H. ny Sun

Noncoding effects of circular RNA CCDC66 promote colon cancer growth and metastasis

Circular RNA (circRNA) is a class of noncoding RNA whose functions remain mostly unknown. Recent studies indicate circRNA may be involved in disease pathogenesis, but direct evidence is scarce. Here, we characterize the functional role of a novel circRNA, circCCDC66, in colorectal cancer. RNA-Seq data from matched normal and tumor colon tissue samples identified numerous circRNAs specifically elevated in cancer cells, several of which were verified by quantitative RT-PCR. CircCCDC66 expression was elevated in polyps and colon cancer and was associated with poor prognosis. Gain-of-function and loss-of-function studies in colorectal cancer cell lines demonstrated that circCCDC66 controlled multiple pathological processes, including cell proliferation, migration, invasion, and anchorage-independent growth. In-depth characterization revealed that circCCDC66 exerts its function via regulation of a subset of oncogenes, and knockdown of circCCDC66 inhibited tumor growth and cancer invasion in xenograft and orthotopic mouse models, respectively. Taken together, these findings highlight a novel oncogenic function of circRNA in cancer progression and metastasis. Cancer Res; 77(9); 2339-50. ©2017 AACR.

In silico identification of oncogenic potential of fyn-related kinase in hepatocellular carcinoma

MOTIVATION Cancer development is a complex and heterogeneous process. It is estimated that 5-10% of human genes probably contribute to oncogenesis, whereas current experimentally validated cancer genes only cover 1% of the human genome. Thus hundreds of cancer genes may still remain to be identified. To search for new genes that play roles in carcinogenesis and facilitate cancer research, we developed a systematic workflow to use information saved in a previously established tumor-associated gene (TAG) database. RESULTS By exploiting the information of conserved protein domains from the TAG, we identified 183 potential new TAGs. As a proof-of-concept, one predicted oncogene, fyn-related kinase (FRK), which shows an aberrant digital expression pattern in liver cancer cells, was selected for further investigation. Using 68 paired hepatocellular carcinoma samples, we found that FRK was up-regulated in 52% of cases (P < 0.001). Tumorigenic assays performed in Hep3B and HepG2 cell lines revealed a significant correlation between the level of FRK expression and invasiveness, suggesting that FRK is a positive regulator of invasiveness in liver cancer cells. CONCLUSION These findings implied that FRK is a multitalented signal transduction molecule that produces diverse biological responses in different cell types in various microenvironments. In addition, our data demonstrated the accuracy of computational prediction and suggested that other predicted TAGs can be potential targets for future cancer research. AVAILABILITY The TAG database is available online at the Bioinformatics Center website: http://www.binfo.ncku.edu.tw/TAG/.

Transcriptional repression of human cad gene by hypoxia inducible factor-1α

De novo biosynthesis of pyrimidine nucleotides provides essential precursors for DNA synthesis and cell proliferation. The first three steps of de novo pyrimidine biosynthesis are catalyzed by a multifunctional enzyme known as CAD (carbamoyl phosphate synthetase-aspartate carbamoyltransferase-dihydroorotase). In this work, a decrease in CAD expression is detected in numerous cell lines and primary culture human stromal cells incubated under hypoxia or desferrioxamine (DFO)-induced HIF-1α accumulation. A putative hypoxia response element (HRE) binding matrix is identified by analyzing human cad-gene promoter using a bioinformatic approach. Promoter activity assays, using constructs harboring the cad promoter (−710/+122) and the −67/HRE fragment (25-bases), respectively, demonstrate the suppression of reporter-gene expression under hypoxia. Suppression of cad-promoter activity is substantiated by forced expression of wild-type HIF-1α but abolished by overexpression of dominant-negative HIF-1α. A chromatin immunoprecipitation assay provides further evidence that HIF-1α binds to the cad promoter in vivo. These data demonstrate that the cad-gene expression is repressed by HIF-1α, which represents a functional link between hypoxia and cell-cycle arrest.

Targeted methylation of two tumor suppressor genes is sufficient to transform mesenchymal stem cells into cancer stem/initiating cells

Although DNA hypermethylation within promoter CpG islands is highly correlated with tumorigenesis, it has not been established whether DNA hypermethylation within a specific tumor suppressor gene (TSG) is sufficient to fully transform a somatic stem cell. In this study, we addressed this question using a novel targeted DNA methylation technique to methylate the promoters of HIC1 and RassF1A, two well-established TSGs, along with a two-component reporter system to visualize successful targeting of human bone marrow-derived mesenchymal stem cells (MSC) as a model cell system. MSCs harboring targeted promoter methylations of HIC1/RassF1A displayed several features of cancer stem/initiating cells including loss of anchorage dependence, increased colony formation capability, drug resistance, and pluripotency. Notably, inoculation of immunodeficient mice with low numbers of targeted MSC resulted in tumor formation, and subsequent serial xenotransplantation and immunohistochemistry confirmed the presence of stem cell markers and MSC lineage in tumor xenografts. Consistent with the expected mechanism of TSG hypermethylation, treatment of the targeted MSC with a DNA methyltransferase inhibitor reversed their tumorigenic phenotype. To our knowledge, this is the first direct demonstration that aberrant TSG hypermethylation is sufficient to transform a somatic stem cell into a fully malignant cell with cancer stem/initiating properties.

A 2·6 Mb interval on chromosome 6q25.2–q25.3 is commonly deleted in human nasal natural killer/T-cell lymphoma

Summary. Natural killer (NK)/T‐cell lymphoma is a special subtype of rare malignant lymphoma that is more prevalent in Asia than in America and Europe. This newly characterized haemato‐lymphoid malignancy is highly aggressive and frequently present in nasal and upper aerodigestive sites. Several studies have reported the commonly deleted region of chromosome 6q21–25 in this particular type of lymphoma. To refine the smallest region of overlapping (SRO) deletion for localization of potential tumour suppressor (TS) genes, we performed loss of heterozygosity (LOH) and homozygosity mapping of deletion (HOMOD) analyses on 37 nasal and nasal‐type NK/T‐cell lymphoma patients using a panel of 25 microsatellite markers, covering the 6q21–q25 region. In all patients studied, LOH was detected in eight (89%) paired‐sample patients, while hemizygous deletion was detected in three (11%) single‐sample patients. Combination of the LOH and HOMOD results defined a distinct 3 Mb SRO on chromosome 6q25. Quantitative multiplex polymerase chain reaction analysis of 10 sequence‐tagged sites further refined the putative TS‐gene‐containing region to a 2·6 Mb interval between TIAM2 and SNX9. Eighteen known genes/Unigene clusters and 25 hypothetical genes are located within this 2·6 Mb region, but none are previously identified TS genes. These results provide a framework for future positional cloning of novel TS gene(s) at 6q25.2–q25.3.

Prostaglandin E2 Induces Fibroblast Growth Factor 9 via EP3-Dependent Protein Kinase Cδ and Elk-1 Signaling

ABSTRACT Fibroblast growth factor 9 (FGF-9) is a potent mitogen that controls the proper development of many tissues and organs. In contrast, aberrant expression of FGF-9 also results in the evolution of many human diseases, such as cancers and endometriosis. Despite its vital function being reported, the cellular and molecular mechanisms responsible for the regulation of FGF-9 expression are mostly unknown. We report here that prostaglandin E2 (PGE2) induces expression of FGF-9, which promotes endometriotic stromal cell proliferation, through the EP3 receptor-activated protein kinase Cδ (PKCδ) signaling pathway. Activation of PKCδ leads to phosphorylation of ERK1/2, and the transcription factor Elk-1 thereby promotes transcription of FGF-9. Two Elk-1 cis-binding sites located at nucleotides −1324 to −1329 and −1046 to −1051 of the human FGF-9 promoter are identified as crucial for mediating PGE2 actions. Collectively, we demonstrate, for the first time, that PGE2 can directly induce FGF-9 expression via a novel signaling pathway involving EP3, PKCδ, and a member of the ETS domain-containing transcription factor superfamily in primary human endometriotic stromal cells. Our findings may also provide a molecular framework for considering roles for PGE2 in FGF-9-related embryonic development and/or human diseases.

Presence of DAZL transcript and protein in mature human spermatozoa

OBJECTIVE To identify the DAZL transcript and protein location in human spermatozoa. DESIGN In vitro experiment. SETTING University-based reproductive genetics laboratory. PATIENT(S) A fertile volunteer. INTERVENTION(S) Reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunostaining for DAZL. MAIN OUTCOME MEASURE(S) Expression of DAZL in human spermatozoa. RESULT(S) The DAZL-specific primers yield a 128 bp product in ejaculate. A protein of approximately 33.5 kDa was detected by Western blot analysis. Immunofluorescence staining showed strong homogeneous staining in the midpiece of spermatozoa and weak staining in the principal piece. A speckled-type distribution was found in the head region. CONCLUSION(S) The DAZL transcript and protein are present in human spermatozoa. The roles of DAZL protein in sperm motility and in the sperm-oocyte interaction await further investigation.

Association of NRAMP 1 Gene Polymorphism with Susceptibility to Tuberculosis in Taiwanese Aboriginals

BACKGROUND/PURPOSE The human homologue of mice natural-resistance-associated macrophage protein 1 (Nramp 1) gene, NRAMP 1, has been reported to play a role in susceptibility to tuberculosis in humans. The aboriginal population in Taiwan has a five-fold higher prevalence of tuberculosis than people of Han ethnicity. Whether genetic factors such as NRAMP 1 polymorphism play a role in the prevalence of tuberculosis in Taiwanese aboriginals should be clarified. METHODS NRAMP 1 polymorphism was studied using a case-control design of patients with tuberculosis, including subjects of Han (Hans) and aboriginal ethnicity in Hualien, eastern Taiwan. The polymorphisms of NRAMP 1 at loci INT4, D543N, 77-385C/T, 3-UTR (CAAA) deletion and 5-(CA)n microsatellite markers were assessed by polymerase chain reaction on tissue DNA isolated from 105 aborigines and 110 Hans with tuberculosis. Comparable numbers of ethnically-matched controls were studied simultaneously. RESULTS Two NRAMP 1 polymorphisms, INT4 and 5-(CA)n, were significantly associated with susceptibility to tuberculosis in aboriginals (p = 0.0070 and p = 0.0031, respectively). However, no association was detected at the five loci of NRAMP 1 polymorphisms among Hans (p > 0.08). CONCLUSION Genetic variation in NRAMP 1 may affect susceptibility to and increase risk for tuberculosis in Taiwanese aboriginals. Although environmental factors play an important role in tuberculosis infection, genetic factors such as NRAMP 1 polymorphism may also contribute to the high prevalence of tuberculosis in Taiwanese aboriginals.

Fibroblast growth factor 9 stimulates steroidogenesis in postnatal Leydig cells.

Fibroblast growth factor 9 (FGF9) is a potent mitogen and survival factor required for morphogenesis during embryonic development and numerous biological functions at adulthood. The reproductive phenotype of mice lacking Fgf9 gene exhibits male to female sexual reversal, suggesting a crucial role of Fgf9 in male sex determination. Our previous study showed that polymorphic microsatellite of FGF9 genes is associated with 46XY female with ambiguous genitalia, implying that the aberrant expression of FGF9 might affect androgen secretion. In this study, we aimed to investigate the effect of FGF9 on testosterone production in mouse Leydig cell and to study the signalling pathways by which FGF9 modulate steroidogenesis. Our results show that mRNAs of Fgf9 and Fgfr isoforms (Fgfr2IIIc, Fgfr3 and Fgfr4) were all expressed in mouse Leydig cells. FGF9 significantly stimulates mouse Leydig cell testosterone production in a dose- and time-dependent manner. Ras-MAPK, PI3K and PKA signalling pathways are involved in the FGF9-induced steroidogenesis. These results provide supportive evidence linking the aberrant expression of FGF9 to human gonadal dysgenesis and suggest a role of FGF9 in postnatal testicular development.

Human DDX3 Interacts with the HIV-1 Tat Protein to Facilitate Viral mRNA Translation

Nuclear export and translation of intron-containing viral mRNAs are required for HIV-1 gene expression and replication. In this report, we provide evidence to show that DDX3 regulates the translation of HIV-1 mRNAs. We found that knockdown of DDX3 expression effectively inhibited HIV-1 production. Translation of HIV-1 early regulatory proteins, Tat and rev, was impaired in DDX3-depleted cells. All HIV-1 transcripts share a highly structured 5’ untranslated region (UTR) with inhibitory elements on translation of viral mRNAs, yet DDX3 promoted translation of reporter mRNAs containing the HIV-1 5’ UTR, especially with the transactivation response (TAR) hairpin. Interestingly, DDX3 directly interacts with HIV-1 Tat, a well-characterized transcriptional activator bound to the TAR hairpin. HIV-1 Tat is partially targeted to cytoplasmic stress granules upon DDX3 overexpression or cell stress conditions, suggesting a potential role of Tat/DDX3 complex in translation. We further demonstrated that HIV-1 Tat remains associated with translating mRNAs and facilitates translation of mRNAs containing the HIV-1 5’ UTR. Taken together, these findings indicate that DDX3 is recruited to the TAR hairpin by interaction with viral Tat to facilitate HIV-1 mRNA translation.