Visualization and processing of data

The Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov/) databases [17] was utilized to conduct an analysis of the expression levels of YY1 and DEPDC1 in all normal and HCC tissues, The genes that exhibited differential expression were obtained from TCGA. RNA sequencing profiles from TCGA were downloaded for TCGA-LIHC clinical observations. To conduct profiling RNA sequencing data, the software GraphPad 9.0 was utilized.

Clinical specimens

Between July 2018 and December 2022, a cohort of 32 HCC tissues and their corresponding nontumor tissues were procured from the Affiliated Hospital of Guizhou Medical University. Prior to surgery, none of the patients underwent chemotherapy or radiotherapy. This study was approved by the hospital ethics committee, and prior to participation, informed consent was obtained from each patient.

Cell culture and antibodies

The Shanghai Cell Bank (Shanghai, China) provided the Huh7 and Hep3B and cell line THLE-3 derived from a normal liver. The cells were subjected to incubation at 37 °C by a humidified incubator containing 5% CO2, and cell lines were confirmed to be devoid of mycoplasma contamination.

In this study, the primary antibodies employed were rabbit antihuman DEPDC1 (dilution 1:1000; cat. no. PA5-34864; Thermo Fisher Scientific, Inc); and mouse anti-HNRNPF (dilution 1:1000; cat. no. 67701-1-Ig; ProteinTech Group, Inc); mouse anti-lgG (dilution 1:1000; cat. no. #5946; CST); rabbit anti-N-cadherin (dilution 1:1000; cat. no. ab202030; Abcam); rabbit anti-Vimentin (dilution 1:1000; cat. no. ab217673; Abcam); mouse antihuman GAPDH (dilution 1:5000; cat. no. #97166; CST); rabbit anti-Ago2 (dilution 1:1000; cat. no. 10686-1-AP; ProteinTech Group, Inc); Secondary antibodies included rabbit anti-goat IgG (H + L) (dilution 1:5000; cat. no. SA00001-4; ProteinTech Group, Inc) and goat anti-mouse (dilution 1:5000; cat. no.SA00001-1; ProteinTech Group, Inc).

Cell transfection

Genechem (Shanghai, China) designed and synthesized Lentivirus expressing shDUXAP8 (Lv-shDUXAP8) or shDEPDC1 (Lv-shDEPDC1), as well as Lentivirus overexpressing DUXAP8 (Lv-DUXAP8) and Lv-DEPDC1. Stable cell lines (Hep3B and Huh7) were generated via lentiviral transduction. Corves (Nanjing, China) designed and synthesized siRNA targeting HNRNPF, as well as plasmids for HNRNPF and DEPDC1 overexpression. The miR-7-5p mimic, inhibitor and negative controls were procured from RiboBio (Guangzhou, China). Using Lipofectamine 2000 reagent (Invitrogen) in accordance with the guidelines of manufacturer.

qRT-PCR

The StepOne Plus RT PCR System (Thermo, USA) was utilized to conduct qRT-PCR, employing SYBR Green (Vazyme, China) as the detection method after total RNA extraction using Trizol Reagent (Sigma, St. Louis, MO, USA). Utilizing the 2-ΔΔCT method by GAPDH and U6 as an internal parameter, we repeat three biological replicates of comparative qRT-PCR. This investigation used a variety of primers, as shown in Table 1.

Table 1 Sequences of PCR primers used in this studyWestern blot

The cells underwent lysis in RIPA lysis buffer, and the quantification of protein concentrations was performed through employment of the BCA method. The membranes were subjected to incubation with primary and secondary antibodies. The protein bands were detected through the utilization of Detection by enhanced chemiluminescence (Pharmacia, Piscataway, NJ, USA). All Western blots were repeated at least 3 times. The density of the immunoreactive bands was quantified using ImageJ software.

Cytosolic and nuclear fractionation

The Hep3B and Huh7 cells underwent two washes with PBS and were subsequently subjected to incubation with hypotonic buffer at 0 °C for a duration of 10 min. The cells underwent centrifugation at a force of 5000 × g for a duration of 5 min, resulting in the collection of the supernatants. The resuspension of pellets was carried out in a nucleus resuspension buffer comprising of 20 mM HEPES, pH 7.9, 400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF for a duration of 30 min. The samples underwent centrifugation at 12,000 × g for a duration of 10 min, following which the nuclear fraction was obtained by collecting the supernatant. During qRT-PCR analysis, GAPDH and U6 mRNA were employed as markers for the cytosolic and nuclear compartments, respectively.

Luciferase reporter assay

The pmirGLO, pmirGLO-WT, or pmirGLO-MUT (Promega, Madison, WI, USA) for DUXAP8 and DEPDC1 were co-transfected with miR-7-5p mimic or the negative control (mimic NC) into Hep3B and Huh7 cells by using Lipofectamine-mediated gene transfer. The Dual Luciferase Reporter Gene system (Promega) was employed to detect luciferase activities 48 h post-transfection, in accordance with the manufacturer's instructions.

RNA pull-down assay

To generate probe-coated beads, streptavidin magnetic beads (RioBio, China) were incubated with a biotin-labeled DUXAP8, the DEPDC1 3′UTR and the control probe (100 pmol) (RiboBio, China) at room temperature for 2 h. The lysates obtained from Hep3B and Huh7 cells were subjected to an overnight incubation with probe-coated beads at a temperature of 4 °C. Subsequently, the beads underwent a washing procedure, followed by the analysis of the extracted miRNA through qRT-PCR.

RNA immunoprecipitation

The Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) was utilized to incubate anti-HNRNPF, anti-IgG and anti-AGO2 by magnetic beads for a duration of 30 min at room temperature, following the manufacturer's instructions. Subsequently, the antibody-bead complexes were subjected to an overnight incubation at 4 °C with cell lysates obtained from Hep3B and Huh7 cells. The RNAs that were bound were subjected to elution, followed by reverse transcription to cDNA, and ultimately detected through qRT-PCR.

Chromatin immunoprecipitation assay

ChIP assay was performed using EZ-Magna ChIP A/G (17–10086, Upstate, Millipore, MA, USA) kit according to the manufacturer’s instructions. The cells were immobilized using a 1% formaldehyde solution, followed by quenching with glycine by the ChIP assay (Millipore, USA). The resulting crosslinked chromatin was subsequently fragmented to a size range of 200–1000 bp through sonication. The lysates were subjected to incubation with the respective antibodies and Magna ChIP Protein G Magnetic beads at a temperature of 4 °C for an extended period of time with mild agitation. The ChIP complexes were subjected to elution and subsequent analysis via qRT-PCR. The resulting data were computed utilizing the 2-ΔΔCT method.

CCK‑8 assay

In this experiment, cells were sown at a density of 2.0 × 103cells/well in 96-well plates. The assessment of cell proliferation was conducted in accordance with CCK-8 assay (Dojindo, Japan). For the subsequent five days, added to each well by a mixture of 10 µl of CCK-8 reagent and 90 µl of medium. Following a 2-h incubation period at 37 °C under light-excluded conditions, it quantified in each well using a microplate reader at 450 nm.

EdU assay

The Zeiss fluorescence photomicroscope (Carl Zeiss, Oberkochen, Germany) was utilized to obtain the results, which were subsequently quantified by counting a minimum of five randomly selected fields utilizing the EdU kit (Roche, Indianapolis, IN, USA).

Transwell assay

The Transwell chamber (Corning, NY, USA) was utilized to conduct migration assays. The upper chambers were utilized for the placement of cells. A medium comprising 10% serum was introduced into the lower chambers. Following a 24-h incubation period and subsequent fixation with 4% formaldehyde, the cells that had migrated to the lower surface were subjected to staining with 0.1% crystal violet. The cells were visualized and enumerated utilizing an Olympus microscope.

Statistical analysis

The experiments were conducted independently on a minimum of three occasions, and statistical analyses were carried out utilizing GraphPad Prism 9.0 (GraphPad, La Jolla, CA, USA). The statistical significance of the variances between experimental groups was assessed through the utilization of Student's t-tests, while differences among multiple groups were evaluated via one-way ANOVA. The presentation of quantitative data is in the form of mean ± SD (standard deviation) derived from triplicate measurements. A statistically significant difference was deemed to exist if *p < 0.05, **p < 0.01, or ***p < 0.001.