The human hepatocellular carcinoma cell line HepG2 (MD, USA) was cultured with RPMI-1640 medium (Gibco Invitrogen Corp.) containing 10% fetal bovine serum (FBS), 100 U/ml penicillin and streptomycin. The cells were cultured in a cell incubator at 37°C, 5% CO₂, and saturated humidity. The cell seeding density was 3 × 10⁴ cells/cm². After 24 h of culture, the cells were cultured in RPMI-1640 medium without FBS for another 24 h. The HSD17B4 expression plasmid was transfected 24 h before VK2 administration.

HSD17B4 cDNA was cloned into the pLL3.7 vector (Addgene, Inc., Cambridge, MA, USA) (15). HepG2 cells (5 × 10⁶) were seeded in a six-well plate and cultured for 24 h at 37°C and 5% CO₂. Cells were transfected when the cells have grown to about 70% confluence. The cells were washed twice with serum-free RPMI-1640, and 1 ml serum-free Opti-MEM I (Gibco; Thermo Fisher Scientific, Inc.) was added to each well. The DNA–Lipofectamine 2000 complex was prepared according to the manufacturer’s instructions (Invitrogen; Thermo Fisher Scientific, Inc.). A total of 6 μg transfection plasmid and 30 μl Lipofectamine reagent were added to each reaction. The empty vector control is indicated by “−, ” and the HSD17B4 plasmid is highlighted with a “+” below. Cells were placed in an incubator at 37°C for 6 h. The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 1% FBS for 24 h, and the transfection efficiency was determined by Western blotting.

The specific small interfering RNA (siRNA) against HSD17B4 (siHSD17B4), containing 5′-GUACCUUUGUAUUUGAGGAdTdT-3′ and 5′-UCCUCAAAUACAAAGGUACdTdT-3′, and the non-specific siRNA (siNC), containing 5′-UUCUCCGAACGUGUCACGUTT-3′ and 5′-ACGUGACACGUUCGGAGAATT-3′, all were purchased from Sigma (15). Transfection was performed using the Lipofectamine reagent following the manufacturer’s instructions. Transfection was conducted after 24 h, and the cells were treated with 50 μM of VK2 for the indicated times, harvested and lysed for tests.

Cell proliferation assays were performed using a 3-(4,5-diethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-etrazolium, inner salt (MTS) cell proliferation assay kit (Promega) according to the manufacturers’ recommendations. The cells were treated with MTS for 3 h before the termination of HSD17B4 plasmid incubation. The OD readings were performed to determine the number of viable cells at 490 nm. All groups were evaluated in an average of three separate wells per experiment.

According to the instructions, total RNA was isolated from cells using TRIzol reagent (Invitrogen). SuperScript Reverse Transcription Kit (Invitrogen) was used to synthesize cDNA, which was used as a template for real-time quantitative PCR using the SYBR Green PCR Master Mix Kit (TaKaRa). Quantitative real-time PCR was performed using the Rotor-Gene 3000 detection system (Gene Biosystems). The PCR cycle consisted of an initial denaturation step at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s, annealing at 55°C for 30 s, and extension at 72°C for 20 s. As an internal control, 18S rRNA was used for RNA template standardization. All PCRs were performed in triplicate. The relative expression level of mRNA was 2^−ΔCt (Ct sample −Ct control). The primer pairs are shown in Table 1 .

Cells are lysed in lysis buffer A for cytoplasmic protein extraction, and then, the cells were used for nucleoprotein extraction in lysis buffer B (15). After the lysates were incubated on ice for 30 min, nuclear protein was obtained by centrifugation at 8,000 g for 10 min, or cytoplasmic protein was obtained by centrifugation at 12,000 g for 20 min. The supernatant was used for testing. The same amount of proteins (100–200 μg) were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electro-transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was sealed with 5% skim milk powder for 1 h at room temperature and incubated overnight at 4°C with primary antibodies: rabbit anti-HSD17B4 (Homemade), 1:2,000 (Jiang et al., 1996); rabbit anti-cyclin D1 (Cell Signaling, Inc., catalog no. 2922S), 1:1,000; rabbit anti-PCNA (Cell Signaling, Inc., catalog no. 13110), 1:1,000; mouse anti-β-actin (Santa Cruz Biotechnology, Inc., catalog no. sc-47778), 1:1,000; rabbit anti-p-STAT3 (Santa Cruz Biotechnology, Inc., catalog no. sc-135649), 1:500; rabbit anti-STAT3 (Santa Cruz Biotechnology, Inc., catalog no. sc-482), 1:500; rabbit anti-p-Akt (Santa Cruz Biotechnology, Inc., catalog no. sc-7985), 1:500; rabbit anti-Akt (Santa Cruz Biotechnology, Inc., catalog no. sc-8312), 1:500; rabbit anti-p-MEK (Santa Cruz Biotechnology, Inc., catalog no. sc-101733), 1:500; rabbit anti-MEK (Santa Cruz Biotechnology, Inc., catalog no. sc-9259), 1:500; rabbit anti-p-ERK (Santa Cruz Biotechnology, Inc., catalog no. sc-23759), 1:500; and rabbit anti-ERK (Santa Cruz Biotechnology, Inc., catalog no. sc-292838), 1:500. After incubation with appropriate fluorescent secondary antibodies at room temperature for 1 h, the anti-antigen complexes were imaged using chemiluminescence Plus Protein Western blot assay kit (Santa Cruz Biotechnology). The expressions of various genes were expressed in relative grayscale, and β-actin was used as the protein loading control.

HepG2 cells with HSD17B4 overexpression were treated with different concentrations of VK2 for 48 h. The cell lysates interacted with anti-HSD17B4 antibody; the precipitate was collected for the determination of the content of VK2 that bound to HSD17B4 in cells. Subsequently, a cell lysates assay for anti-VK2 antibody was performed by commercial ELISA kit (BlueGene Biotech) according to the manufacturer’s instructions. The optical density (OD) was measured at 450 nm. The results were expressed as pg/ml according to a calibrator curve.

In order to test the inhibitory effects of VK2 on tumor growth in vivo, 2 × 10⁶ transduced HepG2 cells were injected into BALB/c nude mice aged 4 weeks. Six weeks post-inoculation, the animals were sacrificed, and the volume of the tumor was recorded.

The results were expressed as mean ± standard deviation (SD). Statistical differences among groups were analyzed using one-way ANOVA. At the p < 0.05 level, the difference was considered statistically significant. All statistical analyses were performed using SPSS 13.0 software.

To test the effect of VK2 on HCC cells proliferation induced by HSD17B4, we measured the activation of cells by MTS assay. VK2 (50 μM) showed a time-dependent inhibition on the proliferation of HepG2 cells with HSD17B4 overexpression ( Figure 1A ). It was statistically significant from 24 h (p < 0.05).

To further verify that VK2 can directly inhibit proliferation of HCC cells with HSD17B4 overexpression, we measured the activation of cells that were transfected with expression plasmid of HSD17B4 or siHSD17B4 to increase or knock down HSD17B4 expression by MTS assay. VK2 showed a dose-dependent inhibition on the proliferation in HSD17B4 overexpressed HepG2 cells ( Figure 1B ). However, there was no significant difference in a dose-dependent inhibition on transfection with siHSD17B4 ( Figure 1C ). These results showed that VK2 could suppress cell proliferation induced by HSD17B4, and the inhibition of proliferation by VK2 requires the presence of HSD17B4.

It has been reported that VK2 can bound to HSD17B4 in HepG2 cells (17). To further prove that VK2 can bind to HSD17B4 directly, we used anti-HSD17B4 antibody to precipitate HSD17B4 in the lysates of HepG2 cells and then detect VK2 content in the precipitate. The VK2 concentrations in HepG2 cells were assayed by ELISA. HepG2 cells were transfected with the empty or HSD17B4 expression plasmid. Then, these cells were treated with VK2. As shown in Figure 2A , VK2 showed a dose-dependent increase in the cells transfected with HSD17B4 expression plasmids, but there was no significant increase in VK2 on the HepG2 cells transfected with the empty plasmid. These results demonstrated that VK2 binds to HSD17B4 to exert its inhibitory effect on HepG2 cells.

Furthermore, we tested the change in HSD17B4 expression in mRNA and protein by VK2 treatment. VK2 did not affect the HSD17B4 expression of mRNA and protein in HepG2 cells by transfecting with a HSD17B4 expression plasmid ( Figures 2B, D ). At the same time, VK2 also did not affect the mRNA and protein expression of HSD17B4 by siRNA to knockdown HSD17B4 expression ( Figures 2C, E ). These results indicated that VK2 bound to HSD17B4 directly and did not affect the expression of HSD17B4.

Our recent studies have found that upregulation of HSD17B4 promoted HepG2 cell proliferation by enhancing cyclin D1 and proliferating cell nuclear antigen (PCAN) expression (16). To determine whether VK2 could reduce the HSD17B4-induced overexpression of proliferation genes, we investigated the expression of cyclin D1 and PCNA on the cells that were transfected with HSD17B4 expression plasmids or siHSD17B4. As shown in Figure 3 , HSD17B4 overexpression increased the protein levels of cyclin D1 and PCNA, and VK2 not only suppressed the expression of cyclin D1 and PCNA on cells that were transfected with empty plasmids but also suppressed their increased expression induced by HSD17B4 overexpression ( Figure 3A ). However, there was no significant decrease in the expression of cyclin D1 and PCNA on the HepG2 cells that were transfected with siHSD17B4 to knockdown HSD17B4 expression ( Figure 3B ). These results indicated that VK2 inhibited the proliferation genes expression of HSD17B4-increased protein of cyclin D1 and PCNA in HepG2 cells.

To further investigate the mechanism by which VK2 suppressed proliferation induced by HSD17B4 overexpression in HepG2 cells, we detected the activation of STAT3 pathway that was directly related to proliferation of HCC cells. HSD17B4 overexpression increased the phosphorylation level of STAT3, and VK2 not only suppressed phosphorylation of STAT3 on cells that were transfected with the empty plasmids but also suppressed their increased activation by induced HSD17B4 overexpression ( Figure 3C ). However, there was no significant decrease in the phosphorylation of STAT3 on the HepG2 cells that were transfected with siHSD17B4 to knockdown HSD17B4 expression ( Figure 3D ). These results indicated that VK2 suppressed expression of proliferation genes by downregulation of HSD17B4-increased phosphorylation of STAT3 in HepG2 cells.

STAT3 phosphorylation can be mediated through the activation of upstream signaling pathway, including Akt and MEK/ERK, that were involved in the proliferation of HCC cells. To further investigate whether VK2 was involved in the downregulation of HSD17B4-increased activation of upstream signaling pathway of STAT3 in HepG2 cells, we detected the phosphorylation of Akt and MEK/ERK. HSD17B4 overexpression increased the phosphorylation levels of Akt, MEK, and ERK, and VK2 not only suppressed their phosphorylation on cells that were transfected with the empty plasmids but also suppressed their increased activation by induced HSD17B4 overexpression ( Figure 4A ). However, there was no significant decrease in the phosphorylation of Akt, MEK, and ERK on the HepG2 cells that were transfected with siHSD17B4 to knockdown HSD17B4 expression ( Figure 4B ). These results indicated that VK2 inhibited the downregulation of HSD17B4-induced phosphorylation of Akt and MEK/ERK in HepG2 cells.

Typically, a single nodular tumor was found mostly localized within the injected site and was observed in animals implanted with HepG2 cells. The xenograft tumors are shown in Figure 5A . To compare the differences in tumor volume, we measured the volume of the tumor ( Figure 5A ) and compared the mean volume of tumors in each group ( Figure 5B ). Evidently, the tumors volumes were increased in HSD17B4 plasmids transfection but were reduced significantly by VK2 treatment. To further preclude the interaction of VK2 with HSD17B4, the inhibitory rate of tumor growth by VK2 showed that there was no significant difference between control (empty mean/empty + VK2 mean) and HSD17B4 overexpression (HSD17B4 mean/HSD17B4 + VK2 mean) ( Figure 5C ). Thus, the results exhibited that VK2 can inhibit the tumor growth by HSD17B4 overexpression.