Clinical samples
All clinical samples were from the Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital, Wuhan University. This study passed the ethical approval [kelun 2022181K] of Zhongnan Hospital of Wuhan University and obtained the informed consent of patients.
Cell culture
Five human liver cancer cell lines (HepG2, Huh-7, HCCLM3, Hep3B, Li-7), the normal liver cell line THLE-2 and HEK-293T cell line were obtained from National Collection of Authenticated Cell Culture (Shanghai, China). All cell lines were cultured in Dulbecco's modified eagle medium (Gibco) containing 10% fetal bovine serum (Gibco) at 5% CO2 at 37 °C and they were recently authenticated and tested for mycoplasma contamination.
RNA extraction and Quantitative real-time polymerase chain reaction (qRT-PCR)
RNA from the tissues and cells were extracted using TRIzol reagent (Invitrogen 15596026, USA), and qualified by NanoDrop ND2000 (Thermo Scientific). HiScript® II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme R223-01, China) was used to reverse transcriptase RNA into cDNA. ChamQ Universal SYBR qPCR Master Mix(Vazyme Q712-02, China) was used to perform qPCR on CFX96TM Real-Time system (Bio–Rad, USA). Relative gene expression was calculated by the cycle threshold (Ct) method (2−ΔΔCt) and was internally referenced by β-actin. All assays were repeated three times. The primer sequences are detailed in Supplementary Table 1.
Western blotting
Tissues or cells were lysated with RIPA (Biosharp BL651A, China) to extract proteins and quantified with BCA Protein Assay Kit (Thermo Scientific 23227, USA). Equal amounts of sample proteins were separated by electrophoresis on a 10% sodium lauryl sulfate polyacrylamide gel and then transferred to a polyvinylidene fluoride (PVDF) membrane. After 1 h of blocking with 5% skim milk, the membrane was incubated with the primary antibody overnight at 4 °C and then with the secondary antibody for 1 h at room temperature. Finally, the positive protein bands were visualized with Tanon-5200 imager(Shanghai, China) by ECL chemiluminescence reagent (Biosharp BL520B, China). The antibodies are listed in Supplementary Table 2.
RNA interference
Small interfering RNA transfections were achieved by GenMute (SignaGen, Maryland, USA) in accordance with the manufacturer’s protocol. SiRNAs were synthesized by GENECREATE (Wuhan, China). The primer sequences of si-RNA are detailed in Supplementary Table 3.
Plasmid and lentiviral constructions, and cellular transfection
The cloned full-length gene of interest sequence was inserted into the expression vector p-CMV (American Invitrogen) for overexpression. The transcripts of EEF1D was NM_032378.7, the transcripts of SNHG6 was NR_002599.2, the transcripts of COPS5 was NM_006837.3. The sh-SNHG6 recombinant plasmids, which was designed by GeneCopoeia, were incorporated into 293T cells using lentiviral vectors and the PEI(Biosharp PR40001, China) reagent for viral supernatant solution preparation. Lentiviruses were transfected into Huh7 cells, and following 24 hours of incorporation, puromycin was used for cell selection and establishment of stable cell lines.
EdU cell proliferation experiments
The cells were incubated with 10 µM EdU(Beyotime C0078S, China) for 2 h, fixed with a paraformaldehyde at room temperature for 15 min, then the cells were permeabilized with 0.5% TritonX-100 for 10 mins and then incubated with click reaction solution for 30 mins. Next the cells were counterstained with Hoechst 33342 and observed under the fluorescence microscope. After staining, the nucleus is blue and the red is EdU-positive cells, and the cell proliferation ratio is calculated according to the percentage of pink cells after synthesis.
CCK8 cell proliferation experiments
We plate 5000 cells/well in 96-well plates, and then add introduction of 10μl CCK8 solution (Biosharp BS350B, China) after 6 h. After 1 h incubation at 37 °C, absorbance was determined at 450nm via a microplate reader.
Flow cytometry
The cells were harvested and stained with the Cell Cycle Staining Kit (Multi Sciences CCS012, China) according to the manufacturer’s instructions. The stained cells were acquired by flow cytometry (FACSCalibur, BD Biosciences, USA), and the percentage of the cell cycle distribution were analyzed.
RNA pulldown, silver staining and mass spectrometry
RNA pulldown kits (Thermo Scientific 20164, USA) and Celerity Silver Staining Kit (Beyotime Biotechnology, Shanghai, China) were used to perform according to the manufacturer’s instructions. Later, gels were used for MS-based analysis.
Co-Immunoprecipitation(CO-IP)
Collect cells in a 10 cm dish and use 1 ml of IP buffer (20 mM Tris-HCl, pH 7.4, 150 mM sodium chloride, 1 mM EDTA pH 8, 1% NP-40, 1× protease, and phosphatase Inhibitor cocktail(meilunbio MB2678-1, China) lyse at 4 °C for 30 min, take the lysate supernatant and add protein A/G magnetic beads (Thermo Scientific 88802, USA) and antibody, then incubate at 4 °C for 6 h. Finally, the interaction protein was pulled down by the magnetic beads. That magnetic beads were resuspended in SDS-loading buffer and then heated up to 96 °C for 10 min to perform Western blottingting experiment. The antibodies are listed in Supplementary Table 2. The procedures of silver staining and mass spectrometry were the same as mentioned above.
RNA immunoprecipitation and DNA Electrophoresis
Cells were lysed in RIP buffer (150 mM KCL, 25 mM Tris pH 7.4, 5 mM EDTA, 0.5 mM DTT, 0.5% NP-40, 100 U/ml RNase inhibitors) for 30 min at 4 °C. After centrifugation to obtain the supernatant lysate, 10% of the total RNA was extracted by trizol method as the Input group. Then the remaining lysate was divided into two groups, protein A/G magnetic beads and antibody was added to the reaction system, incubated at 4°C for 6 hours, and the total RNA was extracted by trizol method after washing. The antibodies are listed in Supplementary Table 2. Levels of RNA of interest are analyzed by qPCR. The DNA sample was added to a 1% agarose gel containing 1/10000 Ethidium Bromide together with loading buffer, electrophoresis with constant pressure at 120 V in TAE buffer, and finally observed under UV light.
In vivo experiments
4-week-old male BALB/c nude mice were purchased from the Vettonlihua Animal Center (Wuhan, China) and housed in a specific pathogen-free facility with appropriate ambient temperature and humidity. All mice were divided randomly into the same two groups for subsequent experiments. Stable transfected Huh7 cells knocked down SNHG6 were subcutaneously inoculated according to 1×10^6 cells each to establish a mouse subcutaneous tumor model. We measured the length and width of the tumor with a vernier caliper every 3 days, and calculated the volume (V = 0.52 L × W × W, where V is the volume, L is the length, and W is the width)[18]. After 21 days, the mice were euthanized, their tumor tissue was taken to measure weight and volume, and the relevant proteins were immunohistochemically detected. All animal experiments passed ethical approval [ZN2022163] by the Laboratory Animal Ethics Committee, Zhongnan Hospital of Wuhan University (Hubei province, China), and were in accordance with the Guide for the Care and Use of Laboratory Animals.
Immunohistochemistry (IHC)
The tissue was fixed in paraformaldehyde, embedded in paraffin, and cut into 4 μm thick sections. IHC was performed using the UltraSensitiveTM S-P kit (Maixin, Fuzhou, China) according to the manufacturer's protocol. Then we analyzed the percentage of positive cells. The antibodies are displayed in Supplementary Table 2.
Kaplan–Meier plotter analysis
The TCGA database was selected to analyze the expression of the gene of interest in liver cancer by using the GEPIA (http://gepia.cancer-pku.cn/) online website. According to the median expression values of SNHG6 and COPS5, the patients were divided into two groups: average high expression and average low expression, and the cutoff value of EEF1D was selected as 50%. Subsequently the OS was calculated using Kaplan-Meier Plotter (http://www.kmplot.com).
Statistical analysis
GraphPad Prism 8.0 software and SPSS 25.0 were used for statistical analysis. All data were expressed as the mean ± standard deviation (SD), and analyzed using the Student’s t-test. Lastly, two‐sided *p < 0.05, **p < 0.01, or ***p < 0.001 were considered significant.