Human recombinant TNF-α was obtained from T&L Biological Technology. Transcription factor E2F (E2F), p53, NF-κB, eukaryotic initiation factor 2 α kinase 1 (EIK1), transforming growth factor (TGF)-β), JNK, myc proto-oncogene protein (c-MYC), PI3K/AKT, Wnt, protein giant-lens (Gil), Notch, STAT3 and ETS transcription factor (Elk1) luciferase reporter plasmids and the pHAGE puro vector were gifted by the School of life sciences, Wuhan University, Wuhan, China. These luciferase reporter constructs contain the DNA-binding motifs of transcription factor in these signaling pathways.

PTPRA mRNA data in patients with the breast cancer gene (BRCA) were analyzed using the Gene Expression Profiling Interactive Analysis database (gepia.cancer-pku.cn) with Kaplan Meier analysis and log-rank tests as previously described (15). The expression data of 1,085 tumors and 291 adjacent normal tissues were obtained from The Cancer Genome Atlas (TCGA; www.tcga.org). Group cut-off was at 50% and based on this, patients were divided into the low PTPRA or the high PTPRA group. The overall survival was calculated in low and high PTPRA groups for 250 months according to previous reports (21–23) using the GEPIA database to display the relevance of PTPRA mRNA in patients with breast cancer.

MCF-7 and HEK293T cells were purchased from the American Type Culture Collection. MCF-7 cells were cultured in DMEM/nutrient mixture F12 (1:1) (Gibco; Thermo Fisher Scientific, Inc.) medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. HEK293T cells were cultured in DMEM medium supplemented with 10% FBS. All cells were cultured in a 5% CO₂ chamber at 37°C.

Total RNA was extracted out using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) from MCF-7 cells and reverse transcribed into cDNA using a high capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.) at 4°C according to the manufacturer's protocols. cDNA encoding PTPRA was amplified with PCR with the following primers: Forward: 5′-GATCCGCCACCAUGGATGGATTCCTGGTTCATTCTTGTTC-3′ and reverse: 5′-TCGAGCTTGAAGTTGGCATAATCTG-3′. The PTPRA fragment was gel-purified via BamH I and EcoRI and rejoined to BamH I-EcoRI digested pHAGE puro plasmid. A total of 10 µg Flag-PTPRA expression plasmid were introduced to MCF-7 cells for exogenous overexpression using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. As the control, the empty pHAGE puro plasmid was introduced to MCF-7.

PTPRA knockout plasmids were constructed using the clustered regularly interspaced short palindromic repeat (CRISPR) knockout method (24,25). Briefly, the gRNA targeting sequence was obtained and inserted into CRISPR/Cas9 Plasmid using the Precision X Multiplex gRNA kit (SBI http://systembio.com/products/crispr-cas9-systems/) according to the manufacturer's protocol. The non-specific binding targets of the CRISPR/Cas9 plasmid served as the negative control. In total, 2.5×10³ HEK293T cells per well at 80–90% confluence were transfected with 400 ng CRISPR/Cas9-PTPRA plasmid or the control plasmid for 2 days. Next, MCF-7 cells were transfected with 10 µl lentiviral particles for 72 h at 37°C. The infected MCF-7 cells were screened in the presence of 1 µg/ml puromycin for 7 days. The puromycin-resistant cells were subjected to further confirmation by agarose gel electrophoresis and western blotting. The cells carrying the non-specific binding targets CRISPR/Cas9 plasmid were used as a control.

Collected cells were lysed with RIPA buffer (BioVision, Inc.) and the supernatant was extracted for SDS-PAGE analysis. After quantification using the Bradford assay, protein lysates (10 µg/lane) were separated by 8% SDS-PAGE, transferred to PVDF membranes and blocked with TBS containing 5% non-fat milk at room temperature for 1 h. The membranes were probed with mouse monoclonal anti-Flag antibody (1:3,000; cat. no. 81069; ProteinTech Group, Inc.) or PTPRA antibody (1:2,000; cat. no. 13079–1-AP; ProteinTech Group, Inc.), β-actin antibody (1:2,000; cat. no. Ag27042; ProteinTech Group, Inc.) for 1–2 h at room temperature. The blots were then incubated and reprobed with horseradish peroxidase-conjugated secondary antibody (1:2,000; cat. no. SA00001-1; ProteinTech Group, Inc.) for 1 h at room temperature. Band signals were visualized using an ECL system (GE Healthcare). The following antibodies were diluted in TBS and used for immunoblotting: Anti-FLAG, anti-β-actin and anti-PTPRA.

Overexpressed-PTPRA MCF-7 cells or two PTPRA-knockout independent clones (PTPRA^−/−1# and PTPRA^−/−2#) and their corresponding control MCF-7 cells were harvested and prepared in single-cell suspension (1×10⁴ cells/ml) for the subsequent cell assays.

A colony formation experiment was conducted to estimate the clonogenic activity of breast cancer cells. Prepared cells were seeded in 6-well plates and cultured in an incubator at 37°C. Following 8-day culture, the colonies were fixed using 100% methanol for 15 min and stained using 0.5% crystal violet for 20 min at room temperature before quantification under an inverted light microscope (ECLIPSE TE2000-S; Nikon) at 200× magnification. The indicated colony formation units were recorded in 5 random fields for every replicate and plotted.

In the Cell Counting Kit (CCK)-8 assay, the aforementioned cells were cultured in 96-well plates (1×10³ cells/well). Following incubation for 1, 3, 5 and 7 days, diluted CCK-8 solution (Dojindo Molecular Technologies, Inc.) was supplemented into each well according to the manufacturer's manual. After incubation for another 1–2 h at 37°C, cell proliferation was evaluated spectrophotometrically at a wavelength of 450 nm with an Automated Enzyme Immunoassay Analyzer (Shanghai Dongcao Biotechnology Co., Ltd, Tosoh Corporation; http://www.biomart.cn/infosupply/57204830.htm).

For Transwell migration assay, Transwell inserts (Corning Inc.) with porous polycarbonate membranes were firstly placed in 24-well plates. The lower compartment was filled with 2.6 ml DMEM containing 40% FBS. MCF-7 cells (1×10⁴) were added to the upper compartment and cultured in Transwell plates at 37°C for 2 days. Cell debris that did not migrate through the membrane were removed with a cotton swab. The migratory cells were fixed with 5% glutaraldehyde for 10 min and stained 1% crystal violet in 2% ethanol for 20 min before images were captured and quantification under an inverted light microscope (ECLIPSE TE2000-S; Nikon). All comparison experiments were performed in triplicate.

In order to screen the target signaling pathways of PTPRA, a series of luciferase reporter gene assays were performed to determine the effect of PTPRA on the transcriptional activity of several documented tumor signaling markers: E2F, p53, NF-κB, EIK1, TGF-β, JnK, c-MYC, Wnt, Gil, Notch, STAT3 and Elk1.

A total of 1×10⁴ HEK293T cells were cultured in 24-well plates overnight at 37°C and transfected with the aid of Lipofectamine^® 2000 (Thermo Fisher Scientific, Inc.). Each transfection contained the indicated luciferase reporter vector (200 ng/well) and prl-tk (10 ng/well) empty control plasmid or PTPRA expression plasmid or control vector. Following 36 h incubation at 37°C, the released cells were treated with trypsin and luciferase activity was measured using a Dual-Glo Luciferase Assay kit (Promega Corporation), according to the manufacturer's protocol.

Furthermore, HEK293T cells were transfected with pNFKB-luc and PTPRA plasmids at various diluted concentrations (100, 200 and 400 ng/well) to further confirm the effect of PTPRA on NF-κB transcriptional activity. Luciferase activity was normalized using the Renilla luciferase activity.

Total RNA from cells was extracted from lysed cells using TRIzol^® (Thermo Fisher Scientific, Inc.). Reverse transcription was performed using oligo dT primers using RT kit (Invitrogen) according to the manufacturer's protocol. mRNA of IKBα (one of classic downstream molecules of the NF-κB signaling pathway (26) in and two PTPRA deficient MCF-7 cells(PTPRA^−/−1# and PTPRA-^/−2#) cells and their corresponding control MCF-7 cells (PTPRA^+/+) was assessed upon TNF-α stimulation or not. The relative expression was quantified by the 2^−ΔΔCq method (27).

Statistical analyses were performed using GraphPad Prism software (version 6.0; GraphPad Software). Log-rank test was carried out to calculate significance of PTPRA in predicting overall survival of breast cancer patients using GEPIA according to the creator of this website (28).

For continuous variables, measured data are presented as the mean ± SEM. Unpaired two-tailed Student's t-test was used to compare differences between two groups. One-way ANOVA was used to evaluate the statistical significance among multiple groups followed by Tukey's post hoc corrective test. P<0.05 was considered to indicate a statistically significant difference. All experiments except the analysis from GEPIA were performed at least three times.

The BRCA database from GEPIA was analyzed and used to compare the expression of PTPRA in breast cancer and normal tissues in order to determine the prognostic value of PTPRA in breast cancer. PTPRA expression was significantly increased in breast cancer tissues compared with normal tissues (Fig. 1A). As presented in Fig. 1B, patients with high PTPRA demonstrated worse clinical outcomes compared with patients with PTPRA, while there was no significant difference between groups (P=0.45). These results indicated that PTPRA expression level may serve a putative role in breast cancer malignancy.

A vector containing Flag-PTPRA was constructed and transfected into MCF-7 cells using Lipofectamine 2000 to verify the specific biological function of PTPRA. PTPRA overexpression was confirmed using anti-Flag antibodies via western blotting (Fig. 2A). Furthermore, PTPRA overexpression significantly promoted the colony formation ability of MCF cells (Fig. 2B). A growth curve analysis demonstrated that PTPRA overexpression dramatically enhanced proliferation compared with the control cells (Fig. 2C). Additionally, the Transwell assay demonstrated that the number of migratory cells in the overexpression group was significantly increased compared with the control group (Fig. 2D). These results indicated that PTPRA increased the tumorigenic properties of MCF7 cells in vivo.

PTPRA was further depleted in MCF-7 cells using CRISPR to confirm the effect of PTPRA on cell behaviors. Western blotting revealed that PTPRA was almost completely silenced (Fig. 3A). Furthermore, the colony formation ability and proliferation of PTPRA-deficient MCF-7 cells were significantly decreased compared with that of control MCF-7 cells (PTPRA^+/+) (Fig. 3B and C, respectively). Consistently, MCF7 cells deficient in PTPRA had fewer migratory cells than the control cells (PTPRA^+/+) (Fig. 3D). These results suggested that PTPRA deficiency decreased cell colony formation ability and inhibited tumor cell migration ability.

Oncogenesis, the development and prognosis of tumors, involves complicated pathway networks that are implicated in numerous signal pathways, such as microtubule-associated protein kinase, NF-κB and signal transducer and activator of transcription factor 3 (29). The results of luciferase reporter gene assays demonstrated that NF-κB transcriptional activity was markedly increased compared with controls (Fig. 4A). The luciferase reporter gene assay did not demonstrate any obvious alterations in the expression of the other signaling molecules. NF-κB transcriptional activity was also demonstrated to be increased in a dose-dependent manner (Fig. 4B). These results indicated that PTPRA overexpression in HEK293T cells stimulated NF-κB transcriptional activity.

RT-qPCR was utilized to quantify one of the classic downstream molecules of the NF-κB signaling pathway, IKBα in MCF-7 cells. No transcription of the IKBα gene was detected in PTPRA^−/− and PTPRA^+/+ MCF-7 cells without TNF-α treatment (Fig. 4C). However, the TNF-α stimulus changed these outcomes. IKBα gene transcription in PTPRA^+/+ MCF-7 cells was increased compared with that in PTPRA-deficient MCF-7 cells. These outcomes indicated that TNF-α-mediated PTPRA stimulated the activation of NF-κB and promoted the tumor phenotype of breast cancer cells.