Data for WIPI2 expression and clinical information in the The Cancer Genome Atlas (TCGA) (https://www.cancer.gov/tcga) were extracted and analyzed in the UALCAN platform (http://ualcan.path.uab.edu) (13).

HCC tissue arrays were purchased from the National Engineering Center for Biochips (Shanghai, China). The expression of WIPI2 in the tissues was evaluated by IHC assay using the WIPI2 antibody (dilution 1:150; cat. no. ab105459; Abcam). The tissue sections were collected, dewaxed in xylene, and rehydrated in graded ethanol solutions. All the following steps were carried out in a moist chamber. Then the tissue sections were blocked with 3% peroxide-methanol at room temperature for endogenous peroxidase ablation and incubated with blocking buffer (normal goat serum) at room temperature. After that, the blocking buffer was discarded and the sections were washed with PBS for 3 times. The sections were then incubated with purified primary antibody overnight at 4°C, and incubated with the corresponding secondary antibody (dilution 1:200; cat. no. KGAA35; Nanjing KeyGen Biotech. Co. Ltd.). Finally, the sections were stained with DAB and hematoxylin. The staining was scored according to the staining intensity (no color, 0; faint yellow, 1; claybank, 2; brown, 3) and the proportion of positive cells (0, <5; 1, <5 and <25%; 2, <25 and <50%, 3, <50 and <75%; 4, <75% positive cells), and the final staining scores were calculated as the score of the staining intensity multiplied by the score of the proportion of positive cells [score of 0 indicates negative staining (0+); 1–4 is weak staining (1+); 5–8 is moderate staining (2+); 9–12 scores indicates strong staining (3+)].

Huh7 and Hep3B cells were purchased from the Shanghai Institutes for Biological Sciences (China). The cells were maintained in DMEM (cat. no. 11965; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS), 100 U/ml of penicillin and 100 µg/ml of streptomycin (complete medium) at 37°C in a humidified atmosphere containing 5% CO₂.

The siRNAs targeting the WIPI2 sequence (KD, TACGGAAGATGTGTGCATT) and the non-silencing sequence (NC, TTCTCCGAACGTGTCACGT) were purchased from Guangzhou RiboBio Co., Ltd. Transfection with siRNAs was completed using riboFECT CP transfection reagent (Guangzhou RiboBio Co., Ltd) according to the manufacturer's instructions.

Human cell lines (1×10⁵ cells in 0.2 ml/well) were seeded into 96-well plates and transfected with the siRNA and the non-silencing sequence. The cells were cultured at 37°C for 1–5 days. Then 100 µl (5 mg/ml) of MTT solution was added into each well, followed by incubation at 37°C for 4 h. Subsequently, the supernatant was removed, and 150 µl of dimethyl sulfoxide (DMSO) was added into each well. The plate was oscillated at room temperature for 30 min, and then the absorbance at a wavelength of 490 nm was determined by a multifunctional microplate reader (Tecan). The values were calculated after background subtraction. All the MTT experiments were repeated at least three times.

The cell proliferation was determined by the incorporation of 5-ethynyl-2′-deoxyuridine (EdU) into newly synthesized DNA strands, using a Click-iT EdU microplate assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. The HCC cell lines were resuspended and seeded into a 6-well plate and transfected with the siRNA and negative control, respectively. Two hours before cell collection, EdU was added to the cells at a final concentration of 10 µM. After 2 h, the incorporated EdU in DNA was coupled with Azide 488 dye, and subsequently the nuclei were stained with Hoechst 33342. The cells were observed by fluorescence microscope at ×100 magnification. The experiments were repeated at least three times.

Analysis of cell apoptosis was performed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's instructions. Following transfection for 24 h, the cells were collected and fixed with 40 g/l paraformaldehyde for 30 min at room temperature. The TdT enzyme solution was prepared and incubated with cells for 60 min at 37°C. Next, the cells were washed with PBS and incubated with streptavidin-TRITC for 30 min at 37°C. The cell nuclei were then stained with DAPI (dilution 1:1,000) at room temperature for 10 min. Finally, the cells were mounted in anti-fade mounting medium and were observed with a fluorescence microscope at ×100 magnification. The experiments were repeated at least three times.

Total RNA was extracted from HCC cell lines using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. RT-qPCR was performed as previously reported (14,15). Briefly, 1 µg of total RNA was reversely transcribed into cDNA using random primers and Primescript reverse transcriptase (Takara). RT-qPCR for the indicated genes was carried out using the SYBR Green qPCR kit (Takara Dalian) on a fluorescent temperature cycler (Applied Biosystems ABI^™ Vii7 Real Time PCR System; Thermo Fisher Scientific, Inc.). The following primers were used to detect the expression of WIPI2 (forward, 5′-CCATCGTCAGCCTTAAAGCAC-3′ and reverse, 5′-TCCAGGCATACTATCAGCCTC-3′) and GAPDH (forward, 5′-TGACTTCAACAGCGACACCCA-3′ and reverse, 5′-CACCCTGTTGCTGTAGCCAAA-3′). Briefly, after an initial denaturation step at 95°C for 5 min, amplifications were carried out with 45 cycles at a melting temperature of 95°C for 15 sec and an annealing temperature of 60°C for 1 min. GAPDH was selected as an endogenous control, and the relative gene expression was determined by the comparative Ct method. The experiment was repeated at least three times.

Cells transfected with the siRNA and control were harvested, and washed twice with cold PBS. Then the cells were stained with Annexin V-FITC and PI (eBioscience Inc.) and incubated for 15 min at room temperature, and then determined using a BD FACSCalibur Flow Cytometer (BD Biosciences) and analyzed using FlowJo software (v10.0; TreeStar). The experiment was repeated at least three times.

Cells transfected with siRNA were harvested and total proteins were extracted from tumor cell lines using RIPA buffer containing fresh protease and phosphatase inhibitors. The protein concentration was determined using the BCA assay (Pierce; Thermo Fisher Scientific, Inc.). Briefly, equal amounts of proteins (50 µg) were subjected to 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 3% BSA in 10 mM Tris-HCl (pH 7.4) containing 0.05% Tween-20 and incubated with a primary antibody at 4°C for 12 h. After washing with Tris-HCl buffer for three times, the membranes were incubated with a corresponding peroxidase-conjugated secondary antibody (Abcam). Immunoreactive bands were visualized using Super-Signal West Pico Chemiluminescent Substrate (Pierce; Thermo Fisher Scientific, Inc.). The densitometry of the protein bands was quantified by ImageJ software (1.8.0; National Institutes of Health). The experiment was repeated at least three times. The details of the primary antibodies used in the experiments are documented in Table I.

Total RNA was isolated from Hep3B cells transfected with si-WIPI2 (n=3) and Hep3B cells transfected with si-NC (n=3). RNA samples were analyzed by microarray expression profiling using the Affymetrix Human GeneChip PrimeView (Affymetrix) according to the manufacturer's instructions. Briefly, cDNA target preparation and in vitro transcription were conducted using the GeneChip 3′ IVT PLUS Kit (Affymetrix). Arrays were washed, stained and processed using the GeneChip Hybridization Wash and Stain Kit (Affymetrix), after which they were imaged using the Affymetrix GeneChip Scanner 3000 (Affymetrix) for subsequent generation of raw data. Genes significantly differentially expressed between the Hep3B/KD and Hep3B/NC cells were selected based on a threshold setting of fold change >1.3 and P<0.05. Functional pathway analysis was conducted using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis (16) and Gene Ontology (GO) analysis (17) according to the manufacturer's instructions.

Data are presented as the mean ± standard deviation (SD) of three independent experiments. All statistical analyses were performed using SPSS 18.0 software (SPSS Inc.). P<0.05 was considered statistically significant; P<0.01 was considered statistically very significant. Differences among categorical variables were analyzed using one-way ANOVA/SNK test or independent-sample Student's t-test. The immunoreactive scores for WIPI2 for tissue array were analyzed using non-parametric Mann-Whitney U, Kruskal-Wallis H and Wilcoxon tests.

In all 24 types of cancers included in the UACLAN platform, WIPI2 expression was higher in tumor tissues compared with normal tissues (Fig. 1A and B). The survival rate showed that high WIPI2 expression predicted a poor patient prognosis (Fig. 1C).

WIPI2 expression was then detected in HCC tissue array by IHC assay. The array consisted of 86 HCC tissue pairs, including tumor and corresponding adjacent normal tissues. Among these samples, 65 tissue samples were obtained from male patients, while 21 samples were collected from female patients, with a median age of 67 years (range, 26–88 years). Table II and Fig. 2A show that WIPI2 was highly enriched in HCC tumor tissue samples, evidenced by the fact that 66 tumor tissue samples showed a score of ≥2. However, adjacent normal tissue samples displayed significantly reduced WIPI2 expression compared with tumor tissues (P<0.001). The results also indicated that WIPI2 expression was markedly elevated in patients older than 60 years (Table II). In addition, WIPI2 expression was not associated with other factors. However, the statistical analysis indicated that WIPI2 was significantly higher in tumor tissues than that in the corresponding adjacent normal tissues (P<0.001) (Fig. 2B).

In the present study, siRNA targeting WIPI2 was employed to suppress WIPI2 expression in the HCC cell lines in order to verify its roles in the tumorigenesis of HCC in vitro. After transfection with siRNA, the expression of WIPI2 at the mRNA and protein levels were both decreased in the HCC Huh7 and Hep3B cell lines (Fig. 3A-D).

In order to determine the effect of WIPI2 on the cell proliferation of HCC cells, MTT and EdU assays were used after WIPI2 downregulation. MTT results showed that the proliferation of HCC cells was increased following WIPI2 depletion (si-WIPI2) (Fig. 4A and B). In addition, the EdU assay indicated that cell proliferation was significantly decreased after WIPI2 ablation. The number of EdU-positive cells was significantly reduced upon WIPI2 knockdown (KD) in contrast to the negative control (NC) cells (Fig. 4C-F).

To explore the role of WIPI2 in regulating the growth of HCC cells, we assessed the changes in apoptosis after WIPI2 depletion by TUNEL assay and FCM. The results of TUNEL staining revealed that the percentage of TUNEL-positive cells was significantly increased after WIPI2 knockdown (KD) compared with the negative control (NC) cells (Fig. 5A-D). Consistently, the FCM results indicated that the apoptotic rate of HCC cells was significantly increased after WIPI2 knockdown both in the Huh7 and Hep3B cell lines (Fig. 5E and F). These results showed that WIPI2 ablation could promote the apoptosis of HCC cells.

To uncover the mechanism underlying the WIPI2-induced proliferation and apoptosis, we conducted a microarray analysis to identify differentially expressed genes upon WIPI2 depletion using Hep3B/KD and Hep3B/NC cell lines (Fig. 6). The results showed that a total of 63 genes were upregulated, and 199 genes were downregulated after WIPI2 depletion (P<0.05 and fold change >1.3), and all the details of the 216 gene symbols and the fold changes are shown in Table SI. Supervised analysis was conducted following the KEGG and GO technical route (Fig. 7A and B). We found that the AMPK signaling pathway was markedly modified after WIPI2 depletion.

In regards to the downstream factors of the AMPK pathway, AKT is associated with cell proliferation and apoptosis. Thus, we hypothesized that AKT may be involved in the inhibition of HCC cells. Further verification was conducted by western blot analysis. Phosphorylation of AKT (p-AKT) was significantly decreased, while total AKT remained unchanged after WIPI depletion (Fig. 8A and C) in the Hep3B cells. The results also revealed that the ratios of p-AMPK/AMPK and p-AKT/AKT were reduced after WIPI2 knockdown (Fig. 8E). Cyclin D1 is associated with cell proliferation; thus, we detected the expression of cyclin D1 and found that it was significantly decreased after WIPI2 depletion (Fig 8A and C) in the Hep3B cells. Since TUNEL assay indicated that the apoptosis was increased, we also determined the levels of Bcl2, Bax, caspase-3 and cleaved-caspase 3, key biomarkers of apoptosis (Fig. 8B and D). The results indicated that the Bcl2/Bax ratio was decreased (Fig. 8F) and cleaved-caspase 3 was also evaluated and caspase-3 was not altered (Fig. 8B and D) after WIPI2 knockdown. Similar results were observed in the Huh7 cells (Fig. 8G-L). Taken together, our data revealed that WIPI2 regulated the growth of HCC cells mainly through the AMPK pathway.