Xizhong Shen

Metabolomic profiling of human urine in hepatocellular carcinoma patients using gas chromatography/mass spectrometry

With the technique of metabolomics, gas chromatography/mass spectrometry (GC/MS), urine or serum metabolites can be assayed to explore disease biomarkers. In this work, we present a metabolomic method to investigate the urinary metabolic difference between hepatocellular carcinoma (HCC, n - 20) male patients and normal male subjects (n - 20). The urinary endogenous metabolome was assayed using chemical derivatization followed by GC/MS. After GC/MS analysis, 103 metabolites were detected, of which 66 were annotated as known compounds. By a two sample t-test statistics with p < 0.05, 18 metabolites were shown to be significantly different between the HCC and control groups. A diagnostic model was constructed with a combination of 18 marker metabolites or together with alphafetoprotein, using principal component analysis and receiver-operator characteristic curves. The multivariate statistics of the diagnostic model yielded a separation between the two groups with an area under the curve value of 0.9275. This non-invasive technique of identifying HCC biomarkers from urine may have clinical utility.

Investigation of volatile biomarkers in liver cancer blood using solid-phase microextraction and gas chromatography/mass spectrometry.

Solid-phase microextraction (SPME) followed by gas chromatography/mass spectrometry (GC/MS) was used for the detection of liver cancer volatile biomarkers. Headspace SPME conditions (fiber coating, extraction temperature and extraction time) and desorption conditions were optimized and applied to the determination of volatiles in human blood. Between the liver cancer group (n = 19) and the normal group (n = 18), positive rates of 19 volatile compounds among the total of 47 detected were found to be different with statistical significance (p < 0.05, chi-squared test). We suggested hexanal, 1-octen-3-ol and octane, of the 19 compounds, as biomarkers of liver cancer with clinical diagnostic value. The sensitivity and specificity of 94.7% and 100% for hexanal, 84.2% and 100% for 1-octen-3-ol, and 89.5% and 100% for octane were obtained, respectively, after the cutoff values had been properly established. These results show that SPME-GC/MS is a simple, rapid and sensitive method for the investigation of volatile disease markers in human blood.

Metabolomic investigation of gastric cancer tissue using gas chromatography/mass spectrometry

Gastric cancer screening or diagnosis is mainly based on endoscopy and biopsy. The aim of this study was to identify the difference of metabolomic profile between normal and malignant gastric tissue, and to further explore tumor biomarkers. Chemical derivatization together with gas chromatography/mass spectrometry (GC/MS) was utilized to obtain the metabolomic information of the malignant and non-malignant tissues of gastric mucosae in 18 gastric cancer patients. Acquired metabolomic data was analyzed using the Wilcoxon rank sum test to find the tissue metabolic biomarkers for gastric cancer. A diagnostic model for gastric cancer was constructed using principal component analysis (PCA), and was assessed with receiver-operating characteristic (ROC) curves. Results showed that 18 metabolites were detected differently between the malignant tissues and the adjacent non-malignant tissues of gastric mucosa. Five metabolites were also detected differently between the non-invasive tumors and the invasive tumors. The diagnostic model could discriminate tumors from normal mucosae with an area under the curve (AUC) value of 0.9629, and another diagnostic model constructed for clinical staging was assessed with an AUC value of 0.969. We conclude that the metabolomic profile of malignant gastric tissue was different from normal, and that the selected tissue metabolites could probably be applied for clinical diagnosis or staging for gastric cancer.

Metabolomic study for diagnostic model of oesophageal cancer using gas chromatography/mass spectrometry.

The prognosis for oesophageal cancer is poor. Attempts have been made for the identification of biomarkers for early diagnosis. Metabolomic panel has been evaluated as potential candidate biomarkers. With gas chromatography/mass spectrometry (GC/MS) as a sensitive modality for metabolomics, various tissue metabolites can be detected and identified. We hypothesized that tissue metabolomic biomarkers may be identifiable and diagnostically useful for oesophageal cancer. We present a metabolomic method of chemical derivatization followed by GC/MS to analyze the metabolic difference in biopsied specimens between oesophageal cancer and corresponding normal mucosae obtained from 20 oesophageal cancer patients. The GC/MS data was analyzed using a two sample t-test to explore the potential metabolic biomarkers for oesophageal cancer. A diagnostic model was constructed to discriminate normal from malignant samples, using principal component analysis (PCA) and receiver-operating characteristic (ROC) curves. t-Test showed a total of 20 marker metabolites detected were found to be different with statistical significance (P<0.05). The multivariate logistic analysis yielded a complete distinction between the two groups. The diagnostic model could discriminate tumors from normal mucosae with an area under the curve (AUC) value of 1. Our findings suggest that this assay may potentially provide a new metabolomic biomarker for oesophageal cancer.

A serum metabolomic investigation on hepatocellular carcinoma patients by chemical derivatization followed by gas chromatography/mass spectrometry

The purpose of this study was to investigate the serum metabolic difference between hepatocellular carcinoma (HCC, n = 20) male patients and normal male subjects (n = 20). Serum metabolome was detected through chemical derivatization followed by gas chromatography/mass spectrometry (GC/MS). The acquired GC/MS data was analyzed by stepwise discriminant analysis (SDA) and support vector machine (SVM). The metabolites including butanoic acid, ethanimidic acid, glycerol, L-isoleucine, L-valine, aminomalonic acid, D-erythrose, hexadecanoic acid, octadecanoic acid, and 9,12-octadecadienoic acid in combination with each other gave the strongest segregation between the two groups. By applying these variables, our method provided a diagnostic model that could well discriminate between HCC patients and normal subjects. More importantly, the error count estimate for each group was 0%. The total classifying accuracy of the discriminant function tested by SVM 20-fold cross validation was 75%. This technique is different from traditional ones and appears to be a useful tool in the area of HCC diagnosis.

BIRC6 promotes hepatocellular carcinogenesis: Interaction of BIRC6 with p53 facilitating p53 degradation

The genes that encode inhibitor of apoptosis proteins (IAPs) are frequently overexpressed in human cancers. However, the expression pattern and clinical significance of BIRC6, a member of IAPs, in hepatocellular carcinoma (HCC) remains unclear. Here we investigated the role of BIRC6 in hepatocellular carcinogenesis. We used immunoblot and immunochemical analyses to determine the levels of BIRC6 in 7 hepatoma cell lines and 160 HCC specimens. We evaluated the proognostic value of BIRC6 expression and its association with clinical parameters. A lentivirus‐mediated silencing method was used to knockdown BIRC6, and the biological consequences of BIRC6 silencing in three hepatoma cell lines were investigated in vitro and in vivo. We found that BIRC6 overexpression was significantly correlated with serum ALT level and HCC vascular invasion. Patients with positive BIRC6 expression in tumor tissue had a poor survival and a high rate of recurrence. BIRC6 knockdown remarkably suppressed cell proliferation, caused G1/S arrest and sensitized hepatoma cells to sorafenib‐induced apoptosis in hepatoma cells, which was partly reversed by RNA interference targeting p53. The mechanistic study revealed that BIRC6 interacted with p53 and facilitated its degradation. The in vivo study showed that BIRC6 knockdown inhibited xenograft tumor growth and increased the sensitivity of tumor cells to sorafenib in nude mice. Taken together, these findings demonstate that BIRC6 overexpression in HCC specimens is indicative of poor prognosis and that its interaction with p53 facilitates the degradation of p53, leading to carcinogenesis and an anti‐apoptotic status.

Genome-wide association study: new genetic insights into HBV/HCV-related hepatocellular carcinoma genomes.

Abstract Hepatocellular carcinoma (HCC) is the third common cause of cancer-related death with highest prevalence in developing countries, such as Southeast China and Saharan African. The major pathogenic factors can be categorized into environmental effects and genetic variations, and it is mostly caused by hepatitis B or C virus (HBV and HCV). The geographic prevalence of chronic hepatitis B and C (CHB and CHC) varies, with HBV heavily-infected in developing countries and HCV prevalent in developed countries. The infection of either hepatitis virus B or C causes damage to the liver cells through cellular immune attack by the mechanism of inflammation. However, how liver cell injury progresses to HCC development is still poorly understood. Along with the maturation of genome-wide association study (GWAS) technology, the specific genetic mutations responsible for the progression from CHB or CHC to HCC have been identified. Moreover, the findings of similar studies for these variants are different from each other due to diverse populations. More functional experiments are warranted to confirm the precise roles of these genetic mutations in the correlations between HBV/HCV and HCC for the future clinical application.

Prognostic significance of eukaryotic initiation factor 4E in hepatocellular carcinoma

PurposeAberrant expression of eukaryotic initiation factor 4E (eIF4E) has been observed in human malignancies. However, its role in hepatocellular carcinoma (HCC) remains to be established. The purpose of this study was to detect eIF4E expression and to evaluate its clinical relevance.MethodsThe eIF4E expression was studied in ninety HCC and randomly selected thirty-one non-tumor tissues from the same patient cohort, as well as in normal hepatic and HCC cell lines. The relation between its expression and clinicopathological parameters was also analyzed.ResultseIF4E expression was higher in HCC samples and cell lines compared with that in non-tumor tissues (Pxa0<xa00.001) and hepatocyte LO2, respectively. eIF4E overexpression was significantly associated with tumor number (Pxa0=xa00.005) and incomplete encapsulation (Pxa0=xa00.001). The 5-year overall survival rate and disease-free survival rate for patients with high eIF4E expression were 32.5 and 31.2xa0%, respectively; and for low eIF4E expression, it was 67.9 and 64.4xa0%, respectively (Pxa0<xa00.001). Furthermore, subgroup analysis showed that high eIF4E level predicted poorer overall survival only for incomplete encapsulation (Pxa0=xa00.001) and cirrhosis (Pxa0<xa00.001), but not for complete encapsulation (Pxa0=xa00.804) and non-cirrhosis (Pxa0=xa00.359). Multivariate analysis revealed that eIF4E overexpression was an independent indicator for both overall survival (hazard ratio, 2.015; Pxa0=xa00.043) and disease-free survival (hazard ratio, 2.666; Pxa0=xa00.006).ConclusionseIF4E protein might result in the malignant progression of HCC, and its overexpression may be a powerful prognostic biomarker and therapeutic target for HCC patients.

microRNA-19a-3p promotes tumor metastasis and chemoresistance through the PTEN/Akt pathway in hepatocellular carcinoma

microRNA-19a-3p (miR-19a-3p) has been reported to regulate cell proliferation in hepatocellular carcinoma (HCC), but its role in HCC metastasis remains unknown. In this study, miR-19a-3p was noted to be upregulated in HCC specimens and cell lines. Aberrant expression of miR-19a-3p stimulated HCC cell metastasis, and phosphatase and tensin homolog (PTEN) was shown to be a direct target of miR-19a-3p. miR-19a-3p-mediated HCC metastasis was reversed by restoration of PTEN or could be imitated by silencing of PTEN. Modulation of miR-19a-3p also altered expression of phosphorylated Akt, a downstream mediator of PTEN. Moreover, aberrant expression of miR-19a-3p induced sorafenib resistance by regulating the PTEN/Akt pathway. In conclusion, ectopic expression of miR-19a-3p contributes to HCC metastasis and chemoresistance by modulating PTEN expression and the PTEN-dependent pathways.

Microarray Expression Profiling of microRNAs Reveals Potential Biomarkers for Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) remains a major health problem for delayed diagnosis, inefficient surveillance and poor prognosis. Recent studies have indicated that non-coding RNAs contribute to the development of new strategies for diagnosis and treatment of HCC. In the present study, we employed 18 pairs of HCC and matched non-tumor tissues for the identification of differentially expressed microRNAs (miRNAs) in HCC, among which 7 paired specimens were selected randomly for microarray detection. Totally, twenty-three miRNAs were screened out to have statistically significant differences with the threshold of P < 0.01 and fold-change ≥ 2.0 or ≤ 0.5 using miRNA microarray. In the validation stage, two miRNAs exhibited higher expression levels in the HCC tissues compared with those in the matched non-tumor tissues, whereas the expression levels of ten miRNAs were lower in the HCC tissues than those in the matched non-tumor tissues. In further analysis, eight miRNAs, including miR-4270, miR-125b-5p, miR-199a-3p, miR-10a-5p, miR-424-5p, miR-195-5p, miR-106b-5p and miR-3651, were retained, when another constraint about the signal intensity of microarray probes was established. Among these miRNAs, our study was the first to show the higher expression level of miR-3651 and the lower expression level of miR-4270 in HCC. The areas under the receiver-operating-characteristic curve values of miR-3651 and miR-4270 were 0.730 and 0.967, respectively, indicating their potential diagnostic values. Our results may help provide the context for expanded interpretations of miRNA studies involved in the progression of liver disease, potentially serving as a diagnostic tool of HCC.