Abstract
The objective of the present study was to screen the key genes of ribavirin in hepatocellular carcinoma (HCC) and provide novel therapeutic targets for HCC treatment. The mRNA expression datasets of GSE23031 and GSE74656, as well as the microRNA (miRNA) expression dataset of GSE22058 were downloaded from the Gene Expressed Omnibus database. In the GSE23031 dataset, there were three HCC cell lines treated with PBS and three HCC cell lines treated with ribavirin. In the GSE74656 dataset, five HCC tissues and five carcinoma adjacent tissues were selected. In the GSE22058 dataset, 96 HCC tissues and 96 carcinoma adjacent tissues were selected. The differentially expressed genes (DEGs) and differentially expressed miRNAs were identified via the limma package of R. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed with the Database for Annotation, Visualization and Integrated Discovery. The target mRNAs of DEMs were obtained with TargetScan. A total of 559 DEGs (designated DEG-Ribavirin) were identified in HCC cells treated with ribavirin compared with PBS and 632 DEGs (designated DEG-Tumor) were identified in HCC tissues compared with carcinoma adjacent tissues. A total of 220 differentially expressed miRNAs were identified in HCC tissues compared with carcinoma adjacent tissues. In addition, 121 GO terms and three KEGG pathways of DEG-Ribavirin were obtained, and 383 GO terms and 25 KEGG pathways of DEG-Tumor were obtained. A total of five key miRNA-mRNA regulated pairs were identified, namely miR-183→CCNB1, miR-96→DEPDC1, miR-96→NTN4, miR-183→NTN4 and miR-145→NTN4. The present study indicated that certain miRNAs (including miR-96, miR-145 and miR-183) and mRNAs (including NAT2, FBXO5, CCNB1, DEPDC1 and NTN4) may be associated with the effects of ribavirin on HCC. Furthermore, they may provide novel therapeutic targets for HCC treatment.
Keywords: hepatocellular carcinoma, ribavirin, microarray analysis
Introduction
Hepatocellular carcinoma (HCC) is the fifth most common malignancy and arises most frequently in patients with cirrhosis (1). It is the second most common cause of cancer-associated mortality globally with 1.6 million mortalities per year, and it is hypothesized that the high global incidence rate and late presentation of HCC may be responsible for this (2,3). Additionally, the general prognosis was poor with an overall survival rate between 3 and 5% in 2006 (4). Symptoms of HCC include yellow skin, bloating from fluid in the abdomen, easy bruising from blood clotting abnormalities, loss of appetite, unintentional weight loss, nausea, vomiting and tiredness (5,6). The primary risk factors for HCC were hepatitis C, hepatitis B, alcoholism, aflatoxin and cirrhosis of the liver (7–10). Liver transplantation, tyrosine kinase inhibitors and surgical resection are currently the primary treatment options (11–13). The treatment of HCC has not been fundamentally improved, which may be seen in the increasing morbidity and mortality each year (14). Ribavirin is an anti-viral drug used to treat hepatitis C, respiratory syncytial virus and other viral infections. If infection is persistent, ribavirin is often used in combination with peginterferon α-2b or peginterferon α-2a (15,16). It has been reported that hepatitis C infection was globally associated with 25% of HCC cases in 2006 (15). Therefore, ribavirin, by itself or in conjunction with peginterferon α-2b or pegylated interferon, has been used to treat HCC in patients with viral infections (17–20). Exploration of the genetic changes in HCC cells is necessary for the study of the pathogenesis and progression of HCC, as well as to develop effective treatments. In the present study, a microarray analysis of mRNA and microRNA (miRNA) was performed in the treatment of ribavirin on HCC, in order to identify possible biomarkers and provide novel potential therapeutic targets for HCC.
Materials and methods
Microarray data and data prx10-processing
The mRNA expression datasets of GSE23031 and GSE74656, as well as the miRNA expression dataset of GSE22058, (21–23) were downloaded from the Gene Expressed Omnibus database (http://www.ncbi.nlm.nih.gov/geo/). They were analyzed using the platforms GPL570 [HG-U133_Plus_2] Affymetrix Human Genome U133 Plus 2.0 Array (Thermo Fisher Scientific, Inc., Waltham, MA, USA), GPL16043 GeneChip® PrimeView™ Human Gene Expression Array (with External spikx10-in RNAs; Thermo Fisher Scientific, Inc.) and GPL10457 Rosetta human miRNA qPCR array (Rosetta Inpharmatics; Merck Sharp & Dohme, Hoddesdon, UK), respectively. The mRNA data (GSE23031) contained three HCC cell lines treated with PBS and three HCC cell lines treated with ribavirin. In the GSE74656, five HCC tissues and five carcinoma adjacent tissues were selected for the study. In the GSE22058, 96 HCC tissues and 96 carcinoma adjacent tissues were selected to study. Robust Multi-Array Average (RMA) was an algorithm used to create an expression matrix from Affymetrix data (24). The raw data were converted into a recognizable format by R, and the RMA was used for correction and normalization.
Differential expression analysis
The differentially expressed genes (DEGs) were identified via the limma package V3.32.10 (http://www.bioconductor.org/packages/3.5/bioc/html/limma.html) (25). According to the criteria: P<0.05 and |log(fold change)|>1, the DEGs were identified in HCC cells treated with ribavirin compared with PBS and designated DEG-Ribavirin. With the same criteria, the DEGs were identified in HCC tissues compared with their matched adjacent tissues and designated DEG-Tumor. Additionally, the differentially expressed miRNAs (DEMs) were obtained in HCC tissues compared with carcinoma adjacent tissues with P<0.05 and |log(fold change)|>0.3.
Functional and pathway enrichment analysis
The Database for Annotation, Visualization and Integrated Discovery (https://david.ncifcrf.gov/) (26) is a widely-used web-based tool for functional and pathway enrichment analysis. In the present study, it was used to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEG-Ribavirin and DEG-Tumor data. The GO terms and KEGG pathways were selected with P<0.05.
Comparison of DEGs and screening of miRNA-mRNA regulated pairs
The overlapped DEGs of DEG-Ribavirin and DEG-Tumor were selected, and the overlapped DEGs with opposite expression between DEG-Ribavirin and DEG-Tumor were also selected. The TargetScan database was used to predict biological target mRNAs of miRNAs that matched the seed region of each miRNA (27). The target mRNAs of DEMs were then selected using TargetScan. The key miRNAs, which regulated the overlapped DEGs with opposite expression between DEG-Ribavirin and DEG-Tumor, were identified. Subsequently, the miRNA-mRNA regulated pairs were constructed.
Results
DEGs. A total of 559 DEGs (269 upregulated and 290 downregulated) and 623 DEGs (272 upregulated and 351 downregulated) were identified in DEG-Ribavirin and DEG-Tumor. The heat map of them and the top 30 most significant DEGs are presented in Figs. 1 and 2, Tables I and II, respectively. A total of 220 DEMs were obtained. The 30 most significant DEMs are presented in Table III.
Figure 1.
Heatmap of differentially expressed genes in hepatocellular carcinoma cells treated with ribavirin compared with PBS.
Figure 2.
Heatmap of differentially expressed genes in hepatocellular carcinoma tissues compared with their matched adjacent tissues.
Table I.
Top 30 most significant differentially expressed genes in hepatocellular carcinoma cells treated with ribavirin compared with PBS.
| Gene | logFC | Average expression | P-value |
|---|---|---|---|
| NCF2 | 3.636237 | 10.08812 | 4.01×10−13 |
| TXNIP | 3.04897 | 9.867782 | 5.95×10−12 |
| PRORSD1P | 2.527183 | 6.729178 | 4.91×10−11 |
| NPPB | 3.0072 | 12.09607 | 8.26×10−11 |
| CYR61 | 2.018572 | 10.7489 | 9.19×10−11 |
| PLA2G4C | 1.93915 | 5.61348 | 9.29×10−11 |
| CDKN2B | 2.282063 | 7.523593 | 9.52×10−11 |
| LEAP2 | 2.52559 | 9.937438 | 1.39×10−10 |
| DUSP4 | 1.86831 | 8.419143 | 3.18×10−10 |
| FLVCR1-AS1 | 2.01682 | 8.90536 | 4.17×10−10 |
| ASH1L-AS1 | 1.803117 | 9.666342 | 4.20×10−10 |
| CXCL3 | 1.797497 | 8.092698 | 5.80×10−10 |
| GLIPR2 | 1.688522 | 9.089844 | 5.91×10−10 |
| CYP1A1 | 1.560867 | 11.576 | 7.61×10−10 |
| EID2B | 1.667057 | 8.101158 | 9.18×10−10 |
| JUNB | 1.574027 | 8.65205 | 1.10×10−09 |
| BTG2 | 1.498662 | 9.091506 | 1.41×10−09 |
| PPL | 1.94965 | 8.769882 | 2.08×10−09 |
| ZNF436-AS1 | 2.570947 | 8.267133 | 2.12×10−09 |
| TIGD7 | 1.654807 | 6.61165 | 2.17×10−09 |
| LOC284513 | 1.59588 | 6.487373 | 2.40×10−09 |
| TUFT1 | 1.50993 | 10.31677 | 2.43×10−09 |
| CTSE | 1.439267 | 11.22817 | 2.48×10−09 |
| ERP27 | 1.376543 | 10.6632 | 2.51×10−09 |
| UCA1 | 1.611053 | 10.73581 | 2.60×10−09 |
| HSD3B1 | 1.699597 | 5.372037 | 2.61×10−09 |
| GATA6-AS1 | 1.971443 | 9.336978 | 2.99×10−09 |
| LOC100134822 | 1.45604 | 7.840603 | 3.38×10−09 |
| THUMPD3-AS1 | 1.365701 | 7.84944 | 3.41×10−09 |
| GDA | 1.482742 | 8.376296 | 3.62×10−09 |
logFC, log fold-change.
Table II.
Top 30 most significant DEGs in HCC tissues compared with carcinoma adjacent tissues.
| Gene | logFC | Average expression | P-value |
|---|---|---|---|
| CTHRC1 | 2.856945 | 5.825291 | 6.69×10−07 |
| PEA15 | 1.468145 | 8.69977 | 1.04×10−06 |
| CENPE | 2.043658 | 6.056165 | 1.29×10−06 |
| C21orf56 | 1.237448 | 5.670084 | 1.54×10−06 |
| DBN1 | 1.383559 | 6.53977 | 1.76×10−06 |
| GLA | 1.380323 | 7.172235 | 2.00×10−06 |
| DDX39 | 1.17872 | 7.721654 | 2.63×10−06 |
| MPV17 | 1.190142 | 9.079669 | 2.67×10−06 |
| RFX5 | 1.417855 | 7.558042 | 2.89×10−06 |
| TMEM144 | 1.379456 | 5.655336 | 3.59×10−06 |
| ASNS | 2.515006 | 6.603396 | 3.63×10−06 |
| SLC38A6 | 1.678498 | 7.591919 | 3.93×10−06 |
| GRAMD1A | 1.013492 | 7.228709 | 4.65×10−06 |
| COMMD8 | 1.109678 | 8.353807 | 7.57×10−06 |
| YWHAZ | 1.008572 | 8.92205 | 7.94×10−06 |
| PLXNC1 | 1.451381 | 5.772042 | 1.18×10−05 |
| PLVAP | 1.001818 | 5.796379 | 1.20×10−05 |
| SHCBP1 | 1.327673 | 4.917339 | 1.39×10−05 |
| LAMC1 | 1.104793 | 6.905647 | 1.46×10−05 |
| ANXA2P2 | 1.89594 | 11.61567 | 1.52×10−05 |
| ACTR3 | 1.094921 | 9.606995 | 1.63×10−05 |
| CCDC88A | 1.002649 | 6.195715 | 1.64×10−05 |
| E2F3 | 1.093946 | 6.541921 | 1.75×10−05 |
| FAM118B | 1.006248 | 6.9915 | 1.76×10−05 |
| ZNF354A | 1.069078 | 5.918941 | 1.81×10−05 |
| RASGEF1A | 2.021872 | 4.908126 | 1.84×10−05 |
| NUP37 | 1.362635 | 6.839194 | 2.01×10−05 |
| ESM1 | 1.291572 | 4.762991 | 2.03×10−05 |
| KPNA2 | 2.606398 | 9.341649 | 2.13×10−05 |
| DCUN1D5 | 1.38889 | 8.463629 | 2.34×10−05 |
logFC, log fold-change; HCC, hepatocellular carcinoma; DEGs, differentially expressed genes.
Table III.
Top 30 most significant differentially expressed miRNA in HCC tissues compared with carcinoma adjacent tissues.
| miRNA | logFC | P-value |
|---|---|---|
| hsa-mir-188 | 0.36842 | 2.41×10−39 |
| hsa-mir-106b | 0.32071 | 1.52×10−36 |
| hsa-mir-214 | −0.54861 | 7.34×10−36 |
| hsa-mir-93 | 0.32346 | 1.73×10−35 |
| hsa-mir-10a | −0.51702 | 6.33×10−35 |
| hsa-mir-199a-1 | −0.78765 | 6.28×10−33 |
| hsa-mir-199a-2 | −0.73035 | 5.57×10−31 |
| hsa-mir-301 | 0.5629 | 1.16×10−28 |
| hsa-mir-424 | −0.31246 | 1.72×10−28 |
| hsa-mir-33 | 0.29308 | 7.92×10−26 |
| hsa-mir-324-5p | 0.37868 | 1.01×10−23 |
| hsa-mir-25 | 0.20282 | 1.53×10−22 |
| hsa-mir-125b | −0.37741 | 1.95×10−22 |
| hsa-mir-339 | 0.21825 | 3.36×10−21 |
| hsa-mir-145 | −0.35693 | 4.27×10−21 |
| hsa-mir-148b | 0.20955 | 6.76×10−21 |
| hsa-mir-151 | 0.25728 | 9.53×10−21 |
| hsa-mir-221 | 0.35829 | 1.39×10−20 |
| hsa-mir-18a | 0.41731 | 3.09×10−20 |
| hsa-mir-130b | 0.38724 | 3.75×10−20 |
| hsa-mir-195 | −0.34663 | 6.72×10−20 |
| hsa-mir-99a | −0.38524 | 8.55×10−20 |
| hsa-mir-15b | 0.27273 | 1.80×10−19 |
| hsa-mir-183 | 0.42758 | 2.31×10−19 |
| hsa-mir-222 | 0.30098 | 2.62×10−18 |
| hsa-mir-125a | −0.26924 | 4.47×10−18 |
| hsa-mir-378 | −0.33767 | 7.60×10−18 |
| hsa-mir-101 | −0.24801 | 1.85×10−17 |
| hsa-mir-331 | 0.20462 | 2.33×10−17 |
| hsa-mir-200b | −0.6996 | 2.38×10−17 |
HCC, hepatocellular carcinoma; logFC, log fold-change; mir, microRNA.
GO terms and KEGG pathways
A total of 121 GO terms and 3 KEGG pathways (cell cycle pathway, p53 signaling pathway and glycine, serine and threonine metabolism pathway) of DEG-Ribavirin were obtained. A total of 383 GO terms and 25 KEGG pathways of DEG-Tumor were obtained. The top 20 enriched GO terms of DEG-Ribavirin and DEG-Tumor are presented in Tables IV and V, respectively. The enriched KEGG pathways of DEG-Ribavirin and DEG-Tumor are presented in Tables VI and VII, respectively.
Table IV.
Top 20 enriched GO terms of DEG-Ribavirin.
| Category | GO ID | Go name | Gene number | P-value |
|---|---|---|---|---|
| BP | GO:0022403 | Cell cycle phase | 29 | 1.66×10−06 |
| BP | GO:0007049 | Cell cycle | 43 | 1.66×10−06 |
| BP | GO:0000279 | M phase | 24 | 8.49×10−06 |
| BP | GO:0007067 | Mitosis | 19 | 1.02×10−05 |
| BP | GO:0000280 | Nuclear division | 19 | 1.02×10−05 |
| BP | GO:0000087 | M phase of mitotic cell cycle | 19 | 1.29×10−05 |
| BP | GO:0048285 | Organelle fission | 19 | 1.73×10−05 |
| BP | GO:0000278 | Mitotic cell cycle | 25 | 1.88×10−05 |
| BP | GO:0022402 | Cell cycle process | 32 | 3.23×10−05 |
| BP | GO:0008283 | Cell proliferation | 27 | 3.62×10−05 |
| BP | GO:0031497 | Chromatin assembly | 11 | 5.88×10−05 |
| BP | GO:0065004 | Protein-DNA complex assembly | 11 | 8.65×10−05 |
| BP | GO:0006334 | Nucleosome assembly | 10 | 2.36×10−04 |
| BP | GO:0051726 | Regulation of cell cycle | 21 | 2.43×10−04 |
| BP | GO:0051301 | Cell division | 19 | 4.39×10−04 |
| BP | GO:0034728 | Nucleosome organization | 10 | 5.08×10−04 |
| BP | GO:0006323 | DNA packaging | 11 | 6.79×10−04 |
| CC | GO:0000786 | Nucleosome | 8 | 9.25×10−04 |
| BP | GO:0010033 | Response to organic substance | 33 | 0.001109 |
| CC | GO:0005819 | Spindle | 12 | 0.001114 |
GO, Gene Ontology; DEGs, differentially expressed genes; CC, cellular component; BP, biological process.
Table V.
Top 20 enriched GO terms of DEG-tumor.
| Category | GO ID | Go name | Gene number | P-value |
|---|---|---|---|---|
| BP | GO:0000279 | M phase | 49 | 1.22×10−17 |
| BP | GO:0022403 | Cell cycle phase | 54 | 7.41×10−17 |
| BP | GO:0007067 | Mitosis | 39 | 1.19×10−16 |
| BP | GO:0000280 | Nuclear division | 39 | 1.19×10−16 |
| BP | GO:0000087 | M phase of mitotic cell cycle | 39 | 2.21×10−16 |
| BP | GO:0000278 | Mitotic cell cycle | 50 | 4.09×10−16 |
| BP | GO:0048285 | Organelle fission | 39 | 5.79×10−16 |
| BP | GO:0022402 | Cell cycle process | 61 | 5.37×10−15 |
| MF | GO:0048037 | Cofactor binding | 39 | 3.37×10−14 |
| BP | GO:0016054 | Organic acid catabolic process | 26 | 4.73×10−14 |
| BP | GO:0046395 | Carboxylic acid catabolic process | 26 | 4.73×10−14 |
| CC | GO:0005819 | Spindle | 30 | 7.20×10−14 |
| BP | GO:0007049 | Cell cycle | 71 | 9.42×10−14 |
| BP | GO:0055114 | Oxidation reduction | 62 | 3.81×10−13 |
| BP | GO:0007059 | Chromosome segregation | 21 | 2.69×10−12 |
| MF | GO:0009055 | Electron carrier activity | 34 | 3.23×10−12 |
| CC | GO:0000793 | Condensed chromosome | 26 | 5.97×10−12 |
| CC | GO:0000777 | Condensed chromosome kinetochore | 18 | 1.41×10−11 |
| MF | GO:0050662 | Coenzyme binding | 29 | 6.32×10−11 |
| CC | GO:0000779 | Condensed chromosome, centromeric region | 18 | 1.37×10−10 |
GO, Gene Ontology; DEGs, differentially expressed genes; CC, cellular component; BP, biological process; MF, molecular foundation.
Table VI.
Enriched KEGG pathways of DEG-Ribavirin.
| Category | Pathway name | Gene number | P-value |
|---|---|---|---|
| KEGG_PATHWAY | hsa04110: Cell cycle | 15 | 1.19×10−05 |
| KEGG_PATHWAY | hsa00260: Glycine, serine and threonine metabolism | 5 | 0.011495 |
| KEGG_PATHWAY | hsa04115: p53 signaling pathway | 6 | 0.045851 |
DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes.
Table VII.
Enriched KEGG pathways of DEG-tumor.
| Category | Pathway name | Gene number | P-value |
|---|---|---|---|
| KEGG_PATHWAY | hsa00071: Fatty acid metabolism | 15 | 1.37×10−09 |
| KEGG_PATHWAY | hsa00280: Valine, leucine and isoleucine degradation | 14 | 5.58×10−08 |
| KEGG_PATHWAY | hsa04110: Cell cycle | 21 | 1.08×10−06 |
| KEGG_PATHWAY | hsa00830: Retinol metabolism | 12 | 3.12×10−05 |
| KEGG_PATHWAY | hsa00380: Tryptophan metabolism | 10 | 7.32×10−05 |
| KEGG_PATHWAY | hsa00650: Butanoate metabolism | 9 | 1.33×10−04 |
| KEGG_PATHWAY | hsa00250: Alanine, aspartate and glutamate metabolism | 8 | 4.63×10−04 |
| KEGG_PATHWAY | hsa04114: Oocyte meiosis | 15 | 5.67×10−04 |
| KEGG_PATHWAY | hsa00640: Propanoate metabolism | 8 | 5.69×10−04 |
| KEGG_PATHWAY | hsa03320: PPAR signaling pathway | 11 | 0.001281 |
| KEGG_PATHWAY | hsa00980: Metabolism of xenobiotics by cytochrome P450 | 10 | 0.00174 |
| KEGG_PATHWAY | hsa00910: Nitrogen metabolism | 6 | 0.003688 |
| KEGG_PATHWAY | hsa00590: Arachidonic acid metabolism | 9 | 0.004249 |
| KEGG_PATHWAY | hsa00140: Steroid hormone biosynthesis | 8 | 0.005165 |
| KEGG_PATHWAY | hsa00982: Drug metabolism | 9 | 0.007941 |
| KEGG_PATHWAY | hsa00591: Linoleic acid metabolism | 6 | 0.008896 |
| KEGG_PATHWAY | hsa00340: Histidine metabolism | 6 | 0.010346 |
| KEGG_PATHWAY | hsa04115: p53 signaling pathway | 9 | 0.013645 |
| KEGG_PATHWAY | hsa00260: Glycine, serine and threonine metabolism | 6 | 0.013718 |
| KEGG_PATHWAY | hsa00410: β-Alanine metabolism | 5 | 0.017838 |
| KEGG_PATHWAY | hsa04920: Adipocytokine signaling pathway | 8 | 0.036604 |
| KEGG_PATHWAY | hsa00620: Pyruvate metabolism | 6 | 0.037809 |
| KEGG_PATHWAY | hsa00232: Caffeine metabolism | 3 | 0.039507 |
| KEGG_PATHWAY | hsa05222: Small cell lung cancer | 9 | 0.042544 |
| KEGG_PATHWAY | hsa04512: ECM-receptor interaction | 9 | 0.042544 |
DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes.
miRNA-mRNA regulated pairs
There were 50 overlapped DEGs, with 32 [including N-acetyltransferase (NAT2) and F-box only protein 5 (FBXO5)] exhibiting opposite expression between DEG-Ribavirin, and DEG-Tumor. A heat map of the 32 overlapped DEGs is presented in Fig. 3. Furthermore, three DEMs (miR-96, miR-145 and miR-183) were revealed to correspond to three DEGs (CCNB1, DEPDC1 and NTN4) which were included in the aforementioned 32 overlapped DEGs. Finally, five miRNA-mRNA regulated pairs were selected between the above three DEGs and the three DEMs, namely miR-183→CCNB1, miR-96→DEPDC1, miR-96→NTN4, miR-183→NTN4 and miR-145→NTN4.
Figure 3.
Heatmap of the overlapped DEGs with opposite expression between DEG-Ribavirin and DEG-Tumor. Adj, adjacent; DEG, differentially expressed gene.
Discussion
In the present study, the DEGs in HCC cells treated with ribavirin compared with PBS treated HCC tissue, HCC tissues and carcinoma adjacent tissues, were firstly identified, and 32 overlapped DEGs with opposite expression between DEG-Ribavirin and DEG-Tumor were selected. It was notable that NAT2 and FBXO5 were two mRNAs of them with opposite expression between DEG-Ribavirin and DEG-Tumor. NAT2 serves a function in the metabolic activation and detoxification of aromatic amines, which in turn serves a function in the metabolism of aromatic and heterocyclic amines, and hydrazines via N-acetylation and O-acetylation (28). As early as in 1996, Agúndez et al (29) reported that the slow acetylation was associated with an increased risk of HCC. Furthermore, it has been demonstrated that NAT2 activity is associated with smoking-associated HCC (30–32). A number of previous studies that have investigated the association between NAT2 genotypes and HCC risk have been published (32–36). FBXO5, also known as early mitotic inhibitor-1, is a key cell-cycle regulator that promotes S-phase and M-phase entry by inhibiting anaphasx10-promoting complex/cyclosome activity (37). Zhao et al (38) revealed that FBXO5 was overexpressed in HCC, which is in agreement with the results of the present study, and also reported that FBXO5 may control tumor cell proliferation in HCC. In the present study, it was identified that the expression of NAT2 was lower in HCC cells and HCC tissues. However, expression was increased following treatment with ribavirin. However, FBXO5 was overexpressed in HCC cells and HCC tissues, and decreased following treatment with ribavirin. Therefore, it is suspected that NAT2 and FBXO5 may be biomarkers of ribavirin in the treatment of HCC.
The cell cycle has been demonstrated to be associated with the progression and migration of HCC (39–41), and regulation of the cell cycle is considered an effective strategy for HCC treatment (42–45). The p53 signaling pathway has been heavily studied and is reported to serve a function in the occurrence and development of HCC (45–49). The association between the glycine, serine and threonine metabolism pathway and HCC has been less studied, and the glycine, serine and threonine metabolism pathway was also enriched in DEG-Tumor tissues. In this study, only three KEGG pathways of DEG-Ribavirin were obtained, namely cell cycle, p53 signaling pathway and glycine, serine and threonine metabolism. Cell cycle was the most significantly enriched function in this study, which was identified from the enriched GO terms of DEG-Ribavirin (e.g. cell cycle phase, cell cycle and M phase) and DEG-Tumor (e.g. M phase and cell cycle phase), as well as the enriched KEGG pathways of DEG-Tumor. The results of the present study suggest that these three KEGG pathways may be associated with the pathogenesis and treatment of HCC; however, more in-depth research is required.
In the present study, three DEMs (miR-96, miR-145 and miR-183) were identified to correspond to three overlapped DEGs (CCNB1, DEPDC1 and NTN4) with opposite expression in DEG-Ribavirin and DEG-Tumor, and 5 miRNA-mRNA regulated pairs were selected, namely miR-183→CCNB1, miR-96→DEPDC1, miR-96→NTN4, miR-183→NTN4 and miR-145→NTN4. It has been demonstrated that miR-96 downregulation may suppress the growth of HCC (50), and miR-96 may promote cell proliferation and invasion through targeting ephrinA5 in HCC (51). Chen et al (52) considered serum miR-96 as a promising biomarker for HCC with chronic hepatitis B virus infection. Previous studies have demonstrated that miR-145 may inhibit proliferation, migration and invasion, as well as promote apoptosis in HCC (53–56). MiR-183 may also regulate the growth, invasion and apoptosis of HCC (57–59). It has been identified that these miRNA and mRNA are possible biomarkers of ribavirin in HCC, and they may regulate HCC through the 5 miRNA-mRNA pairs.
In conclusion, a number of miRNAs (e.g. miR-96, miR-145 and miR-183) and mRNAs (e.g. NAT2, FBXO5, CCNB1, DEPDC1 and NTN4) may be associated with the effects of ribavirin on HCC. Furthermore, they may provide novel therapeutic targets for drugs of HCC.
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