Integration of the viral DNA into host chromosomes was found in most of the hepatitis B virus (HBV)–related hepatocellular carcinomas (HCCs). Here we devised a massive anchored parallel sequencing (MAPS) method using next-generation sequencing to isolate and sequence HBV integrants. Applying MAPS to 40 pairs of HBV–related HCC tissues (cancer and adjacent tissues), we identified 296 HBV integration events corresponding to 286 unique integration sites (UISs) with precise HBV–Human DNA junctions. HBV integration favored chromosome 17 and preferentially integrated into human transcript units. HBV targeted genes were enriched in GO terms: cAMP metabolic processes, T cell differentiation and activation, TGF beta receptor pathway, ncRNA catabolic process, and dsRNA fragmentation and cellular response to dsRNA. The HBV targeted genes include 7 genes (PTPRJ, CNTN6, IL12B, MYOM1, FNDC3B, LRFN2, FN1) containing IPR003961 (Fibronectin, type III domain), 7 genes (NRG3, MASP2, NELL1, LRP1B, ADAM21, NRXN1, FN1) containing IPR013032 (EGF-like region, conserved site), and three genes (PDE7A, PDE4B, PDE11A) containing IPR002073 (3′, 5′-cyclic-nucleotide phosphodiesterase). Enriched pathways include hsa04512 (ECM-receptor interaction), hsa04510 (Focal adhesion), and hsa04012 (ErbB signaling pathway). Fewer integration events were found in cancers compared to cancer-adjacent tissues, suggesting a clonal expansion model in HCC development. Finally, we identified 8 genes that were recurrent target genes by HBV integration including fibronectin 1 (FN1) and telomerase reverse transcriptase (TERT1), two known recurrent target genes, and additional novel target genes such as SMAD family member 5 (SMAD5), phosphatase and actin regulator 4 (PHACTR4), and RNA binding protein fox-1 homolog (C. elegans) 1 (RBFOX1). Integrating analysis with recently published whole-genome sequencing analysis, we identified 14 additional recurrent HBV target genes, greatly expanding the HBV recurrent target list. This global survey of HBV integration events, together with recently published whole-genome sequencing analyses, furthered our understanding of the HBV–related HCC.

BackgroundSOX2 is a key gene implicated in maintaining the stemness of embryonic and adult stem cells. SOX2 appears to re-activate in several human cancers including glioblastoma multiforme (GBM), however, the detailed response program of SOX2 in GBM has not yet been defined.ResultsWe show that knockdown of the SOX2 gene in LN229 GBM cells reduces cell proliferation and colony formation. We then comprehensively characterize the SOX2 response program by an integrated analysis using several advanced genomic technologies including ChIP-seq, microarray profiling, and microRNA sequencing. Using ChIP-seq technology, we identified 4883 SOX2 binding regions in the GBM cancer genome. SOX2 binding regions contain the consensus sequence wwTGnwTw that occurred 3931 instances in 2312 SOX2 binding regions. Microarray analysis identified 489 genes whose expression altered in response to SOX2 knockdown. Interesting findings include that SOX2 regulates the expression of SOX family proteins SOX1 and SOX18, and that SOX2 down regulates BEX1 (brain expressed X-linked 1) and BEX2 (brain expressed X-linked 2), two genes with tumor suppressor activity in GBM. Using next generation sequencing, we identified 105 precursor microRNAs (corresponding to 95 mature miRNAs) regulated by SOX2, including down regulation of miR-143, -145, -253-5p and miR-452. We also show that miR-145 and SOX2 form a double negative feedback loop in GBM cells, potentially creating a bistable system in GBM cells.ConclusionsWe present an integrated dataset of ChIP-seq, expression microarrays and microRNA sequencing representing the SOX2 response program in LN229 GBM cells. The insights gained from our integrated analysis further our understanding of the potential actions of SOX2 in carcinogenesis and serves as a useful resource for the research community.

SOX2 is an important stem cell marker and plays important roles in development and carcinogenesis. However, the role of SOX2 in Epithelial-Mesenchymal Transition has not been investigated. We demonstrated, for the first time, that SOX2 is involved in the Epithelial-Mesenchymal Transition (EMT) process as knock downof SOX2 in colorectal cancer (CRC) SW620 cells induced a Mesenchymal-Epithelial Transition (MET) process with recognized changes in the expression of key genes involved in the EMT process including E-cadherin and vimentin. In addition, we provided a link between SOX2 activity and the WNT pathway by showing that knock down of SOX2 reduced the WNT pathway activity in colorectal cancer (CRC) cells. We further demonstrated that SOX2 is involved in cell migration and invasion in vitro and in metastasis in vivo for CRC cells, and that the process might be mediated through the MMP2 activity. Finally, an IHC analysis of 44 cases of colorectal cancer patients suggested that SOX2 is a prognosis marker for metastasis of colorectal cancers.

Glioblastoma is the most common and aggressive primary brain tumor. MicroRNAs (miRNAs) are a set of noncoding RNA of about 20*22 nt in length and they play regulatory roles such as regulating the expression of proteins. Altered miRNA expression is related to cancers, including glioblastoma. In this report, we used deep sequencing to explore the miRNA profiles of glioblastoma and normal brain tissues. We found 875 and 811 known miRNA and miRNA* in glioblastoma and normal brain tissue, respectively, representing the largest characterization of the miRNAs in GBM so far. 33 of them were upregulated in glioblastoma, including miR-21, which is well known as an oncomir, while 40 of them were downregulated. Using miR-10b, miR-124, miR-433, and miR-92b as examples, we verified the data by quantitative RT-PCR, suggesting that deep sequencing was able to capture the expression profiles of miRNAs. In addition, we found 18 novel miRNA and 16 new miRNA* in glioblastoma and normal brain tissues. This report provides a useful resource for future studies of the roles of miRNAs in the pathogenesis and early detection of glioblastoma.

Glioblastoma (GBM) proliferation is a multistep process during which the expression levels of many genes that control cell proliferation, cell death, and genetic stability are altered. MicroRNAs (miRNAs) are emerging as important modulators of cellular signaling, including cell proliferation in cancer. In this study, using next generation sequencing analysis of miRNAs, we found that miR-127-3p was downregulated in GBM tissues compared with normal brain tissues; we validated this result by RT-PCR. We further showed that DNA demethylation and histone deacetylase inhibition resulted in downregulation of miR-127-3p. We demonstrated that miR-127-3p overexpression inhibited GBM cell growth by inducing G1-phase arrest both in vitro and in vivo. We showed that miR-127-3p targeted SKI (v-ski sarcoma viral oncogene homolog [avian]), RGMA (RGM domain family, member A), ZWINT (ZW10 interactor, kinetochore protein), SERPINB9 (serpin peptidase inhibitor, clade B [ovalbumin], member 9), and SFRP1 (secreted frizzled-related protein 1). Finally, we found that miR-127-3p suppressed GBM cell growth by inhibiting tumor-promoting SKI and activating the tumor suppression effect of transforming growth factor-b (TGF-b) signaling. This study showed, for the first time, that miR-127-3p and its targeted gene SKI, play important roles in GBM and may serve as potential targets for GBM therapy.