In total, 7 HBV-positive HCC liver tissue samples with HBsAg (+), HBeAg (+), HBcAb (+), and HBV-DNA > 104 IU/ml and 7 HBV-negative HCC liver tissue samples were used for microarray sequence analysis by KangChen Biotech (Shanghai, China). The clinical characteristics of participants were presented in Table S1. Total RNA was isolated from each tissue specimen using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. In addition, sample labeling and array hybridization were performed as per the manufacturer’s standard protocols for Agilent One-Color Microarray-Based Gene Expression Analysis and miRNA Microarray System with miRNA Complete Labeling and Hyb Kit (Agilent Technologies, Palo Alto CA, USA). After the removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre), mRNA was purified from the total RNA. Each sample was then amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3′ bias utilizing a random priming method (Arraystar Flash RNA Labeling Kit, Arraystar). The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen, USA). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000 Spectrophotometer (Thermo Fisher, USA). The total miRNA from each sample was labeled with Cyanine 3-pCp under the action of T4 RNA ligase. After hybridization, washing, and fixation, 100 μl of hybridization solution was dispensed into the gasket slide to be assembled to the gene and miRNA expression microarray slide, which is scanned with Agilent DNA Microarray Scanner (part number G2505C). The array images were obtained and analyzed using Agilent Feature Extraction Software v10.7. The low intensity of mRNA and miRNA were discarded followed by normalization of signal intensities through GeneSpring GX v11.5.1 (Agilent Technologies, Palo Alto CA, USA). The differential expression patterns of mRNA and miRNA between HBV-positive and HBV-negative HCC samples were identified by volcano plot filtering. Thereafter, the significantly differential expression of mRNA and miRNA was verified by paired t-test, and fold change ≥2.0 with P-value ≤0.05 of mRNA and fold change ≥1.0 with P-value ≤0.05 of miRNA were being the threshold for statistical significance. Further, the significantly differential expression of mRNAs and miRNAs was clustered using the heatmap R package, and the biological function of the Kyoto Encyclopedia of Genes and Genomes (KEGG) in significantly differential mRNAs was analyzed via clusterProfiler R package. KEGG (http://www.kegg.jp/) is as an integrated database resource for biological interpretation of genomics, transcriptomics, proteomics, metagenomics, and other high-throughput data by pathway mapping [17]. Subsequently, the microarray data were uploaded to the Gene Expression Omnibus (GEO) database with an access number of GSE151441 for mRNA and GEO140400 for miRNA.

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