The HepG2 and SSMC-7721 liver cancer cell lines (American Type Culture Collection, Manassas, VA, USA) were maintained in the laboratory at Jinan Central Hospital Affiliated to Shandong University (Shandong, China) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (HyClone, GE Healthcare Life Sciences, Little Chalfont, UK), 1% penicillin and 1% streptomycin (Gibco-BRL, Invitrogen Life Technologies, Carlsbad, CA, USA).

The cells were planted in 48-well plates and subsequent to a 24-h resting period, the Skp2 p45 siRNA and scramble siRNA was transfected with Lipofectamine 2000 (Invitrogen Life Technologies). Following 8 h of transfection, the DMEM medium was replaced and the lysates were prepared subsequent to transfection for 48 h. The Skp2 p45 siRNA was purchased from Santa Cruz Biotechnology, Inc. (sc-36499; Dallas, TX, USA) and scramble siRNA was synthesized by Shanghai Jima Company (Shanghai, China). Rabbit polyclonal immunoglobulin G (IgG) Skp2 p45 antibody (200 μg/ml; sc-7164), mouse monoclonal IgG₁ p27 antibody (200 μg/ml; sc-53906) and mouse monoclonal IgG₁ β-actin (AC-15) antibody (100 μg/ml; sc-69879), as well as horseradish peroxidase-conjugated goat anti-mouse secondary antibody (sc-2005) were all obtained from Santa Cruz Biotechnology, Inc.

Liver tissue samples were obtained from Jinan Central Hospital Affiliated to Shandong University. The subjects included 50 cases of hepatocellular carcinoma and 40 paratumor tissues with an average age of 58±13 years. The subjects were recruited from March 2011 to December 2013. The specimens were obtained during surgery. There were 52 male and 38 female patients during the experiment. Written informed consent was also obtained from the patients’ families. The study was approved by the ethics committee of Jinan Central Hospital Affiliated to Shandong University (Jinan, China). The liver tissues were fixed in neutral buffered paraformaldehyde and processed for hematoxylin and eosin (H&E) staining. Immunohistochemical staining was performed, as previously described (21,22). Briefly, the paraffin-embedded sections were dewaxed, rehydrated, inhibited and incubated overnight with the primary antibodies (anti-p27 and anti-Skp2). Subsequently, the samples were incubated with goat anti-mouse and goat anti-rabbit secondary antibodies (Santa Cruz Biotechnology) for 1 h. The slides were stained, dehydrated and cleared and the stained slides were mounted in Permount and visualized using a Nikon Eclipse Ti microscope (Nikon, Tokyo, Japan). Images were captured using an FE330 digital camera (Olympus Corporation, Tokyo, Japan), which was attached to the microscope.

The whole cell extracts were prepared and separated by PAGE, as previously described (23–25) using buffer and gel obtained from Shanghai Qcbio Science and Technologies Co., Ltd. (Shanghai, China) and the machine (Mini-Protean^® Tetra Cell) from obtained from Bio-Rad (Hercules, CA, USA). The proteins of the sample were separated using gel electrophoresis and were subsequently transferred to a polyvinylidine difluoride membrane (Millipore, Billerica, MA, USA). Subsequently, blocking of non-specific binding was achieved by placing the membrane in a solution of 5% bovine serum albumin. Following blocking, a dilute solution of the appropriate primary antibody was incubated with the membrane at 4°C overnight. The antibodies used were as follows: Anti-p27, anti-Skp2, anti-β-actin and horseradish peroxidase-conjugated goat anti-mouse secondary antibody.

An MTT assay was performed, as described previously (26–28). The cells (5×10⁵) were seeded into 48-well plates. Following culture for 24, 48 or 72 h, the plates were read on a microplate reader (Varioskan Flash Multimode Reader; Thermo Fisher Scientific, Waltham, MA, USA) at a test wavelength of 490 nm and a reference wavelength of 570 nm.

Immunoprecipitation was performed, as previously described (29–31). The cells were lysed and the sample was passed over beads alone or bound to a relevant antibody to absorb any proteins that non-specifically bind to the immunoprecipitation components. For immunoprecipitation analysis, the samples were immunoprecipitated with mouse monoclonal hemagglutinin (HA)-tag antibodies (A012244-100; GenScript, Nanjing, China). The immunocomplexes were separated by SDS-PAGE and treated with the secondary antibody. The complexes of Skp2 or ubiquitin with p27^kip1 were determined by immunoprecipitating the mouse monoclonal anti-Flag antibody (AF519; Beyotime Institue of Biotechnology, Haimen, China) and normalized to the quantity of antibodies. To assess the specificity of the primary antibodies, negative controls were used, in which the antibodies used to immunoprecipitate were incubated for 2 h at room temperature with the respective immunogen peptide.

Cell apoptosis rate was determined by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) (Hangzhou Lianke Biology Technology Limited Company, Hangzhou, China) staining. The one-step staining procedure was selected, which takes only 10 min to perform. Detection was analyzed by flow cytometry (FC 500; Beckman Coulter, Brea, CA, USA), which differentiates between apoptosis and necrosis following Annexin V-FITC and PI staining.

Student’s t-test was used to evaluate statistical significance. Data were analyzed using SPSS 19.0 (IBM SPSS, Armonk, NY, USA) Data are expressed as the mean ± standard deviation. P<0.05 were considered to indicate a statistically significant difference.

In the present study, 90 registered patients were obtained from the database of Jinan Central Hospital Affiliated to Shandong University. As shown in Fig. 1, H&E staining of the samples of paratumor liver tissue and hepatocellular carcinoma tissue was compared with normal liver tissues. It is reported that the Skp2-p27^kip1 signaling pathway is closely associated with cancer (32,33). In the present study, the expression levels of Skp2 and p27^kip1 were detected by immunohistochemical analyses in the paratumor and hepatocellular carcinoma tissues. The results revealed that the positive brown staining for Skp2 was predominantly observed in the nuclei with occasional cytoplasmic expression (Fig. 2). Compared with the immnunostaining of the paratumor tissues, nuclear and cytoplasmic localization of Skp2 was observed in the majority of the examined samples in the patients with hepatocellular carcinoma (Table I) with 70.0% of the malignant tumors demonstrating varying levels of positive immunostaining. A significant difference was observed between the two groups (P<0.01).

The present study then detected the expression and localization of p27^kip1 in the hepatocellular carcinoma and paratumor tissues. As shown in Fig. 2, the results of the immunostaining demonstrated that p27^kip1 exhibited a primarily nuclear pattern of expression. As shown in Table II, 84.0% (42/50) of the malignant hepatocellular carcinoma tissues demonstrated negative immunostaining; however, negative immunostaining was observed in only 10% (4/40) of the paratumor tissues, with a significant difference observed between the two groups (P<0.01).

In order to confirm the localization of Skp2 in SSMC 7721 and HepG2 cells, cytoplasmic and nucleic proteins were prepared and western blot analysis was performed. As shown in Fig. 3, the results revealed that the majority of Skp2 was located in the nuclei of the cells; although there was low expression detected in the cytoplasm.

In order to examine whether the degradation of p27^kip1 is regulated by Skp2, 293T cells, which expressed HA-Skp2, Flag-p27 and ubiquitin together were used. After 48 h, a co-immunoprecipitation assay was performed and the data demonstrated that the K48-linked p27^kip1 increased and the K63-linked p27^kip1 decreased. The total quantity of ubiquitinated p27^kip1 was also downregulated (Fig. 4). Taken together, these data demonstrated that p27^kip1 was degraded by the ubiquitin-proteasome pathway, which was mediated by the Skp2 complex.

Skp2 is an oncogene, which is highly expressed in tumor cells. The present study investigated whether interference of Skp2 with specific siRNA induced the apoptosis of liver cancer cells or promoted liver cancer cell death. As shown in Fig. 5, the apoptotic rates of the SSMC-7721 cells transfected with Skp2-siRNA were significantly higher compared with the control cells (P<0.01).

In the MTT assay (Fig. 5C), the optical density at 490 nm in the cells transfected with Skp2-siRNA were significantly lower compared with that of the control cells (P<0.05 and P<0.01). This was consistent with the results observed by fluorescence-activated cell sorting (FACS), suggesting that the proliferation of cancer cells was inhibited following transfection with Skp2-siRNA.

The present study demonstrated that interference of Skp2 induced apoptosis and inhibited proliferation of the SSMC-7721 cells. In order to further examine the mechanism involved and clarify the association with the expression of p27^kip1, the expression of p27^kip1 was examined by western blot analysis. As shown in Fig. 6, following depletion of the expression of endogenous Skp2 by RNA interference, the levels of endogenous p27^kip1 increased in the HepG2 and SSMC-7721 cells suggesting that Skp2-mediated degradation of p27^kip1 was important for proliferation of the tumor cells.