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      A Serum MicroRNA Panel as Potential Biomarkers for Hepatocellular Carcinoma Related with Hepatitis B Virus

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          Abstract

          Background

          The identification of new high-sensitivity and high-specificity markers for HCC are essential. We aimed to identify serum microRNAs (miRNAs) as biomarkers to be used in diagnosing hepatitis B virus (HBV) –related hepatocellular carcinoma (HCC).

          Methods

          We investigated serum miRNA expression in (261 HCC patients, 233 cirrhosis patients, and 173 healthy controls), recruited between August 2010 and June 2013. An initial screening of miRNA expression by Illumina sequencing was performed using serum samples pooled from HCC patients and controls. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to evaluate the expression of selected miRNAs. A logistic regression model was constructed using a training cohort (n = 357) and then validated using an independent cohort (n = 241). The area under the receiver operating characteristic curve (AUC) was used to evaluate the accuracy of the use of the biomarkers for disease diagnosis.

          Results

          We identified 8 miRNAs (hsa-miR-206, hsa-miR-141-3p, hsa-miR-433-3p, hsa-miR-1228-5p, hsa-miR-199a-5p, hsa-miR-122-5p, hsa-miR-192-5p, and hsa-miR-26a-5p) and constructed an miRNA set that provided high diagnostic accuracy for HCC (AUC = 0.887 and 0.879 for training and validation sets, respectively). The miRNAs could also be used to differentiate HCC patients from healthy (AUC = 0.893) and cirrhosis (AUC = 0.892) patients.

          Conclusions

          We identified a serum of miRNA panel that has considerable clinical value in HCC diagnosis.

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          Most cited references27

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          Normalization of microRNA expression levels in quantitative RT-PCR assays: identification of suitable reference RNA targets in normal and cancerous human solid tissues.

          Proper normalization is a critical but often an underappreciated aspect of quantitative gene expression analysis. This study describes the identification and characterization of appropriate reference RNA targets for the normalization of microRNA (miRNA) quantitative RT-PCR data. miRNA microarray data from dozens of normal and disease human tissues revealed ubiquitous and stably expressed normalization candidates for evaluation by qRT-PCR. miR-191 and miR-103, among others, were found to be highly consistent in their expression across 13 normal tissues and five pair of distinct tumor/normal adjacent tissues. These miRNAs were statistically superior to the most commonly used reference RNAs used in miRNA qRT-PCR experiments, such as 5S rRNA, U6 snRNA, or total RNA. The most stable normalizers were also highly conserved across flash-frozen and formalin-fixed paraffin-embedded lung cancer tumor/NAT sample sets, resulting in the confirmation of one well-documented oncomir (let-7a), as well as the identification of novel oncomirs. These findings constitute the first report describing the rigorous normalization of miRNA qRT-PCR data and have important implications for proper experimental design and accurate data interpretation.
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            Essential metabolic, anti-inflammatory, and anti-tumorigenic functions of miR-122 in liver.

            miR-122, an abundant liver-specific microRNA (miRNA), regulates cholesterol metabolism and promotes hepatitis C virus (HCV) replication. Reduced miR-122 expression in hepatocellular carcinoma (HCC) correlates with metastasis and poor prognosis. Nevertheless, the consequences of sustained loss of function of miR-122 in vivo have not been determined. Here, we demonstrate that deletion of mouse Mir122 resulted in hepatosteatosis, hepatitis, and the development of tumors resembling HCC. These pathologic manifestations were associated with hyperactivity of oncogenic pathways and hepatic infiltration of inflammatory cells that produce pro-tumorigenic cytokines, including IL-6 and TNF. Moreover, delivery of miR-122 to a MYC-driven mouse model of HCC strongly inhibited tumorigenesis, further supporting the tumor suppressor activity of this miRNA. These findings reveal critical functions for miR-122 in the maintenance of liver homeostasis and have important therapeutic implications, including the potential utility of miR-122 delivery for selected patients with HCC and the need for careful monitoring of patients receiving miR-122 inhibition therapy for HCV.
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              MicroRNA expression, survival, and response to interferon in liver cancer.

              Hepatocellular carcinoma is a common and aggressive cancer that occurs mainly in men. We examined microRNA expression patterns, survival, and response to interferon alfa in both men and women with the disease. We analyzed three independent cohorts that included a total of 455 patients with hepatocellular carcinoma who had undergone radical tumor resection between 1999 and 2003. MicroRNA-expression profiling was performed in a cohort of 241 patients with hepatocellular carcinoma to identify tumor-related microRNAs and determine their association with survival in men and women. In addition, to validate our findings, we used quantitative reverse-transcriptase-polymerase-chain-reaction assays to measure microRNAs and assess their association with survival and response to therapy with interferon alfa in 214 patients from two independent, prospective, randomized, controlled trials of adjuvant interferon therapy. In patients with hepatocellular carcinoma, the expression of miR-26a and miR-26b in nontumor liver tissue was higher in women than in men. Tumors had reduced levels of miR-26 expression, as compared with paired noncancerous tissues, which indicated that the level of miR-26 expression was also associated with hepatocellular carcinoma. Moreover, tumors with reduced miR-26 expression had a distinct transcriptomic pattern, and analyses of gene networks revealed that activation of signaling pathways between nuclear factor kappaB and interleukin-6 might play a role in tumor development. Patients whose tumors had low miR-26 expression had shorter overall survival but a better response to interferon therapy than did patients whose tumors had high expression of the microRNA. The expression patterns of microRNAs in liver tissue differ between men and women with hepatocellular carcinoma. The miR-26 expression status of such patients is associated with survival and response to adjuvant therapy with interferon alfa. 2009 Massachusetts Medical Society
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                19 September 2014
                : 9
                : 9
                : e107986
                Affiliations
                [1 ]Department of Hepatosis, The Third Hospital of Zhenjiang Affiliated Jiangsu University, Zhenjiang, China
                [2 ]Department of Infectious Diseases, The First Affiliated Hospital of Soochow University, Suzhou, China
                The University of Hong Kong, China
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: YWT GHG. Performed the experiments: YWT TLP XJY DFW. Analyzed the data: LC XBZ. Contributed reagents/materials/analysis tools: XJY JHG. Contributed to the writing of the manuscript: YWT.

                Article
                PONE-D-14-26223
                10.1371/journal.pone.0107986
                4169601
                25238238
                f78acd45-8ffc-4eb4-a12a-ff598d4aa9f7
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 12 June 2014
                : 16 August 2014
                Page count
                Pages: 12
                Funding
                This work was supported by the Natural Science Foundation of Jiangsu Province, China (BK2011151) ( http://www.jstd.gov.cn/), Medical Project of Health Department, Jiangsu Province (H201248) ( http://www.jswst.gov.cn/), and the Social Development Project of Zhenjiang City (SH201346) ( http://kjj.zhenjiang.gov.cn/). The funders played no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Medicine and health sciences
                Gastroenterology and hepatology
                Liver diseases
                Infectious hepatitis
                Hepatitis B
                Infectious Diseases
                Viral Diseases
                Hepatitis
                Oncology
                Cancer Detection and Diagnosis
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. Our dataset is deposited in Gene Expression Omnibus(GEO) under the accession number GSE59418.

                Uncategorized
                Uncategorized

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