculture medium and kept at an atmosphere of 5% CO₂ at 37°C for 2 weeks. After washing twice with phosphate-buffered saline (PBS), fixing with 4% paraformaldehyde for 30 min and staining with 0.1% crystal violet for 30 min, the number of colonies was counted.

2.8 Transwell assay

To evaluate the OC cells’ invasion and migration, 24-well Transwell chambers (Genetimes, Shanghai, China) were adopted. The invasion and migration assay procedures were similar, except that the latter did not perform gelatinization. Briefly, the transfected cells (1 × 10⁵ cells/well) were incubated in 100 μL serum-free RPMI-1640 in the upper chambers, and the lower chambers were supplemented with 500 μL RPMI-1640 plus 10% FBS. Next using the cotton swabs, the residual cells on upper chambers were wiped off when cultured for 24 h. After that, the cells that migrated or invaded to the lower chamber were washed with PBS, fixed and stained for 30 min. Upon being washed with PBS again, the migrated or invaded cells were photographed and counted under a microscope with five random selected fields.

2.9 Statistical analyses

Survival curve was plotted utilizing Kaplan–Meier method with log-rank tests for comparison. Comparison of two groups was performed utilizing Student’s t test, and one-way analysis of variance served to compare the differences among three or more groups, following a Dunnett post hoc test. All statistical analyses were conducted with GraphPad Prism version 6.0 (GraphPad Software, La Jolla, CA, USA) or SPSS version 21.0 (SPSS, Chicago, IL, USA) software. The results of the in vitro experiments were expressed as mean value ± standard deviation from triplicate experiments. A P < 0.05 was indicated as a statistically significant difference.

1. Ethical statement: Ethical approval was not required. No human and animal assays were used in this study.

3.1 PABPC1 is upregulated in OC and has a connection with poor survival

At first, the expression of PABPC1 was evaluated by bioinformatic analysis using GEO data set GSE54388. As shown in Figure 1a, a significant upregulation was observed in serious OC samples in comparison with normal healthy samples (P = 0.0003). Then we analyzed the expression of PABPC1 from TCGA which included 378 OC tissues. Since no normal tissues were observed in the OC-related RNA-Seq data set in TCGA, we downloaded 88 normal ovarian tissue samples from GTEx database as controls. It was shown that PABPC1 expression in OC samples was higher than that in the normal samples (P < 0.001; Figure 1b). Moreover, we found that PABPC1 displayed a higher expression in OC tissues based on the Bonome Ovarian data set and Yoshihara Ovarian data set from Oncomine database when compared with nontumor tissues (P < 0.01; Figure 1c and d). To validate the observations from bioinformatic analyses, we perform RT-qPCR to examine mRNA expression of PABPC1 in three OC cell lines. A significant upregulation of PABPC1 was shown in all OC cell lines compared to the normal cell line IOSE80 (P < 0.01; Figure 1e), which was in agreement with the outcomes by bioinformatic analysis.

Figure 1

Expression of PABPC1 and Kaplan–Meier curves. High expression of PABPC1 was observed in GSE54388 from GEO database (a), TCGA database and GTEx database (b), and Oncomine database (c and d). (e) mRNA expression of PABPC1 in OC cell lines (A2780, OVCAR-3, SK-OV-3) and normal cell line (IOSE80) was examined using RT-qPCR analysis. **P < 0.01 vs IOSE80 cell line. (f) Kaplan–Meier method was utilized to plot the overall survival curve based upon the TCGA database (N = 374).

The correlation between high/low PABPC1 expression and the overall survival of patients with OC was probed through Kaplan–Meier methods. The curve chart revealed that patients with high PABPC1 levels exhibited a worse overall survival compared with those with low PABPC1 level (P = 0.041; Figure 1f).

3.2 Detection gain and loss function of PABPC1 efficiency

Based on the previous RT-qPCR assay, we found that, among OC-related cell lines, mRNA expression of PABPC1 was highest in SK-OV-3 cells and lowest in A2780 cells. To make the following tests more optimized, SK-OV-3 was chose to perform the loss of function assay and A2780 was chose as the targeted cells for the next overexpression experiments.

Efficiency of overexpression and knockdown was measured on the mRNA and protein expression aspects. Upon transfection with si-PABPC1#1 and si-PABPC1#2, the results showed that the mRNA and protein levels of PABPC1 were remarkably reduced in SK-OV-3 cells in contrasts with si-con group (P < 0.01; Figure 2a and b). By the way, the sequence of si-PABPC1#1 was selected to carry out the subsequent loss-of-function assay due to its better efficiency than si-PABPC1#2 group. Furthermore, PABPC1 expressions were notably enhanced in A2780 cells after transfecting with pcDNA3.1-PABPC1 in comparison with vector group (P < 0.01; Figure 2c and d).

Figure 2

Detection of knockdown and overexpression of PABPC1 efficacy. The mRNA (a and c) and protein (b and d) levels of PABPC1 in SK-OV-3 (a and b) and A2780 (c and d) cells were tested using RT-qPCR (a and c) and Western blot (b and d) analyses. SK-OV-3 cells were transfected with si-PABPC1#1, si-PABPC1#2 and si-con. A2780 cells were transfected with pcDNA3.1-PABPC1 and pcDNA3.1 empty vector. **P < 0.01 vs si-con or vector group.

3.3 PABPC1 expression impacted OC cell proliferative ability

To examine the action of PABPC1 on OC cell proliferation, CCK-8 and plate clone formation assays were performed. According to the results of CCK-8 assay, the OD values of SK-OV-3 cells were notably reduced after downregulation of PABPC1 by comparison with control at 48 and 72 h (P < 0.01; Figure 3a). In the overexpression tests, the opposite trend appeared, which is based on the evidence that the OD values were remarkably higher in pcDNA3.1-PABPC1 group in comparison with the vector group (P < 0.01; Figure 3b).

Figure 3

Influences of knockdown and overexpression of PABPC1 on proliferation. The SK-OV-3 cells proliferative (a) and clonogenic (c) capacities were detected utilizing CCK-8 (a) and clone formation (c) assays after depletion of PABPC1. The A2780 cells proliferative (b) and clonogenic (d) capacities were detected utilizing CCK-8 (b) and clone formation (d) assays after upregulation of PABPC1. **P < 0.01 vs si-con or vector group.

A similar trend was observed in clone formation assays. As shown in Figure 3c, the visible colonies in si-PABPC1 group was less than that in si-con group in SK-OV-3 cells (P < 0.01; Figure 3c). On the contrary, upregulation of PABPC1 in A2780 cells presented a better cloning efficiency when compared with the control (P < 0.01; Figure 3d).

3.4 PABPC1 expression affected OC cell invasiveness and motility

The action of PABPC1 on the abilities of invasion and migration of OC cells was investigated using Transwell assays. Results of invasion assays revealed that the invaded SK-OV-3 cells in si-PABPC1 group (87.67 ± 17.01) showed a nearly twofold decrease compared with the control (155 ± 38.04; P < 0.01; Figure 4a and b). Similarly, the migrated SK-OV-3 cells in si-PABPC1 group (161.67 ± 30.53) were markedly decreased compared with the si-con group (298 ± 25.63; P < 0.01; Figure 4a and b). On the other hand, after PABPC1overexpression (156.67 ± 24.03), the invaded number of A2780 cells was more than twice as many as that of the vector group (64.33 ± 18.56; P < 0.01; Figure 4c and d). Moreover, the migrated A2780 cells in pcDNA3.1-PABPC1 group (301.33 ± 37.82) were also significantly increased when compared with the vector group (165.67 ± 29.09; P < 0.01; Figure 4c and d).

Figure 4

Impacts of downregulation and upregulation of PABPC1 on invasion and migration. The impacts of PABPC1 knockdown on SK-OV-3 cells (a and b) and the impacts of PABPC1 overexpression on A2780 cells (c and d) invasion and migration were measured by Transwell assays.**P < 0.01 vs si-con or vector group.

3.5 PABPC1 expression was associated with EMT process in OC cells

Our previous functional experiments indicated that PABPC1 could promote OC cells’ movement and invasion. Considering that the EMT is frequently used to explain how cancer cells acquire aggressiveness [21], we investigated the action of PABPC1 on the EMT-related markers using Western blot analysis. The outcomes illustrated that the protein expression of epithelial marker E-cadherin was notably elevated after depletion of PABPC1 in SK-OV-3 cells compared with the control (P < 0.01; Figure 5a), whereas the levels of mesenchymal markers N-cadherin and vimentin and transcription factor snail were all significantly downregulated (P < 0.01; Figure 5a). For overexpression of PABPC1, the levels of these markers in A2780 showed the reverse results, as with restrained E-cadherin levels and elevated the levels of N-cadherin, vimentin and snail (P < 0.01; Figure 5b).

Figure 5

PABPC1 regulated the EMT process. (a and b) Western blot was employed to evaluate the effects of PABPC1 knockdown (a) and PABPC1 overexpression (b) on the protein expression of EMT-related markers in SK-OV-3 (a) and A2780 (b) cells. **P < 0.01 vs si-con or vector group.