GSA: an independent development algorithm for calling copy number and detecting homologous recombination deficiency (HRD) from target capture sequencing.

BACKGROUND: The gain or loss of large chromosomal regions or even whole chromosomes is termed as genomic scarring and can be observed as copy number variations resulting from the failure of DNA damage repair. RESULTS: In this study, a new algorithm called genomic scar analysis (GSA) has developed and validated to calculate homologous recombination deficiency (HRD) score. The two critical submodules were tree recursion (TR) segmentation and filtering, and the estimation and correction of the tumor purity and ploidy. Then, this study evaluated the rationality of segmentation and genotype identification by the GSA algorithm and compared with other two algorithms, PureCN and ASCAT, found that the segmentation result of GSA algorithm was more logical. In addition, the results indicated that the GSA algorithm had an excellent predictive effect on tumor purity and ploidy, if the tumor purity was more than 20%. Furtherly, this study evaluated the HRD scores and BRCA1/2 deficiency status of 195 clinical samples, and the results indicated that the accuracy was 0.98 (comparing with Affymetrix OncoScan™ assay) and the sensitivity was 95.2% (comparing with BRCA1/2 deficiency status), both were well-behaved. Finally, HRD scores and 16 genes mutations (TP53 and 15 HRR pathway genes) were analyzed in 17 cell lines, the results showed that there was higher frequency in HRR pathway genes in high HRD score samples. CONCLUSIONS: This new algorithm, named as GSA, could effectively and accurately calculate the purity and ploidy of tumor samples through NGS data, and then reflect the degree of genomic instability and large-scale copy number variations of tumor samples.

PC3-secreted microprotein is a novel chemoattractant protein and functions as a high-affinity ligand for CC chemokine receptor 2.

PC3-secreted microprotein (PSMP) or microseminoprotein is a newly discovered secreted protein whose function is currently unknown. In this study, PSMP was found to possess chemotactic ability toward monocytes and lymphocytes, and its functional receptor was identified as CCR2B. PSMP was identified as a chemoattractant protein from a PBMC chemoattractant platform screen that we established. The mature secreted PSMP was able to chemoattract human peripheral blood monocytes, PBLs, and CCR2B-expressing THP-1 cells, but not peripheral blood neutrophils, even though it does not contain the classical structure of chemokines. CCR2B was identified as one receptor for PSMP-mediated chemotaxis by screening HEK293 cells that transiently expressed classical chemokine receptors; results obtained from the chemotaxis, calcium flux, receptor internalization, and radioligand-binding assays all confirmed this finding. To further identify the major function of PSMP, we analyzed its expression profile in tissues. PSMP is highly expressed in benign prostatic hyperplasia and in some prostate cancers, and can also be detected in breast tumor tissue. In response to PSMP stimulation, phosphorylated ERK levels downstream of CCR2B signaling were upregulated in the PC3 cell line. Taken together, our data collectively suggest that PSMP is a chemoattractant protein acting as a novel CCR2 ligand that may influence inflammation and cancer development.

Human TTC5, a novel tetratricopeptide repeat domain containing gene, activates p53 and inhibits AP-1 pathway.

The transcription factor p53 and AP-1 play an important role in cellular proliferation, transformation and death. In this study, we investigated the role of a novel human gene, TTC5 (tetratricopeptide repeat domain 5), in the regulation of cell signaling pathway and cell viability. TTC5 is a member of the TTC family of proteins and has previously been shown to participate in cellular stress response. Here we demonstrate for the first time that TTC5 significantly activates p53 pathway and inhibits AP-1 transcriptional activity. Further investigation revealed that overexpression of TTC5 up-regulated p53 and p21 expression, and significantly inhibited transcriptional activity, expression and phosphorylation of c-Jun. As for the upstream of signaling pathway of AP-1, our study demonstrated that overexpression of TTC5 significantly down-regulated the expression and phosphorylation of JNK/SAPK. Moreover, overexpression of TTC5 repressed cell proliferation and induced S phase cell cycle arrest. These results indicated that TTC5 may regulate cell viability by p53 and AP-1 signaling pathway.

CCDC134 interacts with hADA2a and functions as a regulator of hADA2a in acetyltransferase activity, DNA damage-induced apoptosis and cell cycle arrest.

Human transcriptional adaptor hADA2a is an important component of the general control nonderepressible 5 (GCN5) histone acetyltransferase complex. Here, we report that coiled-coil domain containing 134 (CCDC134), a novel nuclear protein, binds to hADA2a and enhances the stability of the hADA2a protein in unstressed conditions. Furthermore, CCDC134 was found to participate in the p300/CBP-associated factor (PCAF) complex via hADA2a and affect the histone acetyltransferase activity of the complex. We also found that CCDC134 increased the PCAF-dependent K320 acetylation of p53 and p53 protein stability in the presence of hADA2a overexpression. Moreover, we demonstrated the biological significance of the interaction between CCDC134 and hADA2a. CCDC134 showed obvious nuclear accumulation after ultraviolet (UV) irradiation, and the knockdown of endogenous CCDC134 suppressed hADA2a-induced cell apoptosis activity and G1/S cell cycle arrest. Together, our findings indicate that CCDC134 might act as a novel regulator of hADA2a, and plays roles in the PCAF complex via hADA2a to affect its acetyltransferase activity and UV-induced DNA damage repair.

VSTM1-v2, a novel soluble glycoprotein, promotes the differentiation and activation of Th17 cells.

Cytokines are soluble proteins that mediate immune reactions and are responsible for communication among immune cells. CD4(+) T cells are the principle sources of cytokines of adaptive immunity. Cytokines play critical roles in the differentiation and effector function of CD4(+) T cells. They also play key roles in diseases, and some of them have been developed into drugs in the forms of recombinant cytokines, soluble receptors and neutralizing antibodies. Therefore, identifying novel potential cytokines is necessary and beneficial for better understanding immunology and enhancing human health. To find novel potential cytokines, we carried out an integrated bioinformatics analysis on the whole human genome. Cytokine candidates were selected for cDNA cloning, sub-cloning, secretion verification, expression profile analysis and functional study. Here, we report a novel soluble protein, VSTM1-v2, which is a classical secretory glycoprotein mainly expressed in immune tissues, and can promote the differentiation and activation of Th17 cells.

[Construction of recombinant NF-κB reporter adenovirus and activity analysis].

OBJECTIVE: To study the feasibility of adenovirus-based nuclear factor-κB (NF-κB) reporter as a model to screen the upstream signal regulators of NF-κB. METHODS: A type 5 (E1/E3 deficient) adenovirus vector pAdxsi was used to construct the NF-κB reporter adenovirus. Multiple adherent and suspending cell lines were infected by the NF-κB reporter adenovirus, and the luciferase activity of the NF-κB reporter gene was measured. RESULTS: An NF-κB reporter adenovirus (Ad-NF-κB-luc) was successfully constructed. The virus was capable of infecting HepG2, MGC803, THP-1 and U937 cell lines and showed high activities of NF-κB-luc reporter gene when stimulated by tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS). CONCLUSION: The Ad-NF-κB-luc reporter gene transfer system can effectively infect those cells hard-transfected by conventional transfection reagents. It also produces a high activity of NF-κB-luc reporter gene with stability and reliability. Our study expands the application of NF-κB reporter gene.

Genotypes of Papillary Thyroid Carcinoma With High Lateral Neck Metastasis in Chinese Population.

Papillary Thyroid Carcinoma (PTC) is one of the most commonly diagnosed cancer types in China, characterized by its early age at diagnosis and high 25-year survival rate. Distinct mutational patterns in PTC have been linked to activation of the mitogen-activated protein kinase (MAPK) signaling pathway. To explore the clinical significance of genomic variation patterns in Chinese patients with thyroid carcinoma, we investigated the genomic variants in 83 PTC cases with complete clinical records. The mutational patterns were evaluated using a 688-gene panel which covered known driver genes in PTC tumorigenesis, and featured genetic markers in various PTC-related pathways. We evaluated the relationship between mutational landscape and various clinical information in PTC patients with lateral lymph node metastasis. BRAF V600E was the most common mutation. Mutations in NF1, CDC27, PMS2 and PPP4R2 were more common in men, and mutations in NF1, PMS2 and TERT were related to lateral lymph node metastasis. According to the clustering of mutational patterns, we show that the underline driving mechanisms in lateral lymph node metastasis can be divided into two major groups (BRAF-TERT pathway, and NF1-PMS2 pathway). When combined with the TERT mutations, the BRAF mutation group was prone to lateral lymph node metastasis, particularly in elderly women. The NF1 mutations usually co-existed with PMS2 mutations, and this group included more men and young patients who had a high tumor mutational burden and lateral lymph node metastasis rate.

Human SBK1 is dysregulated in multiple cancers and promotes survival of ovary cancer SK-OV-3 cells.

Protein kinases are involved in comprehensive cellular processes and also implicated in many human diseases. SH3-binding domain kinase 1 (SBK1) was first cloned and characterized in rat and the human cDNA was cloned in our lab in 2006, but the expression and function of endogenous protein have not been well studied in human. In this follow up study, we screened a panel of cell lines and tissues, as well as a tumor tissue array for SBK1 expression at both RNA and protein levels. To detect the protein, we generated the first rabbit polyclonal antibody against human SBK1. We show that the SBK1 is expressed in most of the cells and tissues examined, and the protein is highly up-regulated in ovarian serous adenocarcinoma while down-regulated in esophagus squamous cell carcinoma and stomach adenocarcinoma. When over-expressed in an ovarian cancer cells SK-OV-3 by adenovirus infection, SBK1 protected the cells from apoptosis induced by the viral infection, therefore promoting cancer cell survival. Given that a missense mutation K92E in human SBK1 was identified recently from ovarian mucinous carcinoma, together, these results suggest that the wide-spread expression pattern of human SBK1 may predict a broad cellular function, and its dysregulated in certain cancers suggests an involvement of the protein in the pathogenesis of human cancers.

AC3-33, a novel secretory protein, inhibits Elk1 transcriptional activity via ERK pathway.

The transcription factor AP-1 plays an important role in cellular proliferation, transformation and death. In this study, we report a novel human gene, AC3-33 (GenBank name: c3orf33, FLJ31139), which encodes a secretory protein that can inhibit Elk1 transcriptional activity via ERK1/2 pathway. The AC3-33 mRNA encodes a protein of 251 amino acids, which is a classical secretory protein. Functional investigation reveals that overexpression of AC3-33 significantly inhibit AP-1 activity and DNA-binding ability. Further investigation indicated that overexpression of AC3-33 significantly inhibit transcriptional activity of Elk1 and c-jun, but not c-fos. As for the upstream of signaling pathway of Elk-1, our study demonstrated that overexpression of AC3-33 significantly down-regulates phosphorylation of ERK1/2, but not JNK/SAPK or p38 MAPK. These results clearly indicate that AC3-33 is a novel member of the secretory family and inhibits Elk1 transcriptional activity via ERK1/2 MAPK.

[Study on the mechanism of C17orf62 induced cell death].

OBJECTIVE: To isolate the long coding sequence of human novel gene C17orf62 which is named as C17orf62-L by us, and analyze its effects on cell viability, subcellular localization, and expression profile in multiple cell lines. METHODS: RT-PCR (reverse transcription polymerase chain reaction ) was used to clone C17orf62-L which encoded 187 amino acids from human multi-tissue cDNA library. We used bioinformatics analysis to identify structural characteristics of C17orf62-L, and RT-PCR to detect its expression. By Laser Scanning Confocal Microscopy we identified its subcellular localization of C17orf62-L. Furthermore, flow cytometry experiment was used to validate whether overexpression of C17orf62-L could influence cell phenotypes and Western blot was used to study related mechanisms. RESULTS: C17orf62-L was cloned and constructed into the pcDNA-and pEGFP-expression plasmids. C17orf62-L had signal peptide and transmembrane domain.C17orf62-L was widely expressed in multiple cell lines and was validated partial co-localization with Golgi apparatus. Functional studies showed C17orf62-L could induce cell death accompanied with rising of cleaved PARP(poly ADP-ribose polymerase). CONCLUSION: Human C17orf62 is a novel cell death inducing gene.

Characterization of genomic clones by targeted deep sequencing of ctDNA to monitor liver cancer.

BACKGROUND: In recent years, the morbidity and mortality of cancer patients have continued to increase in China, and there is an urgent need to develop an effective method to monitor tumor dynamics and measure tumor burden. Derived from the cell-free fraction of blood in cancer patients, circulating tumor DNA (ctDNA) has been regarded as a promising surrogate for tumor tissue biopsies. With the development of sequencing technology, ctDNA has been recognized as a specific and highly sensitive biomarker, and it has become a hot research spot in recent years. METHODS: In this paper, we investigated clonal changes before and after surgery in liver cancer patients using ctDNA. RESULTS: First, we evaluated the accuracy and stability of the method in ctDNA detection using virtual tumor samples with known mutations. The results showed that our method detected variants with an allelic frequency of at least 0.5%. We then applied this method to 34 liver cancer patients. A total of 266 clinically relevant mutations were identified in the pretreatment plasma samples. Through the analysis of plasma DNA samples at different treatment time points, we also investigated the possibility of using ctDNA as a prognostic factor to reflect tumor dynamics and to evaluate clinical responses. CONCLUSIONS: The results demonstrated that targeted high-depth next-generation sequencing can be used in ctDNA detection. Compared to traditional biopsy, the detection of ctDNA provides more information for human liver cancer, which is essential to guide the selection of therapy and predict prognosis.

RNF122: a novel ubiquitin ligase associated with calcium-modulating cyclophilin ligand.

BACKGROUND: RNF122 is a recently discovered RING finger protein that is associated with HEK293T cell viability and is overexpressed in anaplastic thyroid cancer cells. RNF122 owns a RING finger domain in C terminus and transmembrane domain in N terminus. However, the biological mechanism underlying RNF122 action remains unknown. RESULTS: In this study, we characterized RNF122 both biochemically and intracellularly in order to gain an understanding of its biological role. RNF122 was identified as a new ubiquitin ligase that can ubiquitinate itself and undergoes degradation in a RING finger-dependent manner. From a yeast two-hybrid screen, we identified calcium-modulating cyclophilin ligand (CAML) as an RNF122-interacting protein. To examine the interaction between CAML and RNF122, we performed co-immunoprecipitation and colocalization experiments using intact cells. What is more, we found that CAML is not a substrate of ubiquitin ligase RNF122, but that, instead, it stabilizes RNF122. CONCLUSIONS: RNF122 can be characterized as a C3H2C3-type RING finger-containing E3 ubiquitin ligase localized to the ER. RNF122 promotes its own degradation in a RING finger-and proteasome-dependent manner. RNF122 interacts with CAML, and its E3 ubiquitin ligase activity was noted to be dependent on the RING finger domain.

NSA2, a novel nucleolus protein regulates cell proliferation and cell cycle.

NSA2 (Nop seven-associated 2) was previously identified in a high throughput screen of novel human genes associated with cell proliferation, and the NSA2 protein is evolutionarily conserved across different species. In this study, we revealed that NSA2 is broadly expressed in human tissues and cultured cell lines, and located in the nucleolus of the cell. Both of the putative nuclear localization signals (NLSs) of NSA2, also overlapped with nucleolar localization signals (NoLSs), are capable of directing nucleolar accumulation. Moreover, over-expression of the NSA2 protein promoted cell growth in different cell lines and regulated the G1/S transition in the cell cycle. SiRNA silencing of the NSA2 transcript attenuated the cell growth and dramatically blocked the cell cycle in G1/S transition. Our results demonstrated that NSA2 is a nucleolar protein involved in cell proliferation and cell cycle regulation.

Human TMEM174 that is highly expressed in kidney tissue activates AP-1 and promotes cell proliferation.

Mitogen-activated protein kinase (MAPK) cascades play an important role in regulation of AP-1 activity through the phosphorylation of distinct substrates. In the present study, we identified a novel protein, TMEM174, whose RNA transcripts are highly expressed in human kidney tissue. TMEM174 is comprised of 243 amino acids, and contains two predicted transmembrane helices which determine its subcellular localization in endoplasmic reticulum and influences its functions. Over-expression of TMME174 enhanced the transcriptional activity of AP-1 and promoted cell proliferation, whereas the truncated mutant TMEM174DeltaTM without the transmembrane regions did not retain these functions. The possible mechanism of activation of AP-1 by TMEM174 was further examined. Our results suggest the potential role of TMEM174 in renal development and physiological function.

Discovery of novel human transcript variants by analysis of intronic single-block EST with polyadenylation site.

BACKGROUND: Alternative polyadenylation sites within a gene can lead to alternative transcript variants. Although bioinformatic analysis has been conducted to detect polyadenylation sites using nucleic acid sequences (EST/mRNA) in the public databases, one special type, single-block EST is much less emphasized. This bias leaves a large space to discover novel transcript variants. RESULTS: In the present study, we identified novel transcript variants in the human genome by detecting intronic polyadenylation sites. Poly(A/T)-tailed ESTs were obtained from single-block ESTs and clustered into 10,844 groups standing for 5,670 genes. Most sites were not found in other alternative splicing databases. To verify that these sites are from expressed transcripts, we analyzed the supporting EST number of each site, blasted representative ESTs against known mRNA sequences, traced terminal sequences from cDNA clones, and compared with the data of Affymetrix tiling array. These analyses confirmed about 84% (9,118/10,844) of the novel alternative transcripts, especially, 33% (3,575/10,844) of the transcripts from 2,704 genes were taken as high-reliability. Additionally, RT-PCR confirmed 38% (10/26) of predicted novel transcript variants. CONCLUSION: Our results provide evidence for novel transcript variants with intronic poly(A) sites. The expression of these novel variants was confirmed with computational and experimental tools. Our data provide a genome-wide resource for identification of novel human transcript variants with intronic polyadenylation sites, and offer a new view into the mystery of the human transcriptome.

Overexpression of CHMP6 induces cellular oncosis and apoptosis in HeLa cells.

Cell death can proceed via at least two distinct pathways, apoptosis and oncosis. Apoptosis is an energy-dependent process characterized morphologically by cell shrinkage, whereas oncosis is defined as a prelethal pathway leading to cell death associated with cellular swelling, organelle swelling, and increased membrane permeability. In this study, we found that overexpression of chromatin modifying protein 6 (CHMP6) induced cell death by a series of experiments, including morphological observation, intracellular ATP determination, caspase-3 activity, and flow cytometry. Typical morphological characteristics consistent with oncosis were observed by transmission electron microscopy. Simultaneously, we obtained some results that indicated apoptosis, but the anti-apoptotic gene Bcl-xL and caspase family inhibitor Z-VAD-FMK had little effect on CHMP6-induced cell death. These results suggest that CHMP6 overexpression can cause cell death, predominantly via oncosis and to a certain extent via apoptosis, and that CHMP6 might be a novel regulator involved in both oncosis and apoptosis.

Variations in Oral Microbiota Composition Are Associated With a Risk of Throat Cancer.

In this study, a next-generation sequencing strategy on 16S ribosomal RNA (16S rRNA) gene was employed to analyze 70 oral samples from 32 patients with throat cancer, nine patients with vocal cord polyp, and 29 healthy individuals (normal controls). Using this strategy, we demonstrated, for the first time, that the salivary microbiota of cancer patients were significantly different from those of patients with a polyp and healthy individuals. We observed that the beta diversity of the cancer group was divergent from both the normal and polyp groups, while alpha-diversity indices such as the Chao1 estimator (P = 8.1e-05), Simpson (P = 0.0045), and Shannon (P = 0.0071) were significantly reduced in cancer patients compared with patients containing a polyp and normal healthy individuals. Linear discriminant analysis (LDA) and Kruskal-Wallis test analyses and real-time quantitative polymerase chain reaction (qPCR) verification test revealed that the genera Aggregatibacter, Pseudomonas, Bacteroides, and Ruminiclostridium were significantly enriched in the throat cancer group compared with the vocal cord polyp and normal control groups (score value >2). Finally, diagnostic models based on putatively important constituent bacteria were constructed with 87.5% accuracy [area under the curve (AUC) = 0.875, 95% confidence interval (CI): 0.695-1]. In summary, in this study we characterized, for the first time, the oral microbiota of throat cancer patients without smoking history. We speculate that these results will help in the pathogenic mechanism and early diagnosis of throat cancer.

High-throughput cell-based screening reveals a role for ZNF131 as a repressor of ERalpha signaling.

BACKGROUND: Estrogen receptor alpha (ERalpha) is a transcription factor whose activity is affected by multiple regulatory cofactors. In an effort to identify the human genes involved in the regulation of ERalpha, we constructed a high-throughput, cell-based, functional screening platform by linking a response element (ERE) with a reporter gene. This allowed the cellular activity of ERalpha, in cells cotransfected with the candidate gene, to be quantified in the presence or absence of its cognate ligand E2. RESULTS: From a library of 570 human cDNA clones, we identified zinc finger protein 131 (ZNF131) as a repressor of ERalpha mediated transactivation. ZNF131 is a typical member of the BTB/POZ family of transcription factors, and shows both ubiquitous expression and a high degree of sequence conservation. The luciferase reporter gene assay revealed that ZNF131 inhibits ligand-dependent transactivation by ERalpha in a dose-dependent manner. Electrophoretic mobility shift assay clearly demonstrated that the interaction between ZNF131 and ERalpha interrupts or prevents ERalpha binding to the estrogen response element (ERE). In addition, ZNF131 was able to suppress the expression of pS2, an ERalpha target gene. CONCLUSION: We suggest that the functional screening platform we constructed can be applied for high-throughput genomic screening candidate ERalpha-related genes. This in turn may provide new insights into the underlying molecular mechanisms of ERalpha regulation in mammalian cells.

DCUN1D3, a novel UVC-responsive gene that is involved in cell cycle progression and cell growth.

DCUN1D3 (DCN1, defective in cullin neddylation 1, domain containing 3) was found during the process of high throughput screening of novel human genes associated with serum response element (SRE) pathway activation. The DCUN1D3 gene is highly conserved among vertebrates. Human DCUN1D3 complementary DNA (cDNA) encodes 304 amino acids with an apparent molecular mass of 34 kDa. However, there has been no report about the function of DCUN1D3. This study detected that DCUN1D3 was broadly expressed in several tumor tissues and cultured cell lines; however, UVC irradiation of different doses significantly increased DCUN1D3 expression level in these cancer cell lines. Over-expression of the DCUN1D3 inhibits cell growth in HeLa. When the DCUN1D3 gene was silenced by siRNA in UVC-treated HeLa, the cell cycle in S phase was remarkably blocked; furthermore, the UVC-induced cell death was inhibited. In addition, DCUN1D3 localized mainly in the cytoplasm and perinuclear, but after UVC treatment, the DCUN1D3 gradually entered the nucleus. All the results above indicate that DCUN1D3 is a novel UVC-response gene involved in cell cycle regulation and cell survival.

URP2SF, a FERM and PH domain containing protein, regulates NF-kappaB and apoptosis.

The transcription factor NF-kappaB plays an important role in inflammation and cell survival. Here, we report the functional characterization of URP2SF (UNC-112 related protein 2 short form), which is a novel protein containing FERM and PH domains. The protein is conserved in evolution across different species. Northern blot and RT-PCR analysis revealed that the expression of URP2SF appeared to be primarily confined to immune system. URP2SF protein localized to the whole cell. Over-expression of URP2SF strongly repressed NF-kappaB activity in the absence or presence of NF-kappaB stimulus. Importantly, URP2SF over-expression also induced cell apoptosis. Furthermore, suppression of URP2SF expression by small interference RNA significantly activated the NF-kappaB reporter gene activities, as well as attenuated TNF-alpha induced apoptosis. Therefore, our data show for the first time that URP2SF may act as a transcriptional repressor in NF-kappaB signaling pathway and regulate cell apoptotic pathway.