Cell culture
HepAD38 cells, expressing pgRNA under the control of the inducible tetracycline promoter, were cultured in D-MEM/Ham’s F-12 culture medium (Merck, Darmstadt, Germany) supplemented with 10% fetal bovine serum with or without 1 mg/mL of tetracycline to maintain the repression of HBV expression or HBV replication, respectively37, 38. Flag-HBx-overexpressed HepG2 cells were cultured in Dulbecco’s Modified Eagle Medium (Merck, Darmstadt, Germany) supplemented with 10% fetal bovine serum39.
Quantitative real-time PCR
The total RNA of cells was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Quantitative real-time PCR was performed with an MX3000P qPCR system (Stratagene, San Diego, CA, USA) using the Universal Probe Library System (Roche Diagnostics, Mannheim, Germany) or TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) Kit (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s protocols. The sequences of primers for human ELAVL1 and GAPDH were as follows: ELAVL1 (forward 5′-CTGATGAATTCTCCCTTGTTCC-3′, reverse 5′-GGCTTGGCAAATTACACTGAA-3′) and GAPDH (forward 5′-CTGACTTCAACAGCGACACC-3′, reverse 5′-TAGCCAAATTCGTTGTCATACC-3′). The sequences of primers for total HBV mRNA, which amplified the region from nucleotides (nt) 1803 to 1894 in HBV sequences covering all HBV transcripts (3.5-, 2.4-, 2.1-, and 0.7-kb mRNAs), and HBV 3.5-kb mRNA, which amplified the region from nt 2268 to 2390 in HBV sequences, were as follows40: total HBV mRNA (sense 5′-TCACCAGCACCATGCAAC-3′, antisense 5′-AAGCCACCCAAGGCACAG-3′) and HBV 3.5-kb mRNA (sense 5′-GAGTGTGGATTCGCACTCC-3′, antisense 5′-GAGGCGAGGGAGTTCTTCT-3′).
Western blotting
HCC cells were subjected to western blot analyses40. Briefly, the lysates were separated by 8% SDS-PAGE gels (Bio-Rad Laboratories, Inc.) and were then transferred to a polyvinylidene difluoride membrane (Bio-Rad Laboratories, Inc.). The membranes were blotted with primary antibodies against ELAVL1 (Cell Signaling Technology), tubulin (Oncogene Science, Cambridge, MA, USA), and Flag (DYKDDDDK) tag (Wako Pure Chemical Industries, Osaka, Japan) and horseradish peroxidase (HRP)-conjugated secondary antibody (GE Healthcare Life Sciences). The membranes were developed using Immobilon western Chemiluminescent HRP Substrate (EMD Millipore), and the signals were detected using ChemiDoc XRS Systems (Bio-Rad Laboratories, Inc.).
Measurement of HBs antigen levels
The HBs antigen levels in culture media were measured using chemiluminescent enzyme immunoassay (CLEIA, Lumipulse System; Fujirebio, Tokyo, Japan).
Lentiviral production and transduction
Lentiviral vectors (CS-H1-shRNA-EF-1a-EGFP) expressing short hairpin RNA (shRNA) targeting human ELAVL1 (target sequence: sh-ELAVL1-1, 5′-GGTTTGGGCGGATCATCAACT-3′; sh-ELAVL1-2, 5′-GGTTTGGCTTTGTGACCATGA-3′) and luciferase (Luc) were constructed. Recombinant lentiviruses were produced as described previously41. The cells were transduced using a lentiviral vector in the presence of protamine sulfate (10 μg/mL; Sigma-Aldrich, St. Louis, MO, USA).
Cell proliferation assay
Cells (1 × 104 cells/well) were seeded on 96-well plates and cultured for 96 h. MTS assays were performed using the CellTiter Aqueous One Solution Kit (Promega Corporation, Madison, WI, USA) according to the manufacturer’s protocols. Cell viability was assessed at 24, 48, 72, and 96 h and was defined as a percentage of the absorbance measured in the control cells. The dye of the formazan product resulting from MTS reduction was quantified using an automatic multiwell spectrophotometer, namely, InfiniteF50 (Tecan, Männedorf, Switzerland).
Patients and surgical specimens
A total of 77 pairs of tumor and adjacent nontumor tissues were subjected to clinicopathological analyses. Written informed consent was obtained from all patients. Paraffin-embedded tumor sections and the surrounding nontumor tissues were examined via H&E staining and immunohistochemistry with anti-ELAVL1 antibody (Cell Signaling Technology). Based on the ELAVL1 expression in the nuclei of cells, HCC and nontumor tissues were classified as follows: negative, partial expression (<50% of nuclei), and diffuse expression (≥50% of nuclei). HCCs with negative or partial ELAVL1 expression were classified as the ELAVL1low group, whereas HCCs with diffuse ELAVL1 expression were classified as the ELAVL1high group. All patients received postoperative radiological follow-up every 2–6 months. Radiological assessments were evaluated based on the response evaluation criteria in solid tumors42. This study was approved by the research ethics committees of the Graduate School of Medicine, Chiba University (approval number: 3300) and performed according to the Declaration of Helsinki.
Data collection and analysis from The Cancer Genome Atlas (TCGA)–Liver Hepatocellular Carcinoma (LIHC)
RNA sequencing (RNA-seq) datasets (ID: TCGA.LIHC.sampleMap/HiSeqV2) were downloaded using the UCSC Xena Browser (https://xenabrowser.net/). The RNA sequencing dataset shows the gene-level transcription estimates as in normalized log counts per million (logCPM). In total, secondary analyses have been conducted on RNA-seq data from both the normal liver (n = 50) and primary HCC (n = 371).
Statistical analysis
Data are expressed as mean with standard deviation (SD) or median with minimum to maximum and interquartile range (IQR). Statistical differences in the quantitative variables between groups were determined using either Student’s t-test or the Mann–Whitney U test. Chi-squared test was used for categorical variables. The log-rank test was used to analyze survival data. The level of significance was set to p < 0.05. All statistical analyses have been conducted using the SPSS statistical software version 27 (IBM, Chicago, IL, USA).