The c-myc transcriptional suppressor, far-upstream element (FUSE)-binding protein (FBP)-interacting repressor (FIR), is alternatively spliced in colorectal cancer tissue (Matsushita et al., Cancer Res 2006). Recently, the knockdown of SAP155 pre-mRNA-splicing factor, a subunit of SF3b, was reported to disturb FIR pre-mRNA splicing and yield FIRDexon2, an exon 2-spliced variant of FIR, which lacks c-myc repression activity. In the present study, novel splicing variants of FIR, D3 and D4, were also generated by SAP155 siRNA, and these variants were found to be activated in human colorectal cancer tissue. Furthermore, the expression levels of FIR variant mRNA were examined in the peripheral blood of colorectal cancer patients and healthy volunteers to assess its potency for tumor detection. As expected, circulating FIR variant mRNA in the peripheral blood of cancer patients were significantly overexpressed compared to that in healthy volunteers. In particular, the area under the receiving operating characteristic curve of FIR, FIRDexon2 or FIRDexon2 ⁄ FIR, was greater than those of conventional carcinoembryonic antigen or carbohydrate antigen 19-9. In addition, FIRDexon2 or FIR mRNA expression in the peripheral blood was significantly reduced after operative removal of colorectal tumors. Thus, circulating FIR and FIRDexon2 mRNA are potential novel screening markers for colorectal cancer testing with conventional carcinoembryonic antigen and or carbohydrate antigen 19-9. Taken together, our results indicate that overexpression of FIR and its splicing variants in colorectal cancer directs feed-forward or addicted circuit c-myc transcriptional activation. Clinical implications for colorectal cancers of novel FIR splicing variants are also discussed in the present paper. (Cancer Sci 2013; 104: 149-156) S everal non-invasive biomarkers, including DNA alterations, mRNA and peptides ⁄ proteins, are specifically expressed in tumor cells.(1,2) Far-upstream element (FUSE) is a sequence required for the proper expression of the human c-myc gene.(3) FUSE is located 1.5 kb upstream of the c-myc promoter P1 and binds the FUSE binding protein (FBP), a transcription factor that stimulates c-myc expression in a FUSE-dependent manner.(4-6) Yeast two-hybrid analysis has revealed that FBP binds to a protein, the FBP interacting repressor (FIR). FIR represses c-myc transcription by delaying promoter escape. (7,8) FIRDexon2, an exon2-splice variant of FIR that lacks c-myc repression activity, has been frequently identified in human primary colorectal cancers.(9) SAP155, a subunit of SF3b, and FIR have been reported to form a complex.(10) SAP155 is required for FIR pre-mRNA splicing. SAP155 ⁄ FIR ( ⁄ FIRDexon2) forms a complex and is mutually upregulated in cancers.(11) Despite the splice mutation, FIRDexon2 retains binding activity to FUSE and is able to displace repression-competent FIR from FUSE, which thwarts FIR-mediated transcriptional repression by FUSE. (11) Up to 60% of all human genes have at least one alternative splice v...

The HBV vaccine failure rate assessed by using PCR analysis was 3.4% (six of 176) in the Chinese adult population undergoing the HBV vaccination program. Hepatitis B virus variants with missense mutation within the 'a' determinant were responsible in most cases.

Next-generation sequencing (NGS) is a revolutionary sequencing technology for analyzing genomes. However, preprocessing methods for mitochondrial DNA (mtDNA) sequencing remain complex, and it is required to develop an authenticated preprocessing method. Here, we developed a simple and easy preprocessing method based on isothermal rolling circle mtDNA amplification using commercially available reagents. Isothermal amplification of mtDNA was successfully performed using both nanoliter quantities of plasma directly and 25 ng of total DNA extracted from blood or tissue samples. Prior to mtDNA amplification, it was necessary to treat the extracted total DNA with Exonuclease V, but it was not required to treat plasma. The NGS libraries generated from the amplified mtDNA provided sequencing coverage of the entire human mitochondrial genome. Furthermore, the sequencing results successfully detected heteroplasmy in patient samples, with called mutations and variants matching those from previous, independent, Sanger sequencing analysis. Additionally, a novel single nucleotide variant was detected in a healthy volunteer. The successful analysis of mtDNA using very small samples from patients is likely to be valuable in clinical medicine, as it could reduce patient discomfort by reducing sampling-associated damage to tissues. Overall, the simple and convenient preprocessing method described herein may facilitate the future development of NGS-based clinical and forensic mtDNA tests.

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Overexpression of alternative splicing of far upstream element binding protein 1 (FUBP1) interacting repressor (FIR; poly(U) binding splicing factor 60 [PUF60]) and cyclin E were detected in esophageal squamous cell carcinomas (ESCC). Accordingly, the expression of FBW7 was examined by which cyclin E is degraded as a substrate via the proteasome system. Expectedly, FBW7 expression was decreased significantly in ESCC. Conversely, c-myc gene transcriptional repressor FIR (alias PUF60; U2AF-related protein) and its alternative splicing variant form (FIRΔexon2) were overexpressed in ESCC. Further, anticancer drugs (cis-diaminedichloroplatinum/cisplatin [CDDP] or 5-fluorouracil [5-FU]) and knockdown of FIR by small interfering RNA (siRNA) increased cyclin E while knockdown of FIRΔexon2 by siRNA decreased cyclin E expression in ESCC cell lines (TE1, TE2, and T.Tn) or cervical SCC cells (HeLa cells). Especially, knockdown of SAP155 (SF3b1), a splicing factor required for proper alternative splicing of FIR pre-mRNA, decreased cyclin E. Therefore, disturbed alternative splicing of FIR generated FIR/FIRΔexon2 with cyclin E overexpression in esophageal cancers, indicating that SAP155 siRNA potentially rescued FBW7 function by reducing expression of FIR and/or FIRΔexon2. Remarkably, Three-dimensional structure analysis revealed the hypothetical inhibitory mechanism of FBW7 function by FIR/FIRΔexon2, a novel mechanism of cyclin E overexpression by FIR/FIRΔexon2-FBW7 interaction was discussed. Clinically, elevated FIR expression potentially is an indicator of the number of lymph metastases and anti-FIR/FIRΔexon2 antibodies in sera as cancer diagnosis, indicating chemical inhibitors of FIR/FIRΔexon2-FBW7 interaction could be potential candidate drugs for cancer therapy. In conclusion, elevated cyclin E expression was, in part, induced owing to potential FIR/FIRΔexon2–FBW7 interaction in ESCC.

The results of this study clearly indicate that the 5.9 kDa peak is a promising novel biological marker for moderate alcohol consumption.

FUSE-binding protein (FBP)-interacting repressor (FIR) is a c-myc transcriptional suppressor. A splice variant of FIR that lacks exon 2 in the transcriptional repressor domain (FIRΔexon2) upregulates c-myc transcription by inactivating wild-type FIR. The ratio of FIRΔexon2/FIR mRNA was increased in human colorectal cancer and hepatocellular carcinoma tissues. Because FIRΔexon2 is considered to be a dominant negative regulator of FIR, FIR heterozygous knockout (FIR+/−) C57BL6 mice were generated. FIR complete knockout (FIR−/−) was embryonic lethal before E9.5; therefore, it is essential for embryogenesis. This strongly suggests that insufficiency of FIR is crucial for carcinogenesis. FIR+/− mice exhibited prominent c-myc mRNA upregulation, particularly in the peripheral blood (PB), without any significant pathogenic phenotype. Furthermore, elevated FIRΔexon2/FIR mRNA expression was detected in human leukemia samples and cell lines. Because the single knockout of TP53 generates thymic lymphoma, FIR+/−TP53−/− generated T-cell type acute lymphocytic/lymphoblastic leukemia (T-ALL) with increased organ or bone marrow invasion with poor prognosis. RNA-sequencing analysis of sorted thymic lymphoma cells revealed that the Notch signaling pathway was activated significantly in FIR+/−TP53−/− compared with that in FIR+/+TP53−/− mice. Notch1 mRNA expression in sorted thymic lymphoma cells was confirmed using qRT-PCR. In addition, flow cytometry revealed that c-myc mRNA was negatively correlated with FIR but positively correlated with Notch1 in sorted T-ALL/thymic lymphoma cells. Moreover, the knockdown of TP53 or c-myc using siRNA decreased Notch1 expression in cancer cells. In addition, an adenovirus vector encoding FIRΔexon2 cDNA increased bleomycin-induced DNA damage. Taken together, these data suggest that the altered expression of FIRΔexon2 increased Notch1 at least partially by activating c-Myc via a TP53-independent pathway. In conclusion, the alternative splicing of FIR, which generates FIRΔexon2, may contribute to both colorectal carcinogenesis and leukemogenesis.

Based on the genetic background of cancer, we have been trying to develop novel diagnostic and therapeutic strategies against human cancers. c-myc gene activation has been detected in many human cancers, indicating a key role of c-myc in tumor development. Thus targeting c-myc gene suppression is a promising strategy for cancer treatment. Recently, an interaction between FIR (FUSE-Binding Protein-Interacting Repressor) and TFIIH/p89/XPB helicase was found to repress c-myc transcription and so might be important for suppressing tumor formation. Previously, we have shown that the expression of splicing variant of FIR is elevated in colorectal cancer tissues and promotes tumor development by disabling FIR-repression to sustain high levels of c-Myc, opposing apoptosis in cancer cells. In this study, FIR recombinant adenovirus vector induces tumor growth suppression against tumor xenografts in animal model experiment. Together, one clue to the development of cancer diagnosis and therapies directed against c-Myc may go through FIR and its splicing variant.