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The Mir-545/374A Cluster Encoded in the Ftx Lncrna Is Overexpressed in HBV-Related Hepatocellular Carcinoma and Promotes Tumorigenesis and Tumor Progression

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The Mir-545/374A Cluster Encoded in the Ftx Lncrna Is Overexpressed in HBV-Related Hepatocellular Carcinoma and Promotes Tumorigenesis and Tumor Progression The miR-545/374a Cluster Encoded in the Ftx lncRNA is Overexpressed in HBV-Related Hepatocellular Carcinoma and Promotes Tumorigenesis and Tumor Progression Qi Zhao1, Tao Li2, Jianni Qi3, Juan Liu1, Chengyong Qin1* 1 Department of Gastroenterology, Provincial Hospital Affiliated to Shandong University, Jinan, China, 2 Department of Infectious Diseases, Provincial Hospital Affiliated to Shandong University, Jinan, China, 3 Central Laboratory, Shandong Provincial Hospital affiliated to Shandong University, Jinan, China Abstract Hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC). Previous studies have shown several long noncoding RNAs (lncRNAs) play various roles in HCC progression, but no research has focused on the expression pattern of microRNA clusters encoded in lncRNAs. The Ftx gene encodes a lncRNA which harbors 2 clusters of microRNAs in its introns, the miR-374b/421 cluster and the miR-545/374a cluster. To date, no research has focused on the role of the miR-545/374a and miR-374b/421 clusters in HBV-related HCC. In this study, 66 pairs of HBV-related HCC tissue and matched non-cancerous liver tissue specimens were analyzed for the expression of the Ftx microRNA clusters. Our results showed that the miR-545/374a cluster was upregulated in HBV-HCC tissue and significantly correlated with prognosis-related clinical features, including histological grade, metastasis and tumor capsule. Transfection studies with microRNA mimics and inhibitors revealed that miR-545/374a expression promoted in vitro cell proliferation, cell migration and invasion. The wild-type HBV-genome-containing plasmid or full-length HBx protein encoding plasmid was transfected into the Bel-7402 cell line and observed for their influence on miR-545/374a expression. We found that transfection of the HBV genome or HBx alone resulted in an increase in miR-545/374a expression. Next, by monitoring the expression of sera miR-545/374a before and after surgical tumor excision, we found serum miR-545/374a was tumor-derived and exhibited a sharp decrease 25 days after tumor excision. We also examined the gender-based difference in miR-545/374a expression among HCC patients and utilized microRNA target prediction software to find the targets of miR-545/374a. One of these targets, namely estrogen-related receptor gamma (ESRRG) was inversely correlated with miR-545 expression. In conclusion, the overexpression of miR-545/374a cluster located in the Ftx lncRNA is partially responsible for a poor prognosis, and monitoring sera levels of miR-545/374a may be a useful diagnostic marker for HCC. Citation: Zhao Q, Li T, Qi J, Liu J, Qin C (2014) The miR-545/374a Cluster Encoded in the Ftx lncRNA is Overexpressed in HBV-Related Hepatocellular Carcinoma and Promotes Tumorigenesis and Tumor Progression. PLoS ONE 9(10): e109782. doi:10.1371/journal.pone.0109782 Editor: Dong-Yan Jin, University of Hong Kong, Hong Kong Received June 19, 2014; Accepted September 2, 2014; Published October 9, 2014 Copyright: ß 2014 Zhao et al. 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. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by grants from the Shandong Science and Technology Committee of China (2013GG3202192), the Postdoctoral Science Foundation of China (2013M541926), and the Postdoctoral Innovation Project Special Foundation of Shandong Province (201302031). The funders had norolein study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * Email: [email protected] Introduction to HCC still remains obscure [1]. In this study, we provide evidence that overexpression of a microRNA cluster promotes Hepatocellular carcinoma (HCC) is one of the most frequently tumorigenesis and tumor progression in HBV-related HCC. occurring cancers worldwide. A variety of risk factors are RNA has become widely suspected as the culprit behind almost associated with HCC development, including the following most every case of epigenetic regulation in a variety of diseases. Only common risk factors: hepatitis B virus (HBV) infection, hepatitis C 1% of the mammalian genome carries protein-coding potential, virus (HCV) infection, consumption of food contaminated with yet 70 to 90% is transcribed to produce a large transcriptome of aflatoxin B1, heavy alcohol intake, and nonalcoholic fatty liver long noncoding RNA (lncRNA, defined as RNA.100 nucleotides disease [1]. Due to the growing incidence of HCC, intense in length) [2]. Clear roles have emerged for several lncRNAs research efforts are currently being undertaken to understand the which participate in HCC disease progression [3,4]. Mechanisti- cellular and molecular mechanisms of the disease so that novel cally, lncRNAs target a site-specific pathway, and may directly diagnostic markers and therapeutic strategies can be developed. interact with chromatin/mRNAs and recruit histone-modifying The mechanism of HBV induced oncogenesis may involve enzymes to induce epigenetic silencing of target genes [3,5,6]. integration of viral genomic fragments into the human genome. Interestingly, few studies have reported that some of these self- An alternative hypothesis is that HBV produces regulators which functional lncRNAs are also acting as microRNA (miRNA) participate in cellular pathways that lead to carcinogenesis. precursors. The lack of experimental data on these miRNAs However, the exact mechanism by which HBV infection leads PLOS ONE | www.plosone.org 1 October 2014 | Volume 9 | Issue 10 | e109782 Ftx MicroRNA Clusters in HBV-Related HCC leaves open questions about their possible relation to malignant Kit (DaAn Gene Co., China) and amplified in a PE 5700 tumors. fluorescence PCR apparatus (Perkin-Elmer, Norwalk, USA). a- The Ftx transcript is a conserved functional lncRNA encoded fetoprotein (AFP), carcino-embryonic (CEA) and c-glutamyltrans- within the X-inactivation center (Xic). Ftx encodes 4 microRNAs ferase c(-GT) were measured by routine laboratory methods. in its introns [7]. Intron 12 encodes 1 cluster of 2 microRNAs Concrete information is shown in Table 1. No significant (miR-374b and miR-421), which is well conserved in different differences between the HBV-related HCC group and the mammalian species. Intron b encodes 1 related cluster of 2 negative control group with respect to age were found. However, microRNAs (miR-374a and miR-545), which is absent in mouse there were significant differences concerning the gender of the and rat due to mutational changes (figure 1A). A recent study patient and the levels of HBV-DNA, AFP, CEA and c-GT (data showed that miR-374a is upregulated in breast cancer tumors and not shown). promotes breast cancer metastasis by activating the Wnt/b- catenin signaling pathway [8]. It remains unknown whether the 3. Cell culture altered expression of miR-374a occurs independently from the Human hepatocellular carcinoma cell lines Bel-7402, HepG2 other 3 microRNAs or whether the Ftx microRNA clusters are (Keygen Biotech, Nanjing, China), and previously constructed regulated as a group. No studies have explored the expression of stable HBV positive HepG2.2.15 cells were maintained in the other 3 microRNAs and their potential relationship with HyClone’s media containing 10% fetal calf serum, 100 U/ml malignant tumors. This raises a critical question that our research penicillin, and 100 mg/ml streptomycin at 37uC in a humidified addresses: does Ftx microRNA clusters play a role in cancer atmosphere containing 5% CO2. Bel-7402 cells were seeded at a progression? density of 2.06105 cells/mL and starved for 24 hours before Another example of altered microRNA expression occurs in transfection. hand-foot-and-mouth disease (HFMD) [9]. In this case miR-545 expression is upregulated, upon enterovirus 71 (EV71) or 4. Plasmid transfection coxsackievirus 16 (CAV16) infection. Since viral infections can The wild-type HBV genome-containing plasmid (pCDNA3.1- alter miR-545 expression, we explored the possible relationship HBV), and the plasmid encoding full-length HBx protein (pIRSE- between Ftx microRNA cluster expression and HBV infection. HBx) were transfected using Lipofectamine 2000 (Invitrogen, CA, In this study, the expression profiles of the miR-545/374a USA) according to the manufacturer’s protocol. Empty expression cluster and the miR-421/374b cluster derived from the Ftx vectors were transfected as a negative control. Expression of transcript were examined in HBV-related HCC. Based upon plasmids in Bel-7402 cell lines was confirmed by high-sensitivity correlation analysis with clinical features, the microRNAs were fluorescent quantitative PCR and western blotting. assayed for their ability to regulate tumor progression. In vitro cell culture assays were established to confirm the biological function 5. Oligonucleotide transfection of the microRNAs and potential regulating mechanism(s). Next, MicroRNA 545 mimics, miR-545 inhibitor, miR-374a mimics, we followed a ‘‘tissue to serum, preoperative to postoperative’’ miR-374a inhibitor, and negative control duplex (designated as pattern to investigate the
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