Six immune-related promising biomarkers may promote hepatocellular carcinoma prognosis: a bioinformatics analysis and experimental validation.

BACKGROUND: Abnormal miRNA and mRNA expression and dysregulated immune microenvironment have been found to frequently induce the progression of hepatocellular carcinoma (HCC) in recent reports. In particular, the immune-related competing endogenous RNAs (ceRNA) mechanism plays a crucial role in HCC progression. However, the underlying mechanisms remain unclear. METHODS: Differentially expressed immune-related genes were obtained from the Immport, GEO, and TCGA databases. The mRNA and protein expression levels in HCC tissues and adjacent normal tissues were confirmed, and we further investigated the methylation levels of these biomarkers to explore their function. Then, the TIMER and TISCH databases were used to assess the relationship between immune infiltration and hub genes. Survival analysis and univariate and multivariate Cox models were used to evaluate the association between hub genes and HCC diagnosis. Hub gene expression was experimentally validated in six HCC cell lines and 15 HCC samples using qRT-PCR and immunohistochemistry. The hub genes were uploaded to DSigDB for drug prediction enrichment analysis. RESULTS: We identified that patients with abnormal miRNAs (hsa-miR-125b-5p and hsa-miR-21-5p) and their targeted genes (NTF3, PSMD14, CD320, and SORT1) had a worse prognosis. Methylation analysis of miRNA-targeted genes suggested that alteration of methylation levels is also a factor in the induction of tumorigenesis. We also found that the development of HCC progression caused by miRNA-mRNA interactions may be closely correlated with the infiltration of immunocytes. Moreover, the GSEA, GO, and KEGG analysis suggested that several common immune-related biological processes and pathways were related to miRNA-targeted genes. The results of qRT-PCR, immunohistochemistry, and western blotting were consistent with our bioinformatics results, suggesting that abnormal miRNAs and their targeted genes may affect HCC progression. CONCLUSIONS: Briefly, our study systematically describes the mechanisms of miRNA-mRNA interactions in HCC and predicts promising biomarkers that are associated with immune filtration for HCC progression.

Reproductive and Developmental Alterations on Zebrafish (Danio rerio) Upon Long-Term Exposure of Di-ethyl phthalate (DEP), Di-isononyl phthalate (DINP) and Di (2-ethylhexyl) phthalate (DEHP).

Di (2-ethylhexyl) phthalate (DEHP), di-ethyl phthalate (DEP) and di-isononyl phthalate (DINP) are all endocrine disrupting chemicals (EDCs) for organisms. However, little research has been done on the effects of long-term EDC exposure. The present study found that the zebrafish barely grew during the 7 months of DINP exposure. The fecundity rate (%) of female spawning was lower in the DEHP treatment by 4 months compared to other exposure groups. Zebrafish treated with 12.5-25.0 ppm of DEP for 4 months presented no spawning. Gonadal-somatic index (GSI) levels significantly decreased, and there were more oocytes in the atresia and peri-nucleus stage compared to the control group. In addition, the hatching rate of embryos were 71.02%, 56.92%, and 21.70% for females treated with DINP, DEHP and DEP, respectively. There were also abnormal craniofacial chondrogenesis development on 72 hpf embryos upon females treated with the three EDCs. In conclusion, long term exposure of DEHP, DINP, and DEP did not only affect the reproductive capacity of female zebrafish, but the 3 plasticizers also influence craniofacial cartilage development of its offspring.

miR-222 targets ACOX1, promotes triglyceride accumulation in hepatocytes.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is one of the most prevalent chronic liver diseases. However, the exact pathogenesis of NAFLD remains to be elucidated. Despite the association with tumors and cardiovascular diseases, the role of miR-222 in NAFLD remains unclear. The present study was to investigate the role of miR-222 in NAFLD. METHODS: Wild-type C57BL/6 mice were fed a high-fat diet for 12 weeks to induce NAFLD. Normal human liver cell line (L02) was cultured with free fatty acid (FFA)-containing medium to stimulate cell steatosis. The mRNA levels of miR-222 and acyl Coenzyme A xidase 1 (ACOX1) were detected by quantitative-PCR (Q-PCR). The prediction of ACOX1 as the target gene for miR-222 was conducted via TargetScan. The overexpression or inhibition of miR-222 was mediated by miR-222 mimics or antagomir, and intracellular triglyceride levels were measured using a triglyceride kit. Luciferase reporter assays verified ACOX1 as the target gene for miR-222. RESULTS: miR-222 was significantly elevated in both the in vivo and in vitro NAFLD models. Overexpression of miR-222 significantly increased triglyceride content in the L02 cells, while inhibition of miR-222 expression restricted the accumulation of triglyceride. Overexpression of miR-222 significantly inhibited ACOX1 expression. Transient transfection assays verified that ACOX1 3'-UTR luciferase reporter activity could be inhibited by miR-222 overexpression. CONCLUSIONS: The present study suggested that miR-222 promotes the accumulation of triglycerides by inhibiting ACOX1.

Serum microRNA signatures and metabolomics have high diagnostic value in colorectal cancer using two novel methods.

Recently, many new diagnostic biomarkers have been developed for colorectal cancer. We chose 2 methods with high diagnostic efficiency, the detection of serum microRNA and metabolomics based on gas chromatography/mass spectrometry (GC/MS), and aimed to establish appropriate models. We reviewed the diagnostic value of all microRNA identified by previous diagnostic tests. We selected appropriate microRNA to validate their diagnostic efficiency, and determined the optimal combination. We included 85 patients with colorectal cancer (CRC) and 78 healthy controls (HC) and detected the expression of the microRNA. GC/MS analysis was conducted, and we used 3 multivariate statistical methods to establish diagnostic models. The concentrations of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) were detected for comparison with the novel models. Ultimately, 62 published studies and 63 microRNA were included in this review. MiR-21, miR-29a, miR-92a, miR-125b and miR-223 were selected to further validate their diagnostic value. The serum levels of the 5 microRNA in CRC patients were significantly higher than those in the HC. The combination of miR-21, miR-29a, miR-92a and miR-125b had the highest area under the curve (AUC) at 0.952, with a sensitivity of 84.7% and a specificity of 98.7%. The GC/MS analysis exhibited an excellent diagnostic value and the AUC reached 1.0. With regard to traditional biomarkers, the AUC of CEA and CA19-9 were 0.808 and 0.705, respectively. The application prospects are good for microRNA and metabolomics as new diagnostic methods because of their high diagnostic value compared with traditional biomarkers.

Serum microRNA signatures and metabolomics have high diagnostic value in gastric cancer.

BACKGROUND: Many novel diagnostic biomarkers have been developed for gastric cancer (GC) recently. We chose two methods with high diagnostic value, the detection of serum microRNAs and metabolomics based on gas chromatography/mass spectrometry (GC/MS), and aimed to establish appropriate models. METHODS: We reviewed the diagnostic accuracies of all microRNAs identified by previous diagnostic tests. Then appropriate microRNAs and their combinations were validated the diagnostic value. We included 80 patients with GC and 82 healthy controls (HCs) and detected the expression of the microRNAs. GC/MS analysis was conducted, and we used three multivariate statistical analyses to establish diagnostic models. The concentrations of carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) were detected for comparison with the novel models. RESULTS: Sixty-seven published studies and 70 microRNAs were finally included in the systematic review. MiR-18a, miR-19a, miR-21, miR-92a, miR-199a and miR-421 were chosen to further validate their diagnostic efficiencies. Five of those microRNAs in GC patients had significantly different expression. The combination of miR-19a and miR-92a had the highest area under the curve (AUC) at 0.850 with a sensitivity of 91.3% and a specificity of 61.0%. The GC/MS analysis performed an excellent diagnostic value and the AUC reached 1.0. CONCLUSION: There is a good potential for microRNAs and GC/MS analysis as new diagnostic methods in view of their high diagnostic value compared with traditional biomarkers.

Endoscopic cyanoacrylate injection with or without lauromacrogol for gastric varices: A randomized pilot study.

BACKGROUND AND AIM: Current guidelines recommend injection of cyanoacrylate as first-line therapy to prevent gastric variceal rebleeding. The method still poses a risk of ectopic embolism, which possibly correlates with the volume of cyanoacrylate used. In this trial, we evaluated the short-term efficacy and safety of tissue adhesive injection combined with lauromacrogol for treating gastric varices. METHODS: Patients admitted to our hospital for variceal hemorrhage were enrolled and blindly randomized into two treatment groups: lauromacrogol group (lauromacrogol-cyanoacrylate-lauromacrogol) and lipiodol group (lipiodol-cyanoacrylate-lipiodol). Patient follow-up was 6 months. Primary outcome was rebleeds, and secondary outcomes were mortality, gastric varices eradication, and treatment-related adverse events. RESULTS: Between March 6, 2013 and October 16, 2013, 96 patients met the criteria. Two cases were lost to follow-up, and all treated cases were successful. No procedural-related adverse events were observed in either group. Cyanoacrylate volumes used in the lauromacrogol group were significantly less than those of the lipiodol group (0.9 ± 0.5 vs 2.0 ± 1.2 mL, P = 0.000). Eleven patients developed upper gastrointestinal rebleeding, which did not show significant difference between groups. On multivaritate analysis, portal venous thrombosis and fever were potential risk factors of rebleeding. Treatment failure, complications, gastric varices obturation, and survival did not differ between the two groups. CONCLUSION: Tissue adhesives combined with lauromacrogol is a safe therapeutic option for gastric varices, with comparably less cyanoacrylate volume used. Because of the small number of study patients, it cannot be proven to have better efficacy than without lauromacrogol. Multicenter studies with larger patient groups are necessary.

Kruppel-like factor 2 inhibit the angiogenesis of cultured human liver sinusoidal endothelial cells through the ERK1/2 signaling pathway.

Kruppel-like factor 2 (KLF2) is a crucial anti-angiogenic factor. However, its precise role in hepatic angiogenesis induced by liver sinusoidal endothelial cells (LSECs) remain unclear. This study was aimed to evaluate the effect of KLF2 on angiogenesis of LSECs and to explore the corresponding mechanism. Cultured human LSECs were infected with different lentiviruses to overexpress or suppress KLF2 expression. The CCK-8 assay, transwell migration assay and tube formation test, were used to investigate the roles of KLF2 in the proliferation, migration and vessel tube formation of LSECs, respectively. The expression and phosphorylation of ERK1/2 were detected by western blot. We discovered that the up-regulation of KLF2 expression dramatically inhibited proliferation, migration and tube formation in treated LSECs. Correspondingly, down-regulation of KLF2 expression significantly promoted proliferation, migration and tube formation in treated LSECs. Additionally, KLF2 inhibited the phosphorylation of ERK1/2 pathway, followed by the function of KLF2 in the angiogenesis of LSECs disrupted. In conclusion, KLF2 suppressed the angiogenesis of LSECs through inhibition of cell proliferation, migration, and vessel tube formation. These functions of KLF2 may be mediated through the ERK1/2 signaling pathway.

Generational effects and abnormalities in craniofacial chondrogenesis in zebrafish (Danio rerio) embryos upon maternal exposure to estrogen endocrine disrupting chemicals.

Bisphenol A (BPA) and diethyl phthalate (DEP) are estrogenic endocrine disrupting chemicals (EEDCs). The present study reconfirmed that the angle of the ceratohyal cartilage (CH) in embryos were larger from maternal BPA and E2, but smaller from DEP compared to the control. However, it is still unknown whether both the BPA and DEP chemicals disrupted the action of E2 and thereby influence the estrogen signaling pathways. Additionally, it remains unclear whether they also disrupted certain related genes in the migratory pathways of neural crest cells (NCCs) in their offspring. The present data showed that nuclear estrogen receptors and membrane estrogen receptors have different disrupted profiles among female zebrafish exposed to BPA (F-BPA), and DEP (F-DEP), and external E2 (F-E2). However, certain related genes in the migratory pathways of NCCs in embryos from F-BPA and F-E2 such as the sox10, chm1, and tgfbr1a mRNA expressions showed a positive relationship compared with CH angles; the gene expressions of sox9a, smad3, and col2a1a and the CH angles of embryos exhibited an opposite relationship upon F-DEP treatments. Thus, we suggested that the genes involved in NCCs migration were potentially induced by the residual maternal DEP contents. Two sets of genes, chm1/tgfb3 and chm1/gper1, exhibited an identical profile in the ovary and its offspring at 2 h of post fertilization upon F-E2 and F-BPA treatments, respectively. We suggested that the maternal mRNA from female to embryos were transferred before the maternal-to-zygotic transition stage.

Maternal effect and dietary supplementation of estradiol-17ß on female zebrafish (Danio rerio) affects the swimming behavior and stress-coping styles of its offspring.

Extrinsic estradiol-17ß (E2) is an environmental hormone. Female fish exposure to waterborne E2 might affect the development of craniofacial cartilage of its offspring. The present study investigates the effects of maternal E2 on larval craniofacial cartilage development by administering oral feed containing E2 (F-E2) to female zebrafish, and examines whether the swimming behavior and their stress coping style are influenced by maternal E2. The results showed that E2 contents responded to dosage and time in male fish after being fed with a diet containing E2. In addition, the E2 contents in female ovaries showed a significant increase after 250 mg of E2/kg treatment for 14 d. On the other hand, the fecundity rate of F-E2 group was lower around 2 folds than FC (female fed 0 mg of E2/kg) group. Craniofacial chondrogenesis on 72 hpf (hours of post fertilization) of F-E2 larvae were abnormalities, and a recovery to a normal developmental pattern was observed at the 96 hpf stage. The swimming speed was slower for F-E2 larvae compared to the FC larvae; and the F-E2 juvenile seems to be less responsive to cortisol (LRC) after cold stress. According to the results, we suggested that F-E2 larvae might have worse environmental adaptability than FC larvae.

Effects of bisphenol A or diethyl phthalate on cartilage development and the swimming behavior of zebrafish (Danio rerio) through maternal exposure.

Waterborne bisphenol A (BPA) and diethyl phthalate (DEP) are endocrine disruptive chemicals that impact the reproductive system of fish. The present study checks the effectiveness of the reproductive capacity on zebrafish after BPA and DEP exposure, and consequently investigates its effect on their development and the swimming behavior of its offspring. The exposure of BPA and DEP to zebrafish reveals that the levels of ovarian 17ß-estradiol (E2) and relative mRNA expression (RRE) ratios (Treatment/Control) of hepatic vitellogenin (vtg1) could be induced and decreased. Liver RRE levels in estrogen receptors (ERs) are also affected. Among the ERs, esr2a significantly increased upon BPA exposure, and esr1 and esr2b decreased upon DEP exposure. In addition, the ceratohyal cartilage (CH) angle of larvae whose mothers were exposed to BPA (F-BPA) was significantly bigger, but the CH angle of larvae whose mothers were exposed to DEP (F-DEP) was significantly smaller than the control. The swimming performance of larvae from F-DEP was more compromised than the control, but the situation did not appear in the larvae from the F-BPA group. The success rate of larvae hatching from F-BPA and F-DEP was lower than control group. Moreover, the successful rate of female spawns was higher in the control group compared to the treatment groups exposed to BPA and DEP. We suggested that both maternal BPA and DEP disrupt E2 levels, and influence the CH development of larvae, resulting in a decrease in successful hatching. Only the swimming behavior of larvae from maternal DEP was disrupted.

Amorphous flexible covalent organic networks containing redox-active moieties: a noncrystalline approach to the assembly of functional molecules.

The organization states of functional molecules have a significant impact on the properties of materials. A variety of approaches have been studied to obtain well-organized molecular assemblies. The present work shows a new non-organized state of isolated and dispersed functional molecules in amorphous flexible covalent organic networks. Redox-active quinone molecules are embedded in the amorphous network polymers. Consecutive reactions between benzoquinone (BQ) and linker molecules generate random network structures through polymerization at different rates and in multiple directions. The low-crystalline stackings of the amorphous network polymers facilitate the formation of nanoflakes through exfoliation in dispersion media. Enhanced electrochemical performances, one of the highest specific capacities in recent studies, were achieved by efficient redox reactions of the quinone moiety. The present noncrystalline approach, low-crystalline stacking of designer amorphous covalent organic networks, can be applied to construct similar nanostructured polymer materials containing functional units.

Glass-transition-induced color-changing resins containing layered polydiacetylene.

A phase-segregated composite of polystyrene (PSt) and layered polydiacetylene (PDA) was formed through simultaneous polymerization and crystallization. As the motion of PSt chains with glass transition is transferred to that of PDA, the color change was achieved by the shortening of the conjugation length with deformation of the layered structure.

Chronic active Epstein-Barr virus (CAEBV) enteritis.

BACKGROUND: Chronic active Esptein-Barr virus (CAEBV) enteritis if rarely observed in immunocompetent patients and can be often misdiagnosed as inflammatory bowel disease. CASE: We hereby present a case of CAEBV enteritis diagnosed with double-balloon enteroscopy and EBER in-situ hybridization. CONCLUSION: This case demonstrates the clinical presentation and diagnosis of CAEBV enteritis and highlights the importance of early recognition of the disease.

Role of Autophagy in Chronic Liver Inflammation and Fibrosis.

Autophagy entails the removal of dysfunctional components to maintain cellular homeostasis. Over the years, studies of autophagy demonstrated its complex physiological and pathological roles in the liver. Apart from regulation of normal metabolic functions such as glycogenolysis, glycogenesis, and ß-oxidation, autophagy also contributes to the modulation of various liver diseases. In this review, we provide a concise overview of the role of autophagy in regulating hepatic metabolism in healthy conditions and various chronic liver diseases. A well-rounded understanding of the role of autophagy may provide insight for future medical advancements in the field of hepatology.

The protective effect and mechanism of the FXR agonist obeticholic acid via targeting gut microbiota in non-alcoholic fatty liver disease.

Background: It is reported that various diseases such as non-alcoholic fatty liver disease (NAFLD) are associated with imbalance of microbiome. And FXR has been well investigated in liver diseases. Purpose: The objective of this study was to identify the role of farnesoid X receptor agonist obeticholic acid via targeting gut microbiota in NAFLD. Patients and methods: Male C57BL/6 mice were fed either a normal-chow diet or a high-fat diet (HFD). Obeticholic acid(30mg/(kg·d)) and/or a combination of antibiotics were administered orally by gavage to mice for 12 weeks. Gut microbiota profiles were established through 16S rRNA amplicon sequencing. The effects of obeticholic acid on liver inflammation, the gut barrier, endotoxemia, gut microbiome and composition of the bile acid were also investigated. Results: Obeticholic acid treatment can significantly improve obesity, circulation metabolism disorders, liver inflammation and fibrosis, and intestinal barrier damage caused by HFD. Removal of normal commensal bacteria can weaken the effect of obeticholic acid. The gut microbial structure was changed, and abundance of Blautia was increased significantly after treated with obeticholic acid. After obeticholic acid treatment, the concentration of taurine-bound bile acid caused by HFD was reduced in the liver. Conclusion: Taken together, these data suggest that obeticholic acid has aprotective effect on NAFLD via changing the components of gut microbiota, specifically increasing the abundance of Blautia.

Balloon-occluded Retrograde Transvenous Obliteration of Portovenous Shunts During Endoscopic Therapy for the Treatment of Gastric Varices.

INTRODUCTION: To explore the safety and feasibility of balloon-occluded retrograde transvenous obliteration (BRTO) of portovenous shunts during endoscopic cyanoacrylate injection for the treatment gastric varices (E-BRTO) secondary to portal hypertension. PATIENTS AND METHODS: A total of 28 cirrhotic patients with gastroesophageal varices and concurrent gastrorenal or gastrosplenorenal shunt, treated with E-BRTO, were enrolled. Operative details were recorded to evaluate the safety, feasibility, and efficacy of the procedure. Short-term follow-up was conducted to denote any incidence of distant emboli, variceal rebleeding, or mortality (Video, Supplemental Digital Content 1, http://links.lww.com/SLE/A179). RESULTS: All the patients successfully received E-BRTO without intraoperative complications. The average volume of cyanoacrylate was 2.4±1.3 mL. During the 90 days follow-up, none of the patients experienced distant systemic emboli. However, 8 patients suffered from gastrointestinal rebleeding, including one death, while 2 patients were lost to follow-up. The short-term rebleeding rate (intention to treat) was about 36% in E-BRTO for this subset of patients. CONCLUSIONS: BRTO during endoscopic cyanoacrylate injection is an alternative selection for cirrhotic patients with portovenous shunts. The procedure is feasible and procedurally safe, but the associated high rebleeding rate may require a multimodality approach.

Longitudinal monitoring of liver stiffness by acoustic radiation force impulse imaging in patients with chronic hepatitis B receiving entecavir.

BACKGROUND: Acoustic radiation force impulse (ARFI) imaging measures liver stiffness (LS), which significantly correlates with the stage of liver fibrosis in treatment-naive patients with chronic hepatitis B (CHB). AIM: We aimed to prospectively assess the clinical usefulness of ARFI during long-term antiviral therapy in CHB. METHOD: Seventy-one CHB patients were consecutively recruited and paired liver biopsies were performed in 27 patients. LS was assessed by ARFI semiannually during entecavir therapy. RESULTS: LS gradually decreased with treatment and continued to decrease after normalization of alanine aminotransaminase. Overall, 97.2% patients achieved improvement of LS, whereas 19.7% patients had more than 30% reduction in LS values between baseline and week 104. Multivariate linear regression analysis showed that the degree of LS reduction significantly correlated with the baseline levels of LS value, platelet and cholinesterase. In the 27 patients who underwent paired liver biopsies, LS significantly correlated with stage of fibrosis and inflammatory grade at baseline. LS values decreased more significantly in patients with fibrosis regression than those with static histological fibrosis. CONCLUSION: In CHB patients, LS assessed by ARFI was gradually reduced during antiviral therapy. Longitudinal monitoring of LS may be a promising noninvasive assessment of fibrosis regression during long-term antiviral therapy in CHB. Further large sample studies are needed.

Longitudinal monitoring of liver fibrosis status by transient elastography in chronic hepatitis B patients during long-term entecavir treatment.

The correlation between improvement in longitudinal liver stiffness and fibrosis regression has not been properly evaluated during long-term antiviral therapy in chronic hepatitis B (CHB) patients. In this study, liver stiffness was serially performed by FibroScan® every 26 weeks in a prospective cohort of CHB patients receiving entecavir. Results were compared with liver biopsies at baseline and week 78. A total of 120 treatment-naïve CHB patients were analyzed, in which 54 (45%) patients had fibrosis regression at 78 weeks of antiviral therapy. Liver stiffness measurement presented as a rapid-to-slow decline pattern and decreased more significantly in patients with fibrosis regression than those without improvement in fibrosis at week 78 (- 46.4 vs. - 28.6%, P < 0.001). Multivariate analysis revealed that percentage decline of 52-week and 78-week liver stiffness from baseline was independent predictive factors for fibrosis regression (OR = 46.6, P < 0.001; OR = 17.8, P = 0.002, respectively). Moreover, percentage decline of 78-week liver stiffness was moderately predictive of fibrosis regression (AUROC = 0.694, P < 0.001), while the optimal cutoff values were different between non-cirrhosis and cirrhosis patients (38 vs. 45%). Fibrosis regression could be predicted with a high positive predictive value (96%) in non-cirrhosis patients and could be excluded with a high negative predictive value (94%) in cirrhosis patients. In conclusion, serial liver stiffness measurement could be applied for longitudinal monitoring of fibrosis status in CHB patients. Continuous decline of liver stiffness after effective antiviral treatment could partially reflect fibrosis regression at an optimal cutoff value.

Serum microRNA signatures and metabolomics have high diagnostic value in hepatocellular carcinoma.

BACKGROUND: Many new diagnostic biomarkers have been developed for hepatocellular carcinoma (HCC). We selected two methods with high diagnostic value, the detection of serum microRNAs and metabolomics based on gas chromatography/mass spectrometry (GC/MS), and attempted to establish appropriate models. METHODS: We reviewed the diagnostic efficiencies of all microRNAs identified by previous diagnostic tests. Then we chose appropriate microRNAs to validate the diagnostic efficiencies, and determined the optimal combination. We included 66 patients with HCC and 82 healthy controls (HCs) and detected the expression of the microRNAs. GC/MS analysis was performed, and we used three multivariate statistical methods to establish diagnostic models. The concentration of alpha feto-protein (AFP) was determined for comparison with the novel models. RESULTS: 82 published studies and 92 microRNAs were ultimately included in this systematic review. Seven microRNAs were selected for further validation of their diagnostic efficiencies. Among which, miR-21, miR-106b, miR-125b, miR-182 and miR-224 had a significantly different expression in HCC patients. The combination of miR-21, miR-106b and miR-224 had the highest area under the curve (AUC) at 0.950 with a sensitivity of 80.3% and a specificity of 92.7%. The GC/MS analysis exhibited an excellent diagnostic value and the AUC reached 1.0. In comparison, the AUC of the traditional biomarker, AFP, was 0.755. CONCLUSION: MicroRNAs and metabolomics shows promising potential as new diagnostic methods due to their high diagnostic value compared with traditional biomarkers.