HCC tissues (n=22) and adjacent normal tissues (n=22) were obtained from HCC patients with a mean age of 58 years (range, 32–76 years) between September 2015 and November 2017 at The Affiliated Hospital of Xuzhou Medical University (Xuzhou, China); all tissue specimens were maintained at −80°C. The patients had not received any other anticancer treatment before surgery. The study protocol was approved by the Ethics Committee of the Affiliated Hospital of Xuzhou Medical University and written informed consent was obtained from all patients.

HCC cell lines (HCCLM6, Hep3B and Huh-7) and a human normal liver cell line (THLE-3) were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. All cells were cultured in DMEM (Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% FBS and 1% penicillin/streptomycin stock solution (Sigma-Aldrich; Merck KGaA). The cell lines were maintained in a humidified atmosphere containing 5% CO₂ at 37°C. Oxygen concentrations were maintained at 1% to induce hypoxia and hypoxic conditions were established using a humidified variable aerobic workstation with 5% CO₂ at 37°C.

The miR-338 mimic and negative control (NC) mimic, and the miR-338 inhibitor and NC inhibitor, were used to overexpress and inhibit miR-338, respectively. Short hairpin RNA (shRNA/sh) targeting ST8SIA6-AS1 (sh-ST8SIA6-AS1#1 and sh-ST8SIA6-AS1#2) and the NC (sh-NC) were used to knock down the expression levels of ST8SIA6-AS1. Full length methylphosphate capping enzyme (MEPCE) was subcloned into the pcDNA3.1 vector to overexpress MEPCE; an empty pcDNA3.1 vector was used as the control. All the plasmids were purchased from Shanghai GenePharma Co., Ltd. and transfected into Hep3B or Huh-7 cells using Lipofectamine^® 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

Total RNA was extracted from HCC tissues or cell lines using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). An miRNeasy Mini kit (Qiagen GmbH) and the TaqMan^® MicroRNA Reverse Transcription kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) were used to isolate miRNAs and reverse transcribe miRNA into cDNA, respectively. For the quantification of mRNAs, a Prime Script™ RT reagent kit (Takara Bio, Inc.) was used for RT. qPCR was subsequently performed using a TaqMan^® Universal PCR Master mix II (Applied Biosystems; Thermo Fisher Scientific, Inc.). The relative expression levels of RNAs (normalized to GAPDH or U6) were quantified using the 2^−ΔΔCq method.

The distribution of ST8SIA6-AS1 in the cytoplasm or nucleus of HCC cells was determined using a PARIS kit (Thermo Fisher Scientific, Inc.). Briefly, Hep3B or Huh-7 cells were harvested and lysed on ice, and the supernatant was collected following centrifugation. The extracted RNAs were analyzed using RT-qPCR, and GAPDH and U6 were used as the cytoplasmic and nucleic controls, respectively.

A total of 8×10⁴ transfected Hep3B or Huh-7 cells/ml were cultured in RPMI-1640 medium, supplemented with 10% FBS, until 100% confluence. A sterile pipette tip was used to generate single-line scratches and the cell monolayer was subsequently washed twice with PBS. The wound closure was imaged at 0 and 24 h using a light microscope (magnification, ×200).

A total of 1×10⁵ Hep3B or Huh-7 cells suspended in 500 µl serum-free medium were plated into the upper champers of Transwell plates (Corning Inc.). The upper chambers were precoated with Matrigel. Medium supplemented with 5% FBS was plated into the lower chambers. Following incubation for 24 h, the invasive Hep3B or Huh-7 cells were fixed with methanol and stained with 0.1% crystal violet for 20 min at room temperature. The invasive cells were visualized under a light microscope (magnification, ×200; Olympus Corporation).

Full-length MEPCE 3′UTR and ST8SIA6-AS1 were amplified and cloned into pmirGLO vectors (Promega Corp.) to construct the pmirGLO-MEPCE-WT reporter gene plasmid (5′-CAGCAAGGCUGGCUGGUGCUGGA-3′) and pmirGLO-ST8SIA6-AS1-WT reporter gene plasmid (5′-GAACAGAAUCGCUAAUAUGCUGG-3′), respectively. Mutations within the miR-338 binding site were created using the QuikChange II Site-Directed Mutagenesis kit (Stratagene; Agilent Technologies, Inc.), to generate the pmirGLO-MEPCE-MUT (5′-CAGCAAGGCUGGCUGGCAACAAC-3′) reporter gene plasmid and pmirGLO-ST8SIA6-AS1-MUT (5′-GAACAGAAUCGCUAAACCAACAA-3′) reporter gene plasmid. Hep3B and Huh-7 cells were co-transfected with miR-338 mimics or NC mimics and ST8SIA6-AS1-WT (or MEPCE-WT) or ST8SIA6-AS1- Mut (or MEPCE-Mut) using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). Following 48 h of transfection, the relative luciferase activity was analyzed using a Dual-Luciferase Reporter assay system. Firefly luciferase activity was normalized to Renilla luciferase activity (Promega Corp.).

A RIP assay was performed using the Magna RNA-binding protein immunoprecipitation kit (EMD Millipore). Briefly, the cell lysate (Hep3B or Huh-7 cells) was incubated in RIP buffer containing magnetic beads conjugated to human anti-Ago2 antibody. IgG served as the endogenous control. The immunoprecipitated RNA was isolated, purified and analyzed using RT-qPCR.

SPSS 20.0 software (IBM Corp.) was used for statistical analysis and data are presented as the mean ± SD of three independent repeats. Clinicopathological characteristics were evaluated using the χ² test. Comparison between HCC and adjacent normal tissues was performed using a paired Student's t-test, while comparisons between the experimental and control groups was performed using an unpaired Student's t-test. Comparison among multiple groups was performed using one-way ANOVA, followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

According to the TCGA database (https://tcga-data.nci.nih.gov/tcga/), ST8SIA6-AS1 expression levels were significantly upregulated in liver hepatocellular carcinoma (LIHC) tissues (n=198) compared with normal tissues (n=62; Fig. 1A). Additionally, patients with HCC with higher levels of ST8SIA6-AS1 had a lower survival rate compared with patients with low expression levels of ST8SIA6-AS1 (Fig. 1B). Similarly, RT-qPCR analysis revealed that expression levels of ST8SIA6-AS1 were significantly upregulated in HCC tissues (n=22) or cell lines (HCCLM6, Hep3B and Huh-7) compared with the adjacent normal tissues (n=22) or the human normal liver cell line (THLE-3; Fig. 1C and D). Moreover, the clinical data indicated that the ST8SIA6-AS1 expression levels were positively correlated with the histological grade, TNM stage and vein invasion (Table I). Altogether, these results suggest that ST8SIA6-AS1 expression levels may be upregulated in HCC tissues and cell lines, and that the upregulation of ST8SIA6-AS1 expression levels may predict the poor prognosis of HCC.

To investigate the biological role of ST8SIA6-AS1 in HCC, loss-of-function assays were performed. The expression levels of ST8SIA6-AS1 were markedly downregulated following the transfection of sh-ST8SIA6-AS1#1 or sh-ST8SIA6-AS1#2 into Hep3B and Huh-7 cells (Fig. 2A). Subsequently, a wound healing assay demonstrated that the migratory ability of HCC cells was significantly increased following hypoxia induction compared with the control group, while the migratory rate was significantly hindered following the transfection with sh-ST8SIA6-AS1#1 or sh-ST8SIA6-AS1#2 (Fig. 2B). Similarly, the Transwell Matrigel assay revealed that the invasive capacity of HCC cells was enhanced by hypoxia treatment, while the invasive capacity was significantly inhibited by the knockdown of ST8SIA6-AS1 (Fig. 2C). Together, these results indicated that the genetic knockdown of ST8SIA6-AS1 may inhibit the migration and invasion of hypoxia-induced HCC cells.

According to the results obtained from nuclear-cytoplasmic fractionation, the majority of ST8SIA6-AS1 was observed to be distributed in the cytoplasm of Hep3B cells (Fig. 3A). Thus, it was hypothesized that ST8SIA6-AS1 may form a ceRNA network in HCC. miR-338 was predicted to contain a binding site on ST8SIA6-AS1 using starBase (http://starbase.sysu.edu.cn; Fig. 3B). Thus, the expression levels of miR-338 were overexpressed by transfecting miR-338 mimics into Hep3B cells (Fig. 3C). A dual-luciferase reporter assay revealed that miR-338 overexpression induced a marked decrease in pmirGLO-ST8SIA6-AS1-WT relative luciferase activity in Hep3B cells, whereas no differences were observed in pmirGLO-ST8SIA6-AS1-Mut-transfected cells (Fig. 3D). Subsequently, a RIP assay revealed that both ST8SIA6-AS1 and miR-338 expression levels were enriched in anti-Ago2 cells, suggesting that ST8SIA6-AS1 and miR-338 may co-exist in RNA-induced silencing complexes (RISC; Fig. 3E). In addition, ST8SIA6-AS1 knockdown significantly upregulated miR-338 expression levels, while miR-338 overexpression significantly downregulated ST8SIA6-AS1 expression levels (Fig. 3F and G). Furthermore, miR-338 expression levels were revealed to be downregulated in HCC tissues or cells compared with the adjacent normal tissues or THLE-3 cells (Fig. 3H and I). Moreover, the expression levels of ST8SIA6-AS1 and miR-338 in HCC tissues were negatively correlated with other (Fig. 3J). In conclusion, these findings suggest that ST8SIA6-AS1 directly interacts with miR-338 to suppress its expression levels in HCC.

To determine whether miR-338 is an important effector for ST8SIA6-AS1-mediated carcinogenesis and the development of hypoxia-treated HCC cells, an miR-338 inhibitor was transfected into sh-ST8SIA6-AS1-expressing Hep3B cells. The upregulation of miR-338 mediated by ST8SIA6-AS1 silencing was reversed following the transfection of the miR-338 inhibitor (Fig. 4A). Moreover, the wound healing and Transwell assays indicated that the inhibitory effects caused by the genetic knockdown of ST8SIA6-AS1 on cell migration and invasion were abolished following miR-338 inhibition (Fig. 4B and C). Taken together, these findings suggest that miR-338 may partially counteract the effects of ST8SIA6-AS1 in regards to hypoxia-induced HCC progression.

According to the prediction using the TargetScan website (http://www.targetscan.org), MEPCE was discovered to contain a complementary binding site for miR-338 (Fig. 5A). The RIP assay revealed that both miR-338 and MEPCE were abundantly expressed in the Ago2 group, but not in the IgG group (Fig. 5B). A dual-luciferase reporter assay also discovered that the overexpression of miR-338 decreased the relative luciferase activity of the MEPCE-WT reporter in Hep3B cells, while the relative luciferase activity in cells transfected with the MEPCE-Mut reporter remained the same (Fig. 5C). Moreover, MEPCE mRNA expression levels were suppressed following the transfection of miR-338 mimics into Hep3B cells (Fig. 5D). RT-qPCR analysis demonstrated that the expression levels of MEPCE were significantly upregulated in HCC tissues and cells compared with normal tissues and cells (Fig. 5E and F). In addition, a negative correlation was identified between the expression levels of MEPCE and miR-338 in HCC tissues, whereas a positive correlation was revealed between the expression levels of MEPCE and ST8SIA6-AS1 (Fig. 5G). Altogether, these findings indicate that MEPCE expression levels may be upregulated in HCC and may directly interact with miR-338.

To validate that MEPCE mediates the oncogenic role of ST8SIA6-AS1, rescue assays were performed. The downregulation of MEPCE mRNA expression levels induced by ST8SIA6-AS1 inhibition were reversed following the transfection with pcDNA3.1/MEPCE (Fig. 6A). Additionally, both wound healing and Transwell Matrigel assays revealed that the suppressive effect of ST8SIA6-AS1 knockdown on cell migration and invasion was reversed following MEPCE overexpression in hypoxia-treated Hep3B cells (Fig. 6B and C). These findings suggest that ST8SIA6-AS1 may affect the migration and invasion of HCC cells under hypoxia by targeting MEPCE.