2.1 Differential expression analysis of DPP4

Based on TCGA and GTEx database, GEPIA2 (http://gepia2.cancer-pku.cn) bioinformatic tool was used to analyze the change of DPP4 mRNA expression in 426 OV tumor tissues compared with 88 control non-tumor tissues. Select Cutoff as |Log2FC|= 0.5. GSE18520 and GSE26712 datasets were used to verify the results from GEPIA2. UALCAN (http://ualcan.path.uab.edu/) was used to analyze the DDP4 protein expression in OV based on the CPTAC (Clinical Proteomic Tumor Analysis Consortium) database. The basic information of GSE18520 and GSE26712 were list in Table 1.

Table 1 Ovarian cancer survival analysis data set information2.2 Serum DPP4 levels detection by ELISA assay

Ethical approval was obtained from the Ethics Committee of Tangdu Hospital, Fourth Military Medical University. Informed consent was exempted from this study. Serum from 24 OV patients including16 with high-grade serous ovarian cancer (HGSOC, age range: 47–76 years) and 8 with other forms of OV (age range: 45–68 years). Additionally, serum from 23 healthy subjects (age range: 42–75 years) was collected. Peripheral blood was collected and centrifuged at 4000 rpm and 4 °C for 5 min to obtain serum. The serum samples were stored at − 80 °C. Serum DPP4 levels were detected using an ELISA kit according to the operation manual (R&D Systems, DY1180). In short, the capture antibody was coated in a 96-well plate and incubated overnight at room temperature. After blocking for 2 h, serum samples were added to the plate and incubated for 2 h. Then, streptavidin-conjugated detection antibody was added and incubated for 2 h. Then streptavidin-HRP antibody was added and incubated for 20 min. Finally, the TMB substrate was added, and the optical density (OD450nm) was measured. Serum DPP4 levels were calculated according to the standard curves.

2.3 Kaplan–Meier (KM) plotter analysis

The KM plotter bioinformatic tool (http://www.kmplot.com/analysis/index.php?p=service) [11] was used to evaluate the potential prognostic value of DPP4 expression in OV patients. The best performing cutoff value was computed by using “auto select best cutoff” model. The source data was based on TCGA and GEO datasets (GSE18520, GSE26712).

2.4 Cell culture

Human ovarian cancer cell lines, OVCAR-3 and SKOV-3, were purchased from Procell Life Science &Technology Co., Ltd (Wuhan, China). OVCAR-3 and SKOV-3 were used to investigate the effect of DPP4 on the cells’ biological behavior. OVCAR-3 was cultured with RPMI-1640 (Gibco, Carlsbad, NY, USA) supplemented with 20% Fetal Bovine Serum fetal bovine serum (ExCell Bio, Shanghai, China). SKOV-3 was cultured with Dulbecco’s Modified Eagle Medium (Gibco) plus 10% Fetal Bovine Serum (ExCell Bio). The cells were cultured in a 5% CO2 incubator at 37 °C.

2.5 Small-interfering RNA (siRNA) and miR-29a-3p transfection

siRNA (Sangon Biotech, Shanghai, China) was used to knock down the expression of DPP4. miR-29a-3p (Sangon Biotech) was used to investigate the effect of miR-29a-3p on DPP4 expression. Approximately 1 × 105 cells were seeded in 6 well plates and cultured for,

24 h Subsequently siRNA was transfected into OVCAR-3 and SKOV3 cells, and miR-29a-3p mimics were transfected into OVCAR-3 cells. The siRNA and miR-29a-3p transfection were carried out using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The sequences are listed in Table 2.

Table 2 siRNA and miRNA sequences used in the study2.6 Quantitative RT-PCR and western blot

After 48 h of siRNA or miR-29a-3p mimic transfection, RNA and protein were extracted from the cells. Total RNA was extracted from cells using a TRIzol Reagent Kit (GLPBIO, Cat: GK20008) For mRNA, cDNA was prepared using the Evo M-MLV Mix Kit (Accurate Biology AG) and cDNA for micro-RNA was prepared by using the Mir-X™ miRNA First-strand Synthesis Kit (Takara Bio, Japan). By using the Blastaq™ MasterMix kit (ABM, Canada), the reference genes for mRNA and miRNA were GAPDH and U6, respectively Quantitative real-time RT-PCR was used to detect the DPP4 mRNA and miR-29a-3p expression levels. Real-time PCR was performed on a QIAGEN Rotor-gene Q Real-Time PCR system (QIAGEN, Germany). Relative expression was calculated using the ΔΔCt method. U6 primers and miR29a-3p reverse primers were provided by the Mir-X™ miRNA First-Strand Synthesis Kit mentioned above. The primers for RT-PCR were list in Table 3.

Table 3 Prime sequences used for RT-PCR

For Western blotting, cell proteins were separated using SDS-PAGE and transferred onto a nitrocellulose membrane. The membranes were blocked for 2–3 h at room temperature, and then incubated overnight at 4 °C with an anti-DPP4 antibody (Cell Signaling Technology, Danvers, MA, USA). After washing with TBST three times, the membranes were incubated with an HRP labeled secondary antibody (ZSGB-BIO, Beijing, China) for 2 h. Proteins were detected using a Western-blotting luminol reagent (4A Biotech Co., Ltd, Beijing, China), GAPDH was used as the internal standard. Protein bands were visualized using a chemiluminescence detection system (Bio-Rad). The relative density of specific protein expression was determined using Image Lab software.

2.7 CCK8 assay

The CCK8 assay was performed to detect cell proliferation according to the manufacturer's instructions (GLPBIO, Cat: GSK10001). First, 2 × 103 cells were seeded in a 96-well plate. second, at different time points during culture (0, 24, 48, 72 h), 10μL of CCK8 reagent was added into each well and the cells were incubated for 2 h at 37 °C. Then, the OD450nm was measured, and the relative cell viability was calculated. as a percentage using the formula: (mean OD450 of treated cells/mean OD450 of control cells) × 100%.

2.8 Flow cytometry (FCM) analysis

Cells were collected 48 h after siRNA transfection for cell cycle and apoptosis detection. FCM was used to examine the cell cycle and apoptosis. For cell cycle analysis, cells were treated by using a cell cycle detection kit (KeyGENBiotech, Nanjing, China) according to the manufacturer's instructions. In brief, cells were fixed for 2 h with ethanol and stained with propidium iodide (PI) in buffer containing 10 μg RNase A. The cells were then kept in the dark at room temperature for 30 min. Cell cycle distribution was assessed using a FACS Calibur flow cytometer (BD Biosciences, San Jose, USA). Cell apoptosis was detected using the annexin V–FITC apoptosis detection kit (4A Biotech Co, Ltd, Beijing, China, Cat: FXP022). Cells were collected, washed twice with PBS and stained with annexin V-FITC and PI in the dark at room temperature for 15 min. The percentage of apoptotic cells was calculated using CellQuest 6.0 (BD Biosciences).

2.9 Cell migration assay

As previously described [12], a scratch assay was used to investigate the cell’s migration ability. In brief, cells were seeded and cultured in a 6-well plate. The scratch was performed when the cell density reached 80–90%. A monolayer of cells was then scratched with a new 200μL pipette tip across the center of the well, and washed gently twice with PBS, Then, cells were cultured for 24 h without serum. The scratch images were captured using a Cytation1 (BioTek, Vermont, USA). The cell migration ratio = (start distant—end distant)/start distant.

2.10 Luciferase reporter assay

To predict the binding sites between DPP4 and miR-29a-3p, TargetScan Human (https://www.targetscan.org/vert_71/) was used. The pGL3-DPP4-wild-type (WT) and pGL3-DPP4-mutant (MUT) were synthesized by Tsingke Biotechnology Co., Ltd (Beijing, China). Then, HEK293 cells were seeded into a 24-well plate and transiently co-transfected with synthesized plasmids (WT [1 µg] or MUT[1 µg]), pRL-TK-Renilla(20 ng), miR-29a-3p mimics(20 pM) or mimics-NC(20 pM) by using Lipofectamine™ 2000 (Invitrogen). After 48 h, the luciferase activities in cells were detected by using the Dual-Luciferase Reporter Assay Kit (Cat No. E1910, Promega, USA) and the Glomax luminescence detector (Promega). The results were expressed as the relative firefly luciferase activity, which is obtained after normalization to Renilla luciferase activity.

2.11 Statistical analysis

Serum DPP4 levels were expressed as the mean ± standard error. Student’s t-test was used to analyze the difference of serum DPP4 levels between OV patients and controls. The data from cell experiments are expressed as the mean ± standard error. Student’s t-test was used to analyze the difference of the cell proliferation rate, cell migration, cell cycle and apoptosis between different groups. All experiments were confirmed in three biological replicates. p < 0.05 was considered to be statistically significant. Statistical analysis was performed using GraphPad Prism 5.01.