Carboxyl-terminal truncated HBx regulates a distinct microRNA transcription program in Hepatocellular carcinoma development

Background: The biological pathways and functional properties by which misexpressed microRNAs (miRNAs) contribute to liver carcinogenesis have been intensively investigated. However, little is known about the upstream mechanisms that deregulate miRNA expressions in this process. In hepatocellular carcinoma (HCC), hepatitis B virus (HBV) X protein (HBx), a transcriptional trans-activator, is frequently expressed in truncated form without carboxyl-terminus but its role in miRNA expression and HCC development is unclear. Methods: Human non-tumorigenic hepatocytes were infected with lentivirus-expressing full-length and carboxyl-terminal truncated HBx (Ct-HBx) for cell growth assay and miRNA profiling. Chromatin immunoprecipitation microarray was performed to identify the miRNA promoters directly associated with HBx. Direct transcriptional control was verified by luciferase reporter assay. The differential miRNA expressions were further validated in a cohort of HBV-associated HCC tissues using real-time PCR. Results: Hepatocytes expressing Ct-HBx grew significantly faster than the full-length HBx counterparts. Ct-HBx decreased while full-length HBx increased the expression of a set of miRNAs with growth-suppressive functions. Interestingly, Ct-HBx bound to and inhibited the transcriptional activity of some of these miRNA promoters. Notably, some of the examined repressed-miRNAs (miR-26a, -29c, -146a and -190) were also significantly down-regulated in a subset of HCC tissues with carboxyl-terminal HBx truncation compared to their matching non-tumor tissues, highlighting the clinical relevance of our data. Conclusion: Our results suggest that Ct-HBx directly regulates miRNA transcription and in turn promotes hepatocellular proliferation, thus revealing a viral contribution of miRNA deregulation during hepatocarcinogenesis. © 2011 Yip et al.published_or_final_versio

Identification of <em>BRCA1/2</em> Founder Mutations in Southern Chinese Breast Cancer Patients Using Gene Sequencing and High Resolution DNA Melting Analysis

<div><h3>Background</h3><p>Ethnic variations in breast cancer epidemiology and genetics have necessitated investigation of the spectra of <em>BRCA1</em> and <em>BRCA2</em> mutations in different populations. Knowledge of <em>BRCA</em> mutations in Chinese populations is still largely unknown. We conducted a multi-center study to characterize the spectra of <em>BRCA</em> mutations in Chinese breast and ovarian cancer patients from Southern China.</p> <h3>Methodology/Principal Findings</h3><p>A total of 651 clinically high-risk breast and/or ovarian cancer patients were recruited from the Hong Kong Hereditary Breast Cancer Family Registry from 2007 to 2011. Comprehensive <em>BRCA1</em> and <em>BRCA2</em> mutation screening was performed using bi-directional sequencing of all coding exons of <em>BRCA1</em> and <em>BRCA2</em>. Sequencing results were confirmed by in-house developed full high resolution DNA melting (HRM) analysis. Among the 451 probands analyzed, 69 (15.3%) deleterious <em>BRCA</em> mutations were identified, comprising 29 in <em>BRCA1</em> and 40 in <em>BRCA2</em>. The four recurrent <em>BRCA1</em> mutations (c.470_471delCT, c.3342_3345delAGAA, c.5406+1_5406+3delGTA and c.981_982delAT) accounted for 34.5% (10/29) of all <em>BRCA1</em> mutations in this cohort. The four recurrent <em>BRCA2</em> mutations (c.2808_2811delACAA, c.3109C>T, c.7436_7805del370 and c.9097_9098insA) accounted for 40% (16/40) of all <em>BRCA2</em> mutations. Haplotype analysis was performed to confirm 1 <em>BRCA1</em> and 3 <em>BRCA2</em> mutations are putative founder mutations. Rapid HRM mutation screening for a panel of the founder mutations were developed and validated.</p> <h3>Conclusion</h3><p>In this study, our findings suggest that <em>BRCA</em> mutations account for a substantial proportion of hereditary breast/ovarian cancer in Southern Chinese population. Knowing the spectrum and frequency of the founder mutations in this population will assist in the development of a cost-effective rapid screening assay, which in turn facilitates genetic counseling and testing for the purpose of cancer risk assessment.</p> </div

Modulation of LMP2A Expression by a Newly Identified Epstein-Barr Virus-Encoded MicroRNA miR-BART2212

Infection with the Epstein-Barr virus (EBV) is a strong predisposing factor in the development of nasopharyngeal carcinoma (NPC). Many viral gene products including EBNA1, LMP1, and LMP2 have been implicated in NPC tumorigenesis, although the de novo control of these viral oncoproteins remains largely unclear. The recent discovery of EBV-encoded viral microRNA (miRNA) in lymphoid malignancies has prompted us to examine the NPC-associated EBV miRNA. Using large-scale cloning analysis on EBV-positive NPC cells, two novel EBV miRNA, now named miR-BART21 and miR-BART22, were identified. These two EBV-encoded miRNA are abundantly expressed in most NPC samples. We found two nucleotide variations in the primary transcript of miR-BART22, which we experimentally confirmed to augment its biogenesis in vitro and thus may underline the high and consistent expression of miR-BART22 in NPC tumors. More importantly, we determined that the EBV latent membrane protein 2A (LMP2A) is the putative target of miR-BART22. LMP2A is a potent immunogenic viral antigen that is recognized by the cytotoxic T cells; down-modulation of LMP2A expression by miR-BART22 may permit escape of EBV-infected cells from host immune surveillance. Taken together, we demonstrated that two newly identified EBV-encoded miRNA are highly expressed in NPC. Specific sequence variations on the prevalent EBV strain in our locality might contribute to the higher miR-BART22 expression level in our NPC samples. Our findings emphasize the role of miR-BART22 in modulating LMP2A expression, which may facilitate NPC carcinogenesis by evading the host immune response

Difference plot showing the <i>BRCA1</i> recurrent or founder mutations relative to the wild type controls.

<p>The melting profile of a wild type (WT) control is chosen as a horizontal base line and the relative differences in the melting of other samples are plotted relative to this baseline. Each trace represents the amplicon from a different individual's DNA sample. Melt curves of the <i>BRCA1</i> founder mutations (green/red) were plotted against melt curve of the wild type (blue).</p

HRM PCR primer sequences for the <i>BRCA</i> recurrent or founder mutations.

*<p>M13F: TGTAAAACGACGGCCAGT.</p>#<p>M13R: CAGGAAACAGCTATGACC.</p

Spectrum of <i>BRCA</i> pathogenic mutations identified.

<p> <b><i>Abbreviation</i></b><i>: SS, Splice-site mutation; NS, Nonsense mutation; FS, Frame-shift mutation; IFD, In-frame deletion mutation. Recurrent mutations are highlighted in bold.</i></p

Characteristics of all probands with <i>BRCA1</i>/<i>2</i> recurrent/founder mutations, with other mutations and without mutation (N = 451).

<p> <i>Abbreviations: F, Female; M, Male; GD, Guangdong; N/A, Not available; BC, Breast cancer; OC, Ovarian cancer;</i></p