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Cancer-associated fibroblasts (CAFs) contribute to tumor progression, and microRNAs (miRs) play an important role in regulating the tumor-promoting properties of CAFs. The objectives of this study were to clarify the specific miR expression profile in CAFs of hepatocellular carcinoma (HCC) and identify its target gene signatures. Small-RNA-sequencing data were generated from nine pairs of CAFs and para-cancer fibroblasts isolated from human HCC and para-tumor tissues, respectively. Bioinformatic analyses were performed to identify the HCC-CAF-specific miR expression profile and the target gene signatures of the deregulated miRs in CAFs. Clinical and immunological implications of the target gene signatures were evaluated in The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA_LIHC) database using Cox regression and TIMER analysis. The expressions of hsa-miR-101-3p and hsa-miR-490-3p were significantly downregulated in HCC-CAFs. Their expression in HCC tissue gradually decreased as HCC stage progressed in the clinical staging analysis. Bioinformatic network analysis using miRWalks, miRDB, and miRTarBase databases pointed to TGFBR1 as a common target gene of hsa-miR-101-3p and hsa-miR-490-3p. TGFBR1 expression was negatively correlated with miR-101-3p and miR-490-3p expression in HCC tissues and was also decreased by ectopic miR-101-3p and miR-490-3p expression. HCC patients with TGFBR1 overexpression and downregulated hsa-miR-101-3p and hsa-miR-490-3p demonstrated a significantly poorer prognosis in TCGA_LIHC. TGFBR1 expression was positively correlated with the infiltration of myeloid-derived suppressor cells, regulatory T cells, and M2 macrophages in a TIMER analysis. In conclusion, hsa-miR-101-3p and hsa-miR-490-3p were substantially downregulated miRs in CAFs of HCC, and their common target gene was TGFBR1. The downregulation of hsa-miR-101-3p and hsa-miR-490-3p, as well as high TGFBR1 expression, was associated with poor clinical outcome in HCC patients. In addition, TGFBR1 expression was correlated with the infiltration of immunosuppressive immune cells.

The comparative effectiveness and safety of growth hormone monotherapy and combination therapy with herbal medicine and growth hormone in short stature children have yet to be clarified.

Some herbal medicines have anti-inflammatory and anti-diarrheal effects. This study analyzed the modulating effect of gut microbiota of anti-inflammatory herbal medicines on antibiotic-associated diarrhea (AAD). The anti-inflammatory effects of 10 herbal medicines and Lizhong-tang active compounds were studied by measuring the nitric oxide production in an in vitro experiment. This was followed by an in vivo experiment in which the anti-diarrheal effects of Lizhong-tang and Magnolia officinalis in a lincomycin-induced AAD mouse model were measured. Changes in the intestinal microflora were observed using terminal restriction fragment length polymorphism analysis. Both Lizhong-tang and M. officinalis were effective against AAD, with Lizhong-tang’s anti-diarrheal effects being particularly effective. In addition, the active compounds of Lizhong-tang, liquiritin and 6-gingerol, inhibited the expression of inducible nitric oxide synthase and cyclooxygenase-2, thus showing an anti-inflammatory effect. Gut microbiota analysis showed that Lizhong-tang could alter the composition of the gut microbiota and ameliorated imbalance in the gut microbiota in a lincomycin-induced AAD mouse model.

We investigated whether liver stiffness (LS) quantified using magnetic resonance elastography (MRE) could predict the prognosis of advanced hepatocellular carcinoma (HCC) patients treated with sorafenib. We selected 50 sorafenib-treated advanced HCC patients who underwent MRE within 3 months before drug administration from a prospectively maintained cohort of chronic liver disease patients, according to the inclusion and exclusion criteria. Univariate and multivariate analyses were performed to evaluate the prognostic role of laboratory data, tumor characteristics, and MRE-assessed LS for overall survival (OS), progression-free survival (PFS), and significant liver injury (grade ≥ 3) after sorafenib administration. High MRE-assessed LS either as continuous (per kPa, hazard ratio (HR) 1.54; 95% confidence interval (CI) 1.23–1.92, p 7.5 kPa, HR 4.06, 95% CI 1.40–11.79, p 7.5 kPa, HR 10.11, 95% CI 2.41–42.46, p = 0.002). PFS analysis identified higher serum AFP (≥ 400 ng/mL) and advanced tumor stage (mUICC IVb) as significant risk factors for early disease progression, whereas LS was not associated with PFS Higher MRE-assessed LS is a potential biomarker for predicting poor OS and significant liver injury in advanced HCC patients treated with sorafenib. • Higher pretreatment LS by MRE (> 7.5 kPa), higher AFP (≥ 400 ng/mL), and advanced tumor stage (mUICC IVb) were associated with poor OS in advanced HCC patients treated with sorafenib. • Higher pretreatment LS by MRE was associated with developing significant (grade ≥ 3) liver injury during sorafenib treatment, which required termination of the therapy. • Patients with high pretreatment LS by MRE should be monitored carefully for potential liver injury during sorafenib treatment.

Currently, a reliable serum biomarker for hepatocellular carcinoma (HCC) has not been established, particularly for early‐stage HCC (single tumor 0.8. In our validation study, serum exo‐miR‐4661‐5p could diagnose HCC in all stages (AUROC = 0.917), even in early stage (AUROC = 0.923), with a greater accuracy than other candidate serum exo‐miRs and serum AFP. The panel composed of exo‐miR‐4661‐5p and exo‐miR‐4746‐5p was identified as the most accurate biomarker for early‐stage HCC (AUROC = 0.947, 95% confidence interval = 0.889‐0.980, sensitivity = 81.8%, and specificity = 91.7%). In conclusion, exo‐miR‐4661‐5p–based serum panel is a promising diagnostic marker for early‐stage HCC., The present study derived potential serum exosomal microRNA panels for hepatocellular carcinoma (HCC) using a systematic, genome‐wide biomarker discovery approach. Serum exosomal microRNA‐4661‐5p–based panel is a potent diagnostic biomarker for early stage HCC and could also be used as a prognostic indicator in patients with HCC.

Magnetic resonance elastography (MRE) is a non-invasive tool for measuring liver stiffness (LS) with high diagnostic accuracy. This study investigated whether quantified LS by MRE could predict early recurrence of patients with hepatocellular carcinoma (HCC) within the Milan criteria. A prospectively collected cohort, which included the HCC patients who underwent MRE before treatment (an HCC-MRE cohort), was analyzed. In the HCC-MRE cohort, only patients under the Milan criteria, who underwent hepatic resection, radiofrequency ablation (RFA), or transarterial chemoembolization (TACE), were reviewed. We investigated whether LS assessed by MRE was an independent predictor of early recurrence using Cox regressions and Kaplan-Meier analyses. A total of 192 HCC patients under the Milan criteria who underwent hepatic resection (n = 96), RFA (n = 23), or TACE (n = 73) were included. Higher LS ratings (kPa; hazard ratio [HR] = 1.12; 95% confidence interval [CI] = 1.01–1.25; p = 0.040) emerged as an independent risk factor for early tumor recurrence. In the subgroup analysis, higher LS ratings were associated with higher risks of early HCC recurrence in both the resection/RFA group (> 4.5 kPa; HR = 2.95; 95% CI = 1.26–6.94; p = 0.013) and the TACE group (> 6 kPa; HR = 2.94; 95% CI = 1.27–6.83; p = 0.012). LS assessed by MRE was an independent predictor of early recurrence among HCC patients under the Milan criteria after achieving a complete response. • Liver parenchymal stiffness measured by MRE predicts early recurrence of treated HCC under Milan criteria. • A liver stiffness > 5.5 kPa was associated with worse recurrence-free survival. • Patients with high pre-treatment LS may benefit from stringent follow-up.

Background: Hepatocellular carcinoma (HCC) is one of the most common and lethal cancers worldwide. Wiskott-Aldrich syndrome protein family member 2 (WASF2) is an integral member of the actin cytoskeleton pathway that plays a crucial role in cell motility. In this study, we aimed to explore the role of WASF2 in HCC carcinogenesis and its regulatory mechanism. Methods: WASF2 expression in HCC was analyzed using six public RNA-seq datasets and 66 paired tissues from patients with HCC. Role of WASF2 in HCC cell phenotypes was evaluated using small interfering RNA (siRNA) in vitro and in vivo. Epigenetic regulatory mechanism of WASF2 was assessed in the Cancer Genome Atlas liver hepatocellular carcinoma project (TCGA_LIHC) dataset and also validated in 38 paired HCC tissues. Results: WASF2 is overexpressed in HCC and is clinically correlated with prognosis. WASF2 inactivation decreased the viability, growth, proliferation, migration, and invasion of Huh-7 and SNU475 HCC cells by restoring G2/M checkpoint function, inducing cell death, and inhibiting epithelial-mesenchymal transition, and hindering actin polymerization. In addition, WASF2 knockdown using siWASF2 in a xenograft mouse model exerted tumor suppressive effect. Furthermore, we observed a negative correlation between WASF2 methylation status and mRNA expression. The cg24162579 CpG island in the WASF2 5′ promoter region was hypomethylated in HCC compared to matched non-tumor samples. Patients with high WASF2 methylation and low WASF2 expression displayed the highest overall survival.Conclusions: WASF2 is overexpressed and hypomethylated in HCC and correlates with patient prognosis. Moreover, WASF2 inactivation exerts anti-tumorigenic effects on HCC cells in vitro and in vivo, suggesting that WASF2 could be a potential therapeutic target for HCC.

Reverse transcription-quantitative (RT-q) PCR is the most feasible and useful technique for identifying and evaluating cancer biomarkers; however, the method requires suitable reference genes for gene expression analysis. The aim of the present study was to identify the most suitable reference gene for the normalization of relative gene expression in human hepatocellular carcinoma (HCC) tissue and blood samples. First, 14 candidate reference genes were selected through a systematic literature search. The expression levels of these genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, PGK1, PPIA, RPLP0, RPL13A, SDHA, TBP, TFRC and YWHAZ) were evaluated using human multistage HCC transcriptome data (dataset GSE114564), which included normal liver (n=15), chronic hepatitis (n=20), liver cirrhosis (n=10), and early (n=18) and advanced HCC (n=45). From the 14 selected genes, five genes, whose expression levels were stable in all liver disease statuses (ACTB, GAPDH, HMBS, PPIA and RPLP0), were further assessed using RT-qPCR in 40 tissues (20 paired healthy tissues and 20 tissues from patients with HCC) and 40 blood samples (20 healthy controls and 20 samples from patients with HCC). BestKeeper statistical algorithms were used to identify the most stable reference genes, of which HMBS was found to be the most stable in both HCC tissues and blood samples. Therefore, the results of the present study suggest HMBS as a promising reference gene for the normalization of relative RT-qPCR techniques in HCC-related studies.

Protein markers of hepatocellular carcinoma (HCC)-derived exosomes (HEX) have not yet been fully evaluated. Here, we identified novel protein contents of HEX and their clinical significance as biomarkers. Exosomes were isolated from human HCC cell lines and an immortalized normal hepatocyte cell line. Proteomic analyses revealed 15 markedly overexpressed proteins in HEX. The clinical relevance of the 15 proteins was analyzed in public RNA-sequencing datasets, and 6 proteins were selected as candidate of potential biomarkers. Serum CCT8 and CFL1 were markedly overexpressed in test cohort (n = 8). In the validation cohort (n = 224), the area under the curve (AUC) of serum CCT8 and CFL1 for HCC diagnosis was calculated as 0.698 and 0.677, respectively, whereas that of serum alpha-fetoprotein (AFP) was 0.628. The combination of three serum markers (CCT8, CFL1, and AFP) demonstrated the highest AUC for HCC diagnosis. (AUC = 0.838, 95% confidence interval = 0.773–0.876) Furthermore, higher serum CCT8 and CFL1 concentrations were significantly associated with the presence of vascular invasion, advanced tumor stage, poor disease-free survival, and poor overall survival. Cofilin-1 and CCT8, enriched proteins in HEX, were identified as potential diagnostic and prognostic serum biomarkers for HCC patients.

Exosomal microRNAs (exo-miRs) contribute to cancer metastasis. To identify pro-metastatic circulating exo-miRs in hepatocellular carcinoma (HCC), next-generation sequencing-based plasma exo-miR profiles of 14 patients with HCC (eight non-metastatic and six with metastasis within 1 year of follow-up) were analyzed. Sixty-one miRs were significantly overexpressed among patients with metastatic HCC. Candidate miRs were selected through integrative analyses of two different public expression datasets, GSE67140 and The Cancer Genome Atlas liver hepatocellular carcinoma (TCGA_LIHC). Integrative analyses revealed 3 of 61 miRs (miR-106b-5p, miR-1307-5p, and miR-340-5p) commonly overexpressed both in metastasis and vascular invasion groups, with prognostic implications. Validation was performed using stored blood samples of 150 patients with HCC. Validation analysis showed that circulating exo-miR-1307-5p was significantly overexpressed in the metastasis group (p = 0.04), as well as in the vascular invasion and tumor recurrence groups. Circulating exo-miR-1307-5p expression was significantly correlated with tumor stage progression (p &lt, 0.0001). Downstream signaling pathways of miR-1307 were predicted using TargetScan and Ingenuity Pathway Analysis. On comprehensive bioinformatics analysis, the downstream pathway of miR-1307-5p, promoting epithelial&ndash, mesenchymal transition (EMT), showed SEC14L2 and ENG downregulation. Our results show that circulating exo-miR-1307-5p promotes metastasis and helps predict metastasis in HCC, and SEC14L2 and ENG are target tumor suppressor genes of miR-1307 that promote EMT.

Liquid biopsy can provide a strong basis for precision medicine. We aimed to identify novel single-nucleotide variants (SNVs) in circulating tumor DNA (ctDNA) in patients with hepatocellular carcinoma (HCC). Deep sequencing of plasma-derived ctDNA from 59 patients with HCC was performed using a panel of 2924 SNVs in 69 genes. In 55.9% of the patients, at least one somatic mutation was detected. Among 25 SNVs in 12 genes, four frequently observed SNVs, MLH1 (13%), STK11 (13%), PTEN (9%), and CTNNB1 (4%), were validated using droplet digital polymerase chain reaction with ctDNA from 62 patients with HCC. Three candidate SNVs were detected in 35.5% of the patients, with a frequency of 19% for MLH1 chr3:37025749T>A, 11% for STK11 chr19:1223126C>G, and 8% for PTEN chr10:87864461C>G. The MLH1 and STK11 SNVs were also confirmed in HCC tissues. The presence of the MLH1 SNV, in combination with an increased ctDNA level, predicted poor overall survival among 107 patients. MLH1 chr3:37025749T>A SNV detection in ctDNA is feasible, and thus, ctDNA can be used to detect somatic mutations in HCC. Furthermore, the presence or absence of the MLH1 SNV in ctDNA, combined with the ctDNA level, can predict the prognosis of patients with HCC.

We investigated whether liver stiffness (LS) quantified using magnetic resonance elastography (MRE) could predict the prognosis of advanced hepatocellular carcinoma (HCC) patients treated with sorafenib.We selected 50 sorafenib-treated advanced HCC patients who underwent MRE within 3 months before drug administration from a prospectively maintained cohort of chronic liver disease patients, according to the inclusion and exclusion criteria. Univariate and multivariate analyses were performed to evaluate the prognostic role of laboratory data, tumor characteristics, and MRE-assessed LS for overall survival (OS), progression-free survival (PFS), and significant liver injury (grade ≥ 3) after sorafenib administration.High MRE-assessed LS either as continuous (per kPa, hazard ratio (HR) 1.54; 95% confidence interval (CI) 1.23-1.92, p0.001) or categorical ( 7.5 kPa, HR 4.06, 95% CI 1.40-11.79, p0.01) variable was significantly associated with poor OS along with higher serum alpha-fetoprotein (AFP, ≥ 400 ng/mL) and advanced tumor stage (modified Union for International Cancer Control (mUICC) IVb). Higher MRE-assessed LS was also significantly associated with the development of significant liver injury after sorafenib administration (per kPa, HR 1.62, 95% CI 1.21-2.17, p = 0.001; 7.5 kPa, HR 10.11, 95% CI 2.41-42.46, p = 0.002). PFS analysis identified higher serum AFP (≥ 400 ng/mL) and advanced tumor stage (mUICC IVb) as significant risk factors for early disease progression, whereas LS was not associated with PFS CONCLUSION: Higher MRE-assessed LS is a potential biomarker for predicting poor OS and significant liver injury in advanced HCC patients treated with sorafenib.• Higher pretreatment LS by MRE ( 7.5 kPa), higher AFP (≥ 400 ng/mL), and advanced tumor stage (mUICC IVb) were associated with poor OS in advanced HCC patients treated with sorafenib. • Higher pretreatment LS by MRE was associated with developing significant (grade ≥ 3) liver injury during sorafenib treatment, which required termination of the therapy. • Patients with high pretreatment LS by MRE should be monitored carefully for potential liver injury during sorafenib treatment.

Hepatocellular carcinoma (HCC) has a high rate of cancer recurrence (up to 70%) in patients who undergo surgical resection. We investigated prognostic gene signatures for predicting HCC recurrence using in silico gene expression analysis. Recurrence-associated gene candidates were chosen by a comparative analysis of gene expression profiles from two independent whole-transcriptome datasets in patients with HCC who underwent surgical resection. Five promising candidate genes, CETN2, HMGA1, MPZL1, RACGAP1, and SNRPB were identified, and the expression of these genes was evaluated using quantitative reverse transcription PCR in the validation set (n = 57). The genes CETN2, HMGA1, RACGAP1, and SNRPB, but not MPZL1, were upregulated in patients with recurrent HCC. In addition, the combination of HMGA1 and MPZL1 demonstrated the best area under the curve (0.807, 95% confidence interval [CI] = 0.681–0.899) for predicting HCC recurrence. In terms of clinicopathological correlation, CETN2, MPZL1, RACGAP1, and SNRPB were upregulated in patients with microvascular invasion, and the expression of MPZL1 and SNRPB was increased in proportion to the Edmonson tumor differentiation grade. Additionally, overexpression of CETN2, HMGA1, and RACGAP1 correlated with poor overall survival (OS) and disease-free survival (DFS) in the validation set. Finally, Cox regression analysis showed that the expression of serum alpha-fetoprotein and RACGAP1 significantly affected OS, whereas platelet count, microvascular invasion, and HMGA1 expression significantly affected DFS. In conclusion, HMGA1 and RACGAP1 may be potential prognostic biomarkers for predicting the recurrence of HCC after surgical resection.

Exosomal microRNAs (exo-miRs) have been promising cancer biomarkers. MiRs in hepatocellular carcinoma (HCC) cell-derived exosomes (HEX) were analyzed to identify reliable serum biomarkers for HCC. To detect overexpressed miRs in HEX, extracted exosomal small RNAs from human HCC cell lines and normal hepatocytes were sequenced and analyzed. Clinical significance of the overexpressed miRs in HEX was evaluated using quantitative real-time PCR (qRT-PCR) on serum samples of a validation cohort consisting of 28 healthy individuals, 60 with chronic liver disease, and 90 with HCC. We found 49 significantly overexpressed miRs in HEX compared to a normal hepatocyte. Among them, miR-10b-5p, miR-18a-5p, miR-215-5p, and miR-940 were overexpressed in HCC tissues and also associated with prognosis of HCC in the analysis of a public omics database. qRT-PCR analysis of the four serum exo-miRs in the validation cohort revealed serum exo-miR-10b-5p as a promising biomarker for early-stage HCC with 0.934 area under the curve (AUC) (sensitivity, 90.7%, specificity, 75.0%, cutoff value, 1.8-fold). Overexpression of serum exo-miR-215-5p was found to be significantly associated with poor disease-free survival in patients with HCC. Serum exo-miR-10b-5p is a potential biomarker for early-stage HCC, while serum exo-miR-215-5p can be used as prognostic biomarker for HCC.

Magnetic resonance elastography (MRE) is a non-invasive tool for measuring liver stiffness (LS) with high diagnostic accuracy. This study investigated whether quantified LS by MRE could predict early recurrence of patients with hepatocellular carcinoma (HCC) within the Milan criteria.A prospectively collected cohort, which included the HCC patients who underwent MRE before treatment (an HCC-MRE cohort), was analyzed. In the HCC-MRE cohort, only patients under the Milan criteria, who underwent hepatic resection, radiofrequency ablation (RFA), or transarterial chemoembolization (TACE), were reviewed. We investigated whether LS assessed by MRE was an independent predictor of early recurrence using Cox regressions and Kaplan-Meier analyses.A total of 192 HCC patients under the Milan criteria who underwent hepatic resection (n = 96), RFA (n = 23), or TACE (n = 73) were included. Higher LS ratings (kPa; hazard ratio [HR] = 1.12; 95% confidence interval [CI] = 1.01-1.25; p = 0.040) emerged as an independent risk factor for early tumor recurrence. In the subgroup analysis, higher LS ratings were associated with higher risks of early HCC recurrence in both the resection/RFA group ( 4.5 kPa; HR = 2.95; 95% CI = 1.26-6.94; p = 0.013) and the TACE group ( 6 kPa; HR = 2.94; 95% CI = 1.27-6.83; p = 0.012).LS assessed by MRE was an independent predictor of early recurrence among HCC patients under the Milan criteria after achieving a complete response.• Liver parenchymal stiffness measured by MRE predicts early recurrence of treated HCC under Milan criteria. • A liver stiffness 5.5 kPa was associated with worse recurrence-free survival. • Patients with high pre-treatment LS may benefit from stringent follow-up.