The present study was approved in January 2017 by the Ethics Committee of the Tongren Hospital, Shanghai Jiao Tong University School of Medicine. The collection of ovarian cancer specimens was performed in conformity to ethical standards. All participants provided written informed consent. The tissues were obtained during surgery and were fixed with 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd.) for 24 h at room temperature and embedded in paraffin. Specimens were obtained from 78 patients (age, 29–77 years, including 16 benign, 16 borderline and 46 malignant patients) with epithelial ovarian cancer who had not previously undergone treatment; the benign group consisted of patients with a benign ovarian tumor. No distant metastasis was detected in the selected patients prior to surgery. Detailed pathological data, including histological type, tumor size, tumor stage and lymph node metastasis, were obtained and summarized. The tumor stage was defined using the 8th edition of the Union for International Cancer Control-tumor node metastasis classification system (17).

The human ovarian cancer cell lines 293T, HO8910, SKOV3, HEY and TOV-21G were purchased from American Type Culture Collection and were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.).

293T cells (60% confluent) in a 10 cm dish were co-transfected with 5 µg lentiviral constructs (pLenti 7.3; Invitrogen; Thermo Fisher Scientific, Inc.), 5 µg plasmid Δ8.9 (Invitrogen; Thermo Fisher Scientific, Inc.) and 3 µg plasmid vesicular stomatitis virus G (Invitrogen; Thermo Fisher Scientific, Inc.) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were incubated at 37°C and the medium was replaced after 12 h. Virus-containing medium was collected 48 h post-transfection and supplemented with 8 µg/ml polybrene to infect target cells in 6-well dishes (60% confluent) at 37°C. Infected cells were selected with 3 µg/ml puromycin at 37°C for ≥1 week post-infection. The lentiviral shRNA vectors targeting MPZL1 and scrambled control shRNA were purchased from Open Biosystems; Dharmacon Inc. For MPZL1 knockdown, the shRNA sequences were: MPZL1-shRNA-1, 5′-TGACATCACAGATATAGGT-3′; MPZL1-shRNA-2, 5′-TCAAGTGGCATAGCCAATG-3′; and shRNA-negative control (NC), 5′-ACCTCCACCCTCACTCTGCCAT-3′. For MPZL1 overexpression, the coding sequence of MPZL1 was inserted into the lentiviral vector pLenti 7.3.

For Src knockdown, Src-small interfering RNA (siRNA) was used at a final concentration of 25 nM and was transfected into cells (60% confluent) using Lipofectamine^® RNAiMAX reagent (Invitrogen; Thermo Fisher Scientific, Inc.) for 72 h at 37°C, according to the manufacturer's protocol. For Src knockdown, the siRNA sequences (Shanghai GenePharma Co., Ltd.) were: Src-siRNA, 5′-CAGGCUGAGGAGUGGUAUUTT-3′; and siRNA-NC, 5′-TTCTCCGAACGTGTCACGT-3′.

Cells were lysed in RIPA buffer [Tris (pH 7.4), 50 mM; NaCl, 150 mM; 1% NP-40; 0.1% SDS; EDTA, 2 µM] containing proteinase inhibitors (Roche Molecular Diagnostics) and phosphatase inhibitors (Roche Molecular Diagnostics), and the protein concentration was determined using the bicinchoninic acid assay. The cell lysates (20 µg total protein) were subjected to 8–10% SDS-PAGE and immunoblotting. Subsequently, proteins were transferred to nitrocellulose membranes, which were blocked for 1 h with 5% nonfat milk at room temperature, and were then incubated with primary antibodies (1:1,000) at 4°C overnight. The membranes were then incubated for 1 h with horseradish peroxidase-linked anti-rabbit IgG antibody (1:1,000; cat. no. 7074; Cell Signaling Technology, Inc) at room temperature. Blots were visualized with ECL western blotting reagents (Thermo Fisher Scientific, Inc.) using ChemiDoc XRS+ (Bio-Rad Laboratories, Inc.). Semi-quantification was conducted using ImageLab 2.0 software Bio-Rad Laboratories, Inc.). Antibodies against the following proteins were used: p130 (cat. no. 13846), phosphorylated (p)-p130 (cat. no. 4015), cortactin (cat. no. 3502), p-cortactin (cat. no. 4569), Src (cat. no. 2108), p-Src (cat. no. 12432), MPZL1 (cat. no. 9893) and GAPDH (cat. no. 5174) (all Cell Signaling Technology, Inc). Notably, since MPZL1 has three alternatively spliced isoforms, at least three bands can be seen in MPZL1 blots, and all bands were measured when semi-quantifying the blots.

Cell proliferation was assessed using the Cell Counting Κit-8 (CCK-8; Dojindo Molecular Technologies, Inc.). Cells (5,000 cells/well) were seeded in triplicate in 96-well plates. After 24, 48, 72, 96 or 120 h at 37°C, CCK-8 reagent (1/20, volume/volume) was added to the cells for 2 h at 37°C. The absorbance of each well was measured at 450 nm, according to the manufacturer's protocol.

Transwell chambers with 8 µm pore membranes (Corning, Inc.) were placed in 24-well culture plates and were incubated with serum-free RPMI-1640 medium at 37°C for 1 h. A 200 µl suspension of 0.5–1×10⁵ cells was seeded into the upper compartment of the Transwell chambers in FBS-free medium, whereas 600–800 µl RPMI-1640 medium supplemented with 10% FBS was added into the bottom wells; cells were incubated at 37°C for 12–24 h. The migrating cells that were attached to the lower membranes of the Transwell chambers were stained with crystal violet (0.1%) at room temperature for 2 h and images were captured at ×200 magnification under a light microscope. Images of three random fields from three replicate wells were obtained, and the migrated cells were counted. The cell invasion assay was the same as the migration assay; however, the chambers were coated with Matrigel.

Cells were seeded in triplicate in 6-well plates. After 12 days, the cells were washed with PBS, fixed with methanol for 1 h and stained with 0.1% crystal violet for 1 h at room temperature. Subsequently, images of the colonies were captured and counted.

Ovarian cancer specimens were collected from the 78 patients during surgery at Tongren Hospital, Shanghai Jiao Tong University. The tissue specimens were fixed in 4% paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd.) for 24 h at room temperature and embedded in paraffin, before being cut into 5 µm sections. The tissue sections were deparaffinized, treated with 3% H₂O₂ for 10 min at room temperature, autoclaved in 10 mM citric sodium (pH 6.0) for 30 min to unmask antigens and rinsed in PBS. Subsequently, sections were incubated with primary antibodies against MPZL1 (1:200; cat. no. GTX46451; GeneTex, Inc.) at 4°C overnight, followed by incubation with a biotinylated secondary antibody (1:1,000; cat. no. ab6844; Abcam) for 1 h at room temperature. Signal amplification and detection was performed using the DAB system (Dako) according to the manufacturer's instructions.

All cases were analyzed independently with the help of two expert pathologists and were assigned a score according to the following criteria: Strongly positive, 3+; positive, 2+; weakly positive, 1+; and negative, 0.

Statistical analysis was performed using GraphPad Prism 6 software (GraphPad Software, Inc.). Genomic analysis of MPZL1 in ovarian cancer (489 high-grade serous ovarian cancer specimens were surgically resected prior to systemic treatment; all patients received a platinum agent and 94% received a taxane) was performed with The Cancer Genome Atlas (TCGA) copy number portal (www.broadinstitute.org/tcga). In all experiments, comparisons between two groups were conducted using two-sided Student's t-test, and one-way analysis of variance followed by Tukey's multiple comparisons test was used to test for differences among more groups. Fisher's exact test was used to determine differences between stages, as presented in Table I. P<0.05 was considered to indicate a statistically significant difference.

Copy number analysis of TCGA ovarian serous adenocarcinoma samples in the cBioportal database revealed that MPZL1, located at chromosome 1q24 (magnified section in Fig. 1A), was genomically amplified in a considerable proportion of cases (Fig. 1A). Notably, the expression levels of MPZL1 were associated with its amplification status (Fig. 1B), thus indicating that the high expression of MPZL1 in tumor tissues may be regulated by copy number amplification of the MPZL1 gene. To further determine the association of MPZL1 expression with the malignant features of ovarian cancer, MPZL1 protein expression was assessed by immunohistochemistry among benign, borderline and malignant epithelial ovarian cancer patient tissues (Fig. 1C; Table I). The results revealed that MPZL1 expression was almost undetectable in patients with benign ovarian cancer, whereas it was expressed at significantly higher levels in malignant cancer patient tissues compared with in benign/borderline cancer patient tissues (Fig. 1D). In addition, the expression profile of the MPZL1 protein was determined using The Human Protein Atlas (18). MPZL1 was ubiquitously expressed in different human tissues as well as various cancer types. In normal ovarian tissues, MPZL1 was lowly expressed, whereas in some ovarian cancer tissues, MPZL1 was overexpressed (data not shown). Taken together, these data indicated that MPZL1 was overexpressed in a subset of patients with ovarian cancer, suggesting that MPZL1 may serve a pivotal role in ovarian cancer.

To study the biological function of MPZL1, the protein expression levels of MPZL1 were detected in four ovarian cancer cell lines (Fig. 2A). Subsequently, the MPZL1 gene was overexpressed via lentiviral infection in SKOV3 and TOV-21G cells (Fig. 2B); these two cell lines were selected as they exhibited relatively lower endogenous MPZL1 expression. Subsequently, the effects of MPZL1 overexpression on the proliferation of these ovarian cancer cells were determined using the CCK-8 assay. Notably, the results demonstrated that exogenous overexpression of the MPZL1 gene promoted ovarian cancer cell proliferation (Fig. 2C). Similarly, the results of the colony formation assay demonstrated that the number of colonies was significantly increased in MPZL1-overexpressed SKOV3 and TOV-21G cells (Fig. 2D). Furthermore, the effects of MPZL1 overexpression were examined on the migratory and invasive abilities of ovarian cancer cells by Transwell assays; ectopic expression of MPZL1 significantly enhanced the in vitro migration and invasion of ovarian cancer cells (Fig. 2E). These results indicated that MPZL1 may serve an important role in promoting cell migration and metastasis of ovarian cancer.

To further verify the role of MPZL1 in ovarian cancer cell migration, HO8910 and HEY cells were selected as cellular models for loss-of-function studies. Firstly, the MPZL1 gene was stably knocked down in HO8910 and HEY cells using a lentiviral shRNA specifically targeting MPZL1 (Fig. 3A). Consequently, cell proliferation was significantly inhibited under normal growth conditions (Fig. 3B). Consistently, the colony formation assay revealed that the number of colonies was evidently decreased following knockdown of MPZL1 in HO8910 and HEY cells (Fig. 3C). Furthermore, Transwell assays demonstrated that the in vitro migration and invasion of ovarian cancer cells were inhibited by MPZL1 depletion (Fig. 3D). These findings indicated that MPZL1 knockdown attenuated proliferation, migration and invasion of ovarian cancer cells.

Recently, many studies have reported that pro-metastatic proteins, including p130, Src and cortactin, are important for cell migration and tumor metastasis (19–21). Furthermore, p130 and cortactin were originally identified as substrate proteins of the Src family kinases, and a previous report demonstrated that the MPZL1/Src/cortactin signaling cascade functions in the process of hepatocellular carcinoma cell migration (22). However, to the best of our knowledge, the potential role of MPZL1 in the phosphorylation of pro-metastatic proteins has not been determined in ovarian cancer. To investigate whether the MPZL1/Src/cortactin signaling cascade exists in ovarian cancer, the expression levels of pro-metastatic proteins were detected by western blotting. The results indicated that stable overexpression of MPZL1 led to increased phosphorylation of p130, Src and cortactin in SKOV3 cells (Fig. 4A). Furthermore, targeted knockdown of the MPZL1 gene by shRNA reduced phosphorylation of these three proteins in HO8910 cells (Fig. 4B). These findings indicated that MPZL1 overexpression reprogrammed the pro-metastatic signaling network in ovarian cancer.

According to previous studies, Src tyrosine kinases serve critical roles in several cellular signal transduction pathways that regulate cell proliferation, adhesion, migration and invasion (22–25). Additionally, as substrate proteins of Src kinase, p-p130 and p-cortactin participate in cell migration (26). Furthermore, amplification of MPZL1 promotes tumor cell migration via Src-mediated cortactin phosphorylation in hepatocellular carcinoma cells (27). To ascertain whether Src kinase activity is essential for MPZL1-induced phosphorylation of p130 and cortactin in ovarian cancer cells, MPZL1 was overexpressed in SKOV3 cells, and the Src gene was then knocked down by siRNA. Notably, stable overexpression of MPZL1 significantly increased phosphorylation of p130, cortactin and Src in SKOV3-MPZL1 cells, and enhanced the migratory and invasive abilities of SKOV3-MPZL1 cells (Fig. 5A-C). Conversely, targeted knockdown of Src via siRNA resulted in reduced phosphorylation of p130 and cortactin, and suppressed the migration and invasion of SKOV3-MPZL1 cells (Fig. 5A-C). Taken together, these findings indicated that Src kinase activity may be essential for MPZL1-mediated migration and invasion of ovarian cancer cells.