The ovarian carcinoma tissue micro-arrays were obtained from the Xi’an Aomei Biotechnology Co., Ltd. (Xi’an, China). These microarrays (catalog no. C1026) were composed of 80 ovarian carcinoma tissue samples (average age 39), which included duplicate core biopsies (1 mm in diameter) from fixed, paraffin-embedded tumors. Immunohistochemical staining of samples were performed as previously reported (28) and the primary antibody of rabbit anti-HBXIP (1:100, Proteintech Group, Chicago, IL, USA) or the primary antibody of rabbit anti-Skp2 (1:30, Boster Group, Wuhan, China) was used. Immunostained slides were evaluated under a microscope. Categorization of immunostaining intensity was performed by three independent observers. The staining levels of HBXIP and Skp2 were classified into three groups using a modified scoring method based on the intensity of staining (0, negative; 1, low; and 2, high) and the percentage of stained cells (0, 0% stained; 1, 1–49% stained; and 2, 50–100% stained). A multiplied score (intensity score x percentage score) lower than 1 was considered to be a negative staining (−), 1 and 2 were considered to be moderate staining (+), and 4 was considered to be intense staining (++).

Ovarian cancer cell lines, SKOV3 cells (29) were cultured in RPMI-1640 medium (Gibco-BRL, Grand Island, NY, USA), 10% fetal bovine serum (FBS); CAOV3 cells (29) were cultured in DMEM medium (Gibco-BRL), 10% FBS. 100 U/ml penicillin and 100 μg/ml streptomycin in humidified 5% CO₂ at 37°C.

pCMV-tag2B, pGL3-Basic vectors (Promega, Madison, WI, USA), pCMV-HBXIP was maintained in our laboratory (6). The 5′-flanking region (from -1309 to +235 nt) of Skp2 gene was inserted into the KpnI/XhoI site upstream of the luciferase gene in the pGL3-basic vector, termed pGL3-Skp2 promoter. Mutant construction of Skp2 promoter, termed as pGL3-Skp2 promoter mut, carried a series substitution of nucleotides within Sp1 binding site. The complete human Skp2 (GenBank accession no. NC 000005.9) gene was subcloned into pCMV-tag2B vector to generate the pCMV-Skp2 construct. siRNA duplexes targeting human HBXIP (or Skp2) gene and siRNA duplexes with non-specific sequences using as negative control (NC) were synthesized by RiboBio (Guangzhou, China) (3,30). All primers and siRNA sequences are listed in Table I.

One day before transfection, cells were harvested and seeded into 6- or 24-well plates. Cells were transfected with plasmid or siRNAs using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol.

Cells transfected with plasmid of HBXIP, plasmid of HBXIP and siRNAs of Skp2, siRNAs of HBXIP, siRNAs of HBXIP and pCMV-Skp2 were seeded in a 6-well plate and cultured for 24 h to form confluent monolayers. A wound was created by dragging a pipette tip through the monolayer, and plates were washed using pre-warmed PBS to remove cellular debris. Wound images were photographed at 0, 24, 48 and 72 h after wounding. The wound gaps were measured at each time point. For Transwell migration assay using SKOV3 and CAOV3 cells with indicated treatment, 5×10⁵ cells were plated on 8 μm Transwell filters (Corning Incorporated, Corning, NY, USA). The cells were induced to migrate towards medium containing 10% FBS for 20 h. Non-migrating cells were removed with a cotton swab. The remaining cells were fixed, stained with hematoxylin and eosin, and analysed by a bright-field microscope.

Total RNA of cells was extracted using TRIzol reagent (Invitrogen). First-strand cDNA was synthesized by PrimeScript reverse transcriptase (Takara Bio, Dalian, China) and oligo (dT) following the manufacturer’s instructions. The primers are listed in Table I.

Western blot analysis was carried out with standard protocols. Primary antibodies used were rabbit anti-Skp2 (1:300, Boster Group), rabbit anti-HBXIP (1:1,000, Proteintech Group), and mouse anti-β-actin (1:800, Sigma-Aldrich, St. Louis, MO, USA). All experiments were repeated 3 times.

For luciferase reporter gene assays, the ovarian cancer cells were transfected with plasmids encoding HBXIP by Lipofectamine 2000. The luciferase activities were determined 48 h after transfection, and the results are the average of 3 independent repeats. The luciferase activities in the cell lysates were measured by a dual luciferase reporter assay kit (Promega), and the luciferase activity was normalized with renilla luciferase activity.

The ChIP assay was performed using the EpiQuik™ chromatin immunoprecipitation kit from Epigentek Group Inc (Farmingdale, NY, USA) according to the published methods (6,31). Protein-DNA complexes were immunoprecipitated with HBXIP antibodies, whereas rabbit preimmune serum served as a control. DNA from input or immunoprecipitated samples was assayed using SYBR-Green-based quantitative PCR with specific primers designed to amplify the Skp2 promoter around the SREs. ChIP/ReChIP: ChIP was performed as above, binding complexes from the first immunoprecipitation were eluted from the sepharose beads using Re-ChIP buffer. The eluted protein-DNA complexes were diluted in radioimmunoprecipitation buffer and resubjected to ChIP using a different antibody.

Each experiment was repeated at least three times. Statistical significance was assessed by comparing mean values (± SD) using a Student’s t-test for independent groups or pairing χ² for dependent groups and was assumed for ^*P<0.05, ^**P<0.01 and ^***P<0.001.

Our previous reports showed that HBXIP was overexpressed in breast cancer and other cancer cells. However, there is no report concerning the expression of HBXIP in ovarian cancer. In this study, we examined the expression of HBXIP in ovarian cancer. The data revealed that HBXIP was overexpressed in ovarian cancer tissue (Fig. 1). It has been reported that Skp2 is also over-expressed in ovarian cancer tissues (23). Thus, we supposed that overexpression of Skp2 might be correlated with enhanced HBXIP in ovarian cancer. Then, we investigated the expression correlation between HBXIP and Skp2 by IHC using tissue microarrays from the same tissue paraffin block. Our data showed that the positive rate of HBXIP was 75% (60/80) in clinical ovarian cancer tissue samples, and the positive rate of Skp2 was 81.67% (49/60) in the HBXIP-positive specimens (Fig. 1). Pairing χ² analysis showed that there was no significant difference between the positive rate of HBXIP and that of Skp2 in the tissues (P>0.05, Table II), suggesting that the expression of Skp2 is relevant to that of HBXIP in ovarian cancer tissues. Additionally, in this study, IHC staining showed that the expression of HBXIP could be observed in both cytoplasm and nucleus in ovarian cancer tissues (Fig. 1B). We also observed that Skp2 was expressed in both cytoplasm and nucleus in ovarian cancer tissues (Fig. 1C), which is consistent with a previous study (32). Thus, we speculated that HBXIP might be involved in the transcriptional regulation of Skp2.

Cell migration is an essential process in cancer metastasis and HBXIP can promote cell migration in breast cancer cells (6,7). Other reports showed that Skp2 can modulate cell migration of breast cancer, prostate cancer and myxofibrosarcoma (18,21,22,24). Thus, we supposed that Skp2 might be involved in the migration enhanced by HBXIP. Wound-healing and Transwell assays showed that treatment with plasmid encoding HBXIP enhanced the migration of SKOV3 cells, while additionally treated with Skp2 siRNAs abolished the effect (Fig. 2A and B). Furthermore, we performed the same assay using another ovarian cancer cell line CAOV3, and obtained similar results (Fig. 2C and D). We found that the inhibited migration of SKOV3 ovarian cancer cells induced by HBXIP siRNAs, was rescued by the overexpression of Skp2 (Fig. 3A and B). Similar results occurred in CAOV3 ovarian cancer cell line (Fig. 3C and D). Furthermore, as shown in Fig. 4, the RNA interference of HBXIP (or Skp2) decreased the expression of protein levels in SKOV3 and CAOV3 cells. Thus, our data suggest that HBXIP promotes the migration of ovarian cancer cells through Skp2 in vitro.

Next, we evaluated whether HBXIP was able to upregulate Skp2 in ovarian cancer cell lines. After transfection with plasmid encoding HBXIP, we observed that the levels of mRNA and protein of Skp2 were upregulated by HBXIP in SKOV3 and CAOV3 cell lines in a dose-dependent manner (Fig. 5). Thus, we verified that HBXIP was able to upregulate Skp2 in ovarian cancer cells.

Furthermore, to explore the mechanism by which HBXIP upregulated Skp2, we cloned the promoter region of Skp2 (-1309/+235) into pGL3-Basic plasmid. Luciferase reporter gene assays showed that HBXIP could increase the promoter activities of Skp2 in SKOV3 or CAOV3 ovarian cancer cells in a dose-dependent manner (Fig. 6A and B), suggesting that HBXIP is capable of activating the Skp2 promoter in the ovarian cancer cells. Next, we investigated the underlying mechanism by which HBXIP activates Skp2 promoter. To map the HBXIP binding site in Skp2 promoter, we designed a series of primers of Skp2 promoter fragments in 5′-flanking region, including the fragments −776/−567, −640/−443 and −464/−250. Interestingly, ChIP assays showed that HBXIP was able to occupy the Skp2 promoter fragment −640/−443 (Fig. 6C), suggesting that the −640/−443 region of Skp2 promoter is the regulatory target sequence of HBXIP. Then, we used online promoter analysis tool Search Promoter Site (http://alggen.lsi.upc.es/cgibin/promo_v3/promo/promoinit.cgi?dir-DB=TF8.3) to predict the putative transcription factor binding sites in the −640/−443 promoter region of Skp2. Strikingly, we observed a putative Sp1 binding site in the region (Fig. 6D). ChIP/ReChIP assay showed that HBXIP and Sp1 were able to form a transcriptional complex on the Skp2 promoter (Fig. 6E). Luciferase reporter gene assays showed that HBXIP failed to work when the Sp1 binding site in Skp2 promoter was mutated (Fig. 6F and G). Thus, we conclude that HBXIP activates Skp2 promoter activity through the transcription factor Sp1.