Thirty-four normal matched controls (healthy or uterus benign tumor cases), 20 CIN and 100 cervical cancer patients (providing serum or tissue samples) admitted to Qilu Hospital of Shandong University were enrolled in the present study, between 2015 and 2016. The CIN and cervical cancer patients were staged according to the 2009 FIGO staging guidelines (19). For all these patients, the records containing the age, HPV infection history, TCT, colposcopy and pathology were examined. The present study, was approved by the Ethics Committee of Qilu Hospital.

The human cervical carcinoma (HeLa, SiHa and CaSki) and E6/E7 immortalized human cervical epithelial (H8) cell lines were obtained from the Cancer Center Laboratory of Shandong University (Jinan, Shandong, China). The HeLa and H8 cells were maintained in Dulbecco's modified Eagles medium (DMEM), SiHa cells were maintained in minimum essential medium (MEM), CaSki cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium, and all of the media (Hyclone Laboratories, Logan, UT, USA) were supplemented with 10% fetal bovine serum (FBS; Gibco, Sydney, Australia). The four cell lines were incubated in a humidified atmosphere at 37̊C with 5% CO₂. In addition, we collected cervical fall off epithelia tissue from healthy women volunteers as normal uterine cervix (NUC) cells.

The SiHa and HeLa cells were washed there times with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 15 min. After permeation with 0.2% Triton X-100 in PBS for 10 min, the cells were immunostained with rabbit anti-B7-H4 monoclonal antibody (1:100 dilution in PBS; Abcam, Cambridge, MA, USA) at 4̊C overnight. The cells were washed three times with PBS for 10 min each, and incubated for 1 h with a secondary goat anti-rabbit IgG antibody (1:200 dilution in PBS; Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China) at room temperature. Cells were washed in 4,6-diamidine-2-phenylindole dihydrochloride (DAPI) (Yusen Biotech Inc., Shanghai, China) for 3 min away from the light. Microscopic observations were performed using the Olympus IX51 inverted microscope.

The transfected cells were washed three times with PBS and lysed on ice using radio immunoprecipitation assay buffer (RIPA; Beyotime Institute of Biotechnology, Haimen, China) with 1% phenylmethylsulfonyl fluoride (PMSF) and 1% NaF for 30 min. The normal and tumor tissues were cut into pieces using tissue scissors, and homogenized by a tissue grinder (Tiangen, Beijing, China). The reagents were added into the tissue homogenates as aforementioned. Cell and tissue lysis were centrifuged at 12,000 rpm for 10 min at 4̊C and the protein extracts (30–50 µg) were loaded in each lane and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes (Millipore, Darmstadt, Germany). The membranes were blocked with 5% skim milk and probed with anti-E2F (ProteinTech Group, Inc., Chicago, IL, USA), anti-B7-H4, anti-phosphorylated Rb-(pRb), anti-P16, anti-P21, anti-Bcl-2, anti-cleaved PARP, anti-cleaved caspase-3 (Abcam) and anti-GAPDH [Cell Signaling Technology (CST), Danvers, MA, USA] antibodies. The membranes were then incubated with anti-rabbit and anti-mouse IgG (Millipore), and detected by enhanced chemiluminescence (ECL) using ImageQuant LAS 4000 (GE Healthcare Life Sciences, Logan, UT, USA). The results were analyzed by ImageJ software (NIH, Bethesda, MD, USA).

The cellular RNA from the tumor cells and tissue samples was extracted using TRIzol reagent (Life Technologies, Carlsbad, CA, USA) and the cDNA was synthesized from 3–5 µg of RNA using M-MLV reverse transcriptase (Invitrogen, Shanghai, China) according to the manufacturer's instructions. In addition, primer-probe sets for qPCR for each gene were designed using the PrimerBank and are listed in Table I. The data were collected using the StepOnePlus™ software (Applied Biosystems, Shanghai, China) and quantified using the 2^−ΔΔCt method.

The serum samples for the detection of B7-H4 were kept frozen at −80̊C until use. The ELISA kit for the detection of B7-H4 was obtained from LifeSpan Biosciences, Inc. (Seattle, WA, USA) and used according to the manufacturer's recommendations.

The tissues were cut into 4-µm sections. Standard SP immunohistochemistry (Maxim, Fuzhou, China) was performed with the B7-H4 antibody (1:500 dilution in PBS; Abcam) and a Polink-2 Plus Polymer HRP Detection System (ZSGB-BIO, Beijing, China). The detection of B7-H4 in cancer cells was assessed by two independent investigators. Quantifications were recorded as follows: <10% positive cells, 0; 10–25%, 1; 26–50%, 2; 51–75%, 3; >75% positive cells, 4. Staining intensity was scored as: absent, 0; weak, 1; moderate, 2; and strong, 3. The final score was the multiplication of the quantification and staining intensity. A final score of 0–1 was classified as negative, and ≥2 was considered positive.

The small interfering RNA sequences targeting human B7-H4 (Si-B7-H4) and its control sequence (Si-control) were designed by GenePharma Co., Ltd. (Shanghai, China). The SiHa cells were seeded in a 6-well plate with 4×10⁴ cells/ml/well. Twenty-four hours later, cells were transfected with 50 nM of the Si-B7-H4 or control sequences using Lipofectamine 2000 (Invitrogen Life Technologies). The transfected cells were harvested 48 h post-transfection for the follow-up experiments. The Si-B7-H4 and Si-control sequences are listed in Table I.

The B7-H4 plasmid was designed by GenePharma Co., Ltd., and the lentiviral vector pLenti-C-Myc-DDk-IRES-Puro (7.6 kb) under the control of a cytomegalovirus promoter was obtained from Vector Gene Technology Co., Ltd. (Beijing, China). After the transfection and virus package, puromycin dihydrochloride (2 µg/ml; Amresco, Solon, OH, USA) was used to generate HeLa cell lines that stably express B7-H4 (pCMV-B7-H4) and its negative control (pCMV-myc).

Cell viability was assessed using Cell Counting Kit-8 (CCK-8) (Tongren, Shanghai, China). According to the product manual, 2×10³ cells were seeded in each well of a 96-well plate, and incubated for 0, 24, 48, 72 and 96 h, and 10 µl of CCK-8 reagent was added to each well at 4 h before assessing the optical density (OD) at 450 nm using a microplate reader (Infinite 2000; Tecan, Männedorf, Switzerland).

Cells were washed twice using precooling PBS, and then digested in 75% alcohol at 4̊C overnight. Propidium iodide (PI) was added into the cell suspensions and the cell cycle distribution was detected by FACSCalibur flow cytometer (both from BD Biosciences, Franklin Lakes, NJ, USA). As for cell apoptosis, we used two apoptosis kits, one was the Annexin V-PE/PI, the other was the Annexin V-FITC/7-AAD. The results were analyzed following the manufacturer's protocol using the flow cytometer as aforementioned.

After the transfection of Si-B7-H4 or pCMV-B7-H4 as well as their negative controls, 6×10⁴ SiHa or 4×10⁴ HeLa cells were digested and suspended in 100 µl serum-free MEM and DMEM, respectively, then the cells were seeded into the upper Transwell chamber (8.0-µm pore size; Costar, Cambridge, MA, USA), in the absence or presence of 100 µl of Matrigel (1:8 dilution in serum-free medium; Corning, Corning, NY, USA). Medium with 20% FBS was added to the lower chamber as a chemoattractant. After 24 h, the cells passing through the filter were stained with 0.1% crystal violet, and the images were captured by the Olympus IX51 inverted microscope. The number of migrated or invaded cells was counted in five random fields (magnification, ×200) of each chamber under the microscope.

GraphPad Prism version 5.01 (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis. In the present study, data are expressed as the means with standard deviations (SDs), and statistical comparisons were performed using a Student's t-test or a Chi-squared test. P<0.05 was considered to indicate a statistically significant result.

To examine whether B7-H4 is expressed in cervical cancer, the relative protein expression levels of B7-H4 in 19 normal cervical and 20 cervical cancer tissues were assessed by western blotting (Fig. 1A). The protein level of B7-H4 in the cervical cancer samples was significantly higher than that in the normal tissues (P<0.001). We also detected the B7-H4 protein expression in H8 and three cervical cancer cell lines including CaSki, HeLa and SiHa (Fig. 1C). The protein expression level of B7-H4 was the lowest in the NUC cells and the highest in the SiHa cell line. To investigate the cellular location of B7-H4, immunofluorescence staining (Fig. 2A) and immunohistochemical staining (Fig. 2B) were performed and the results suggested that B7-H4 was mainly distributed in the cytoplasm of the cervical cancer cells. Collectively, these data suggest that B7-H4 was highly expressed in the cytoplasm of cervical cancer cells. However, it was not equally expressed in the different cervical cancer cell lines. Thus, knockdown of B7-H4 in the SiHa cell line and overexpression of it in the HeLa cell line was employed to study the functions of B7-H4.

To investigate the expression of B7-H4 in blood, ELISA assay was performed to detect the sB7-H4 in 15 healthy volunteers, 20 CIN and 20 cervical cancer patients. The results (Fig. 3A; Table II) revealed that the level of sB7-H4 in CIN patients was higher than that of the healthy volunteers (Fig. 3A, 2.654±1.533 vs. 1.000±0.557; P=0.0004), and in cervical cancer patients, sB7-H4 was higher than that of the CIN patients (Fig. 3A, 5.042±4.336 vs. 2.654±1.533; P=0.0257) and healthy volunteers (Fig. 3A, 5.042±4.336 vs. 1.000±0.557; P=0.0011). There was no difference in CIN I and CIN II–III (P=0.469) or in stage I and II cervical cancer patients (P=0.205), but the level of sB7-H4 in adenocarcinoma was higher than that in squamous carcinoma patients (Table II, 7.101±7.568 vs. 4.527±3.307; P=0.044). The receiver operating characteristic curve (ROC) is presented in Fig. 3B, and the area under the curve (AUC) was 0.955, and by using the concentration of sB7-H4 ≥1.638 as a critical value to predict CIN and cervical cancer, its sensitivity and specificity were 93.33 and 87.50%, respectively. Collectively, these data demonstrated that sB7-H4 has the potential to become an early diagnostic indicator of cervical cancer.

Next, we performed immunohistochemical staining and the results are listed in Table III. We found that the expression level of B7-H4 in cervical cancer tissues was not correlated with age (P=0.241), histology (P=0.536), tumor differentiation (P=0.419), clinical stage (P=0.540), tumor size (P=0.183), lymph vascular space involvement (LVSI; P=1.000), lymph node metastasis (LNM; P=0.620) and deep stromal invasion (DSI; P=0.993).

The HPV oncoprotein E7 forms a complex with tumor suppressor Rb and inhibits the activities of the proteins in cell cycle regulatory systems, which is essential for immortalization and transformation of human cervical squamous epithelial cells (20,21). To investigate whether B7-H4 could affect E7/Rb at the transcriptional level, we examined the effect of silencing and overexpression of B7-H4 on the mRNA level of E7/Rb. Compared with the control groups, the B7-H4 protein levels were significantly decreased in the SiHa cell line (Fig. 4A and B) and markedly increased in the HeLa cell line (Fig. 4C and D) by 2-fold. In addition, the silencing of B7-H4 in the SiHa cell line resulted in the downregulation of E7 mRNA and the upregulation of Rb mRNA, and the overexpression of B7-H4 in the HeLa cell line led to the opposite effect (Fig. 4E and F). These findings revealed that B7-H4 may take part in the formation of cervical cancer by influencing the E7/Rb pathway.

To examine whether B7-H4 could affect cell viability and the cell cycle, we performed CCK-8 and flow cytometric assays (15). The cell viability of the SiHa Si-B7-H4 group was decreased at 48, 72 and 96 h (Fig. 5A), and the cell viability of the HeLa pCMV-B7-H4 group was increased at 48, 72 and 96 h (Fig. 5B), in comparison with their control groups. We also detected the cell cycle distribution using flow cytometry. Compared with the control groups, the SiHa cells treated with Si-B7-H4 were arrested in the G0/G1 phase (Fig. 5C) and the HeLa cells with overexpression of B7-H4 demonstrated accelerated S to G2/M phase transition (Fig. 5D). We also found that the cell cycle regulatory proteins, including pRB, E2F, P16 and P21, were influenced by B7-H4 expression. Specifically, the protein expression levels of pRB, E2F, P16 and P21 were altered with the expression change of B7-H4. These proteins were downregulated in the SiHa cells with silenced B7-H4 (Fig. 5G) and upregulated in the pCMV-B7-H4 group of the HeLa cell line (Fig. 5H) in comparison with their control groups.

To determine whether B7-H4 expression affects cervical cancer cell death, the knockdown of B7-H4 in the SiHa cell line led to an increase in early apoptosis (PI⁻/Annexin⁺) as well as late apoptosis (PI⁺/Annexin⁺) (Fig. 6A and C), and the overexpression of B7-H4 in the HeLa cell line led to a decrease of early apoptosis (7AAD⁻/Annexin⁺) (Fig. 6B and D) when compared with the control groups. The western blotting results revealed that the apoptosis-related proteins, including cleaved PARP and cleaved caspase-3, were upregulated in the Si-B7-H4 group of the SiHa cell line (Fig. 6E) and downregulated in the pCMV-B7-H4 group of the HeLa cell line (Fig. 6F) in comparison with the control groups, and the anti-apoptosis protein Bcl-2 was in inverse proportion (Fig. 6E and F).

Compared with the control groups, the migrated and invasive number of cells decreased significantly in the SiHa Si-B7-H4 group and increased markedly in the HeLa pCMV-B7-H4 group (Fig. 7A and B). Matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF) have been reported to be associated with the migration and invasion of various cancer cell lines (22–24). To study whether B7-H4 affected MMP-2, MMP-9 and VEGF at the transcriptional level, we examined the effect of the silencing and overexpression of B7-H4 at the mRNA level of MMP-2, MMP-9 and VEGF, and found that these three molecules were regulated with the expression change of B7-H4 as determined by RT-PCR. Specifically, the silencing of B7-H4 in the SiHa cell line decreased the mRNA expression of these molecules (Fig. 7E) while the overexpression of B7-H4 in the HeLa cell line increased their mRNA expression (Fig. 7F).