Sixty-eight HCC tissues and matched tumor-adjacent tissues were obtained from the The First Affiliated Hospital of Xi'an Medical University. Tissue specimens were conserved in 10% formalin for further investigation. No patients received immunotherapy, radiotherapy or chemotherapy before surgery. Tumor staging was based on the seventh Union for International Cancer Control/American Joint Committee on Cancer (UICC/AJCC) staging system (11). All samples were used after obtaining informed consent. The Ethics Committee of The First Affiliated Hospital of Xi'an Medical University approved the protocols according to the Declaration of Helsinki.

The human immortalized normal hepatocyte cell line (LO2) and human HCC cell lines including Hep3B, MHCC97L, MHCC97H and HCCLM3 were obtained from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). All cell lines were cultivated in Dulbeccos minimum essential medium (DMEM; Thermo Fisher Scientific, Waltham, MA, USA) using 10% fetal calf serum (FCS; Gibco, Grand Island, NY, USA) at 37°C in a humidified atmosphere of 5% CO₂.

Precursor miR-134, miR-205 and miR-340, and miR-134 inhibitor as well as corresponding negative control vectors were purchased from GeneCopoeia (Guangzhou, China). ICT1 shRNA, non-targeting (NT) shRNA, ICT1 overexpression vector and empty vector (EV) were synthesized and obtained from Shanghai Genechem Co., Ltd. (Shanghai, China). Vectors were transferred into cells using Lipofectamine 2000 (Thermo Fisher Scientific) in accordance with the manufacturers protocols.

The HCC tissues that were previously formalin-fixed and paraffin-embedded were sliced into 4 µm sections, and underwent deparaffination and then rehydration. Antigen retrieval, suppression of endogenous peroxidase activity and 10% skim milk blocking were performed before primary antibody incubation. ICT1 (AP20382b; Abgent, Inc., San Diego, CA, USA) and Ki-67 primary antibody (ab15580; Abcam, Cambridge, MA, USA) was used for incubation overnight at 4°C. The slides were subsequently incubated with peroxidase conjugated secondary antibody (ZSGB Bio, Beijing, China) for 90 min, and a peroxidase-labeled polymer, DAB solution was used for signal development for 5 min. The sections were counterstained with hematoxylin followed by dehydrating and mounting. Staining intensity was scored as no staining, 0, weak staining, 1, moderate staining, 2, and strong staining, 3. Staining quantity was graded as <25%, 1, 25–75%, 2, and >75%, 3. IHC score was manually confirmed by two independent experienced pathologists using the formula: IHC score = staining intensity × staining quantity. IHC score ≥3 was considered as positive expression of ICT1.

RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA, USA) following the manufacturers protocols. The first strand cDNA was compounded using a Tianscript RT kit (Tiangen Biotech, Co., Ltd., Beijing, China). PCR amplification for ICT1 mRNA was performed with the SYBR Premix Ex Taq™ kit (Takara Bio, Shiga, Japan) in ABI 7300 system (Applied Biosystems, Foster City, CA, USA). GAPDH was employed as a reference gene to normalize the expression of ICT1 mRNA. The primers used for ICT1 and GAPDH were purchased from Sangon Biotech Co., Ltd. (Shanghai, China).

For cell proliferation, colony formation assay was performed. HCC cells transfected with corresponding vectors were seeded in 6-well plates and maintained in cell incubators for 14 days. The formed cell colonies were stained with crystal violet solution. The number of cell colonies was counted to represent the cell proliferation ability of HCC cells.

For cell cycle assay, HCC cells were collected 72 h after transfection. These cells were fixed with 80% ethanol overnight, and then were stained with propidium iodide (50 µg/ml; BD Biosciences, Franklin Lakes, NJ, USA) at room temperature for 30 min. The percentage of cells in each cell cycle was measured with FACSCalibur system (BD Biosciences). For apoptosis assay, HCC cells after transfection were subjected to an apoptosis assay. The percentage of apoptotic HCC cells were measured using Annexin V/propidium iodide kit (BD Biosciences, San Diego, CA, USA) based on the protocol provided by manufactures.

For tumor growth studies, nude mice were injected subcutaneously with 1×10⁶ HepG2 cells transfected with ICT1 shRNA or NT shRNA. Tumor sizes were measured every 4 days after subcutaneous injection. Three weeks later, the mice were sacrificed by cervical dislocation under anesthesia, and tumors were removed for the volume measurement. The protocols for animal experiments were approved by the Ethics Committee of The First Affiliated Hospital of Xi'an Medical University.

Wild-type (wt) or mutant (mt) 3′-UTR of ICT1 was amplified and cloned into pmiR-RB-REPORT™ Luciferase. Then, the 3′-UTR of ICT1 and corresponding miRNA vectors were co-transfected into HepG2 cells, respectively. Forty-eight hours after the co-transfection, the cells were lysed and detected using a Dual-Luciferase^® reporter assay kit (Promega, Madison, WI, USA) based on the manufacturers protocols.

Total proteins were collected with RIPA lysis buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), and 40 µg protein were subjected to 4–20% SDS gel electrophoresis (Sigma-Aldrich) and were then transferred to PVDF membranes (Roche, Indianapolis, IN, USA). Then, 5% milk blocked membranes were incubated with ICT1 (Abgent), CDK1 (ab18; Abcam), cyclin B1 (#12231; Cell Signaling Technology, Beverly, MA, USA), Bax (#5023; Cell Signaling Technology) or Bcl-2 (#15071; Cell Signaling Technology) primary antibody and subsequently incubated with matched secondary antibodies (Cell Signaling Technology). Then, signals for each protein expression were detected with the Bio-Rad Gel imaging system (Bio-Rad Laboratories, Hercules, CA, USA). GAPDH (G8140; US Biological, Swampscott, MA, USA) was used as a loading control.

Data are presented as mean ± SD and analyzed by GraphPad Prism 5 software (GraphPad Software, Inc., San Diego, CA, USA). Chi-squared test was employed to explore the association between two variables. The Student's t-test and analysis of variance (ANOVA) were carried out to analyze continuous variables. Survival curves were constructed and differences among groups were calculated using the Kaplan-Meier method and log-rank test. The value of P<0.05 was considered to have statistical significance.

IHC staining was performed to detect the expression of ICT1 in 68 pairs of HCC and matched tumor-adjacent tissues. Forty-seven of 68 (69.12%) HCC tissues showed positive expression of ICT1, while ICT1 signal was observed in only 27 of 68 (39.71%) tumor-adjacent tissues (P<0.05; Fig. 1A). Our data indicated that the levels of ICT1 in HCC tissues were significantly higher than those in matched non-cancerous tissues (P<0.05; Fig. 1B). TCGA data from R2: Genomics Analysis and Visualization Platform (http://r2.amc.nl) showed that the expression of ICT1 is significantly upregulated in HCC tissues compared to normal liver tissues (P<0.05; Fig. 1C), which is consistent with our results. In addition, the expression of ICT1 in HCC cell lines (SMMC-7721, Hep3B, HepG2 and Huh7) were notably upregulated compared to LO2 cells (P<0.05, respectively; Fig. 1D). Thus, the expression of ICT1 is restrained in HCC.

Next, we investigated the clinical significance of ICT1 in HCC patients. Sixty-eight HCC patients were divided into ICT1 positive expression group (IHC score ≥3) and ICT1 negative expression group (IHC score <3). Clinical association analysis found that the ICT1 positive expression was correlated with larger tumor size and advanced TNM tumor stage (P<0.05, respectively; Table I). Notably, Kaplan-Meier plots indicated that ICT1-positive expression in HCC patients showed a significant reduced overall survival and recurrence-free survival compared to ICT1 negative expression cases (P<0.05, respectively; Fig. 2). These results suggest that ICT1 may act as a promising prognostic marker for HCC patients.

Next, we investigated the biological function of ICT1 in HCC cells. HepG2 cells showed the highest level of ICT1, while Hep3B cells showed the lowest level of ICT1. Thus, HepG2 and Hep3B were used for loss- and gain-of-function experiments, respectively. Loss-of-function experiments were performed in HepG2 cells after ICT1 knockdown (P<0.05; Fig. 3A). Colony formation assays revealed that ICT1 knockdown prevented proliferation of HepG2 cells (P<0.05; Fig. 3B). In addition, ICT1 silencing resulted in cell cycle arrest at G2/M phase and increased apoptosis in HepG2 cells (P<0.05, respectively; Fig. 3C and D). Then, ICT1 overexpression was confirmed by immunoblotting in Hep3B cells (P<0.05; Fig. 4A). Further experiments disclosed that ICT1 restoration contributed to proliferation and cell cycle progression, and reduced apoptosis of Hep3B cells (P<0.05, respectively; Fig. 4B-D).

HepG2 cells that were transfected with NT shRNA or ICT1 shRNA were implanted into nude mice via subcutaneous injection. Tumor growth curves showed that ICT1 knockdown reduced the volume of subcutaneous tumor nodules in nude mice with HCC (P<0.05; Fig. 5A). Furthermore, Ki-67 staining was performed to detect the proliferative index of HCC in vivo. The percentage of Ki-67 positive cancer cells in ICT1 knockdown group was notably reduced compared to control group (P<0.05; Fig. 5B). These results indicate that ICT1 inhibits the progression of HCC via regulating proliferation, cell cycle progression and apoptosis.

Previous studies report that ICT1 regulate cell growth of breast, CRC, prostate and lung cancer by modulating cell cycle and apoptosis-associated proteins including CDK1, cyclin B1, Bax and Bcl-2 (7–10). Thus, western blot analysis was performed to detect the levels of CDK1, cyclin B1, Bax and Bcl-2 after modulating ICT1 expression in HCC cells. ICT1 knockdown reduced the levels of CDK1, cyclin B1 and Bcl-2 and increased Bax expression in HepG2 cells (Fig. 6). In turn, ICT1 overexpression led to upregulation of CDK1, cyclin B1 and Bcl-2 and suppression of Bax in Hep3B cells. Thus, ICT1 regulates the levels of CDK1, cyclin B1, Bax and Bcl-2 in HCC cells.

The mechanism involved in ICT1 overexpresion in HCC has rarely been investigated. Increasing evidence indicates that miRNAs play a critical role in regulating the expression of oncogenes and tumor suppressors in human cancers (12–14). Thus, we used two publicly available databases (TargetScan and miRanda) to search for the potential miRNAs involved in regulation of ICT1 in HCC. Five miRNAs including miR-134-5p, miR-340-5p, miR-205-5p, miR-543 and miR-758-3p were recognized as candidates. miR-758-3p is rarely investigated and miR-543 is reported to be overexpressed in HCC (15). Thus, we checked the regulatory effects of miR-134, miR-340, miR-205 on ICT1 mRNA expression. Our data indicated that only miR-134 overexpression significantly reduced the level of ICT1 mRNA in HepG2 cells (P<0.05; Fig. 7A). As suggested by Fig. 7B, the complementary sequence of miR-134 was found in the 3′-UTR of ICT1 mRNA. Notably, luciferase reporter assays demonstrated that miR-134 overexpression reduced the luciferase activity of wt but not mt 3′-UTR of ICT1 (P<0.05; Fig. 7C). Next, we found that miR-134 inversely regulated the levels of ICT1 protein in HepG2 and Hep3B cells (Fig. 7D). Hence, these results disclose that miR-134 inversely regulates ICT1 abundance in HCC cells.