Comprehensive Analysis and Drug Modulation of Human Endogenous Retrovirus in Hepatocellular Carcinomas.

BACKGROUND: Human endogenous retroviruses (HERVs) play an important role in the development of cancer and many diseases. Here, we comprehensively explored the impact of HERVs on hepatocellular carcinomas (HCCs). METHODS: We employed Telescope to identify HERVs and quantify their expression in the total RNA sequencing data obtained from 254 HCC samples, comprising 254 tumor tissues and 34 matched normal tissues. RESULTS: In total, 3357 locus-specific activations of HERVs were differentially expressed, and 180 were correlated with patient survival. Using these 180 HERVs for classification, we found four subgroups with survival correlation. Higher expression levels of the 180 HERVs were correlated with poorer survival, while age, AFP, some mutations, and copy and structural variants differed among subgroups. The differential expression of host genes in high expression of these 180 HERVs primarily involved the activation of pathways related to immunity and infection, lipid and atherosclerosis, MAPK and NF-kB signaling, and cytokine-cytokine receptor interactions. Conversely, there was a suppression of pathways associated with RNA processing, including nucleocytoplasmic transport, surveillance and ribosome biogenesis, and transcriptional misregulation in cancer pathways. Almost all genes involved in HERV activation restriction, KRAB zinc finger proteins, RNA nucleocytoplasmic transport, stemness, HLA and antigen processing and presentation, and immune checkpoints were overexpressed in cancerous tissues, and many over-expressed HERV-related nearby genes were correlated with high HERV activation and poor survival. Twenty-three immune and stromal cells showed higher expression in non-cancerous than cancerous tissues, and seven were correlated with HERV activation. Small-molecule modulation of alternative splicing (AS) altered the expression of survival-related HERVs and their activation-related genes, as well as nearby genes. CONCLUSION: Comprehensive and integrated approaches for evaluating HERV expression and their correlation with specific pathways have the potential to provide new companion diagnostics and therapeutic strategies for HCC.

Whole genome and RNA sequencing analyses for 254 Taiwanese hepatocellular carcinomas.

BACKGROUND: Comprehensive and integrative analysis of hepatocellular carcinoma (HCC) is important. In this study, we explored Taiwanese HCCs using multi-omics analyses. METHODS: We analyzed 254 HCCs by whole genome sequencing and total RNA sequencing, and then used bioinformatic tools to analyze genomic and transcriptomic alterations in coding and non-coding sequences to explore the clinical importance of each sequence. RESULTS: The frequencies of the five most commonly mutated cancer-related genes were TERT, TP53, CTNNB1, RB1, and ARID1A. Genetic alteration frequencies influenced the etiology of HCC; some alterations were also correlated with clinicopathological conditions. Many cancer-related genes had copy number alterations (CNAs) and structure variants (SVs) that changed according to etiology and exhibited potential associations with survival. We also identified several alterations in histone-related genes, HCC-related long non-coding RNAs, and non-coding driver genes that may contribute to the onset and progression of HCC. Transcriptomic analysis revealed that 229 differentially expressed and 148 novel alternative splicing (AS) genes, as well as the presence of fusion genes, were associated with patient survival. Moreover, somatic mutations, CNAs, and SVs were associated with immune checkpoint gene expression and tumor microenvironment. Finally, we identified relationships among AS, immune checkpoint gene expression and tumor microenvironment. CONCLUSIONS: This study shows that genomic alterations are associated with survival, including DNA-based and RNA-based data. Moreover, genomic alterations and their associations with immune checkpoint genes and the tumor microenvironment may provide novel insights for the diagnosis and treatment of HCC.

Dysregulation of Immune Cell Subpopulations in Atypical Hemolytic Uremic Syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a rare, life-threatening thrombotic microangiopathy. Definitive biomarkers for disease diagnosis and activity remain elusive, making the exploration of molecular markers paramount. We conducted single-cell sequencing on peripheral blood mononuclear cells from 13 aHUS patients, 3 unaffected family members of aHUS patients, and 4 healthy controls. We identified 32 distinct subpopulations encompassing 5 B-cell types, 16 T- and natural killer (NK) cell types, 7 monocyte types, and 4 other cell types. Notably, we observed a significant increase in intermediate monocytes in unstable aHUS patients. Subclustering analysis revealed seven elevated expression genes, including NEAT1, MT-ATP6, MT-CYB, VIM, ACTG1, RPL13, and KLRB1, in unstable aHUS patients, and four heightened expression genes, including RPS27, RPS4X, RPL23, and GZMH genes, in stable aHUS patients. Additionally, an increase in the expression of mitochondria-related genes suggested a potential influence of cell metabolism on the clinical progression of the disease. Pseudotime trajectory analysis revealed a unique immune cell differentiation pattern, while cell-cell interaction profiling highlighted distinctive signaling pathways among patients, family members, and controls. This single-cell sequencing study is the first to confirm immune cell dysregulation in aHUS pathogenesis, offering valuable insights into molecular mechanisms and potential new diagnostic and disease activity markers.

Integrated genomic analyses of hepatocellular carcinoma.

BACKGROUND: Genomic alterations play important roles in the development of cancer. We explored the impact of protein-coding genes and transcriptomic changes on clinical and molecular alterations in Taiwanese hepatocellular carcinoma (HCC) patients. METHODS: We analyzed 147 whole-exome sequencing and 100 RNA sequencing datasets of HCC and compared them with The Cancer Genome Atlas (TCGA)-Liver Hepatocellular Carcinoma cohort and develop a panel of 81 apoptosis-related genes for molecular classification. RESULTS: TERT (50%), TP53 (25%), CTNNB1 (14%), ARID1A (12%), and KMT2C (11%) were the most common genetic alterations of cancer-related genes. ALDH2 and KMT2C mutated at much higher frequencies in our cohort than in TCGA, whereas CTNNB1 was found only in 14% of our Taiwanese patients. A high germline mutation rate of ALDH2 in the APOBEC mutational signature and herb drug-related aristolochic acid-associated signature was also observed. Groups A and B of HCC were identified when we used apoptosis-related genes for molecular classification. The latter group, which had poorer survival outcomes, had significantly more aDC, CD4+ Tem, macrophages M2, NKT, plasma cells, and Th1 cells, and less CD4+ memory T cells, CD8+ Tcm, cDC, iDC, and Th2 cells, as well as more inter-chromosome fusion genes. Metatranscriptomic analysis revealed 54 cases of HBV infection. Moreover, we found that the main target gene of HBV integration is ALB. CONCLUSIONS: Unique genomic alterations were observed in our Taiwanese HCC patients. Molecular classification using apoptosis-related genes could lead to new therapeutic approaches for HCC.

Single-cell RNA sequencing of immune cells in patients with acute gout.

Cell subpopulations in the blood and joint fluid of patients with gout are poorly understood. Single-cell RNA sequencing and bioinformatic tools were used to identify cell subsets and their gene signatures in blood and synovial fluid (SF) cells, determine their relationships, characterize the diversity, and evaluate interactions among specific cell types. We identified 34 subpopulations (5 types of B cells, 16 types of T and natural killer cells, 9 types of monocytes, and 4 other cell types) in the blood of five healthy subjects and seven patients with acute gouty, and the SF of three patients with acute gout. We found that naïve CD4 T cells and classical monocytes cell populations were enriched in patients with gout, whereas plasmacytoid dendritic cells and intermediate monocytes were more abundant in healthy subjects. SF was enriched in Th1/Th17 cells, effector memory CD8 T cells, mucosal-associated invariant T cells, and macrophages. Subclusters of these cell subpopulations showed different compositions between healthy subjects and those with acute gout, according to blood and SF samples. At the cellular level, the inflammation score of a subpopulation or subcluster was highest in SF, following by the blood of acute gout patients and healthy person, whereas energy score showed the opposite trend. We also detected specific cell-cell interactions for interleukin-1, tumor necrosis factor-α, and transforming growth factor-ß1 expression in the cells of patients with acute gout. Our study reveals cellular and molecular insights on inflammatory responses to hyperuricemia or uric crystal and may provide therapeutic guidance to improve treatments for gout.

Molecular Classification of Hepatocellular Carcinoma Using Wnt-Hippo Signaling Pathway-Related Genes.

In Taiwan, a combination of hepatitis B and C infection, economic boom-related food and alcohol overconsumption, and Chinese medicine prescriptions has led to a high rate of hepatocellular carcinoma (HCC). However, the causative factors and underlying tumor biology for this unique HCC environment have not been identified. Wnt and Hippo signaling pathways play an important regulatory role in HCC development, and their functions are generally considered as positive and negative regulators of cell proliferation, respectively. In this study, we characterized the molecular features of HCC using a newly developed classification system based on the expression of the Wnt-Hippo signaling pathway-related genes. RNA sequencing (RNA-Seq) was performed on liver tumor tissues from 100 patients with liver cancer. RNA-Seq data for 272 previously characterized Wnt-Hippo signaling pathway-related genes were used for hierarchical clustering. We analyzed the data in terms of prognostic value, transcriptome features, immune infiltration, and clinical characteristics, and compared the resulting subclasses with previously published classifications. Four subclasses of HCC (HCCW1-4) were identified. Subclass HCCW1 displayed the highest PCDHB4 expression. Subclass HCCW2 displayed lower Edmondson-Steiner grades (I and II) and CTNNB1 mutation frequencies. Subclass HCCW3 was associated with a good prognosis, the highest PCDHGB7 expression, high CD8+ naïve T cells abundance, and relatively low TP53 mutation rates. Subclass HCCW4 was associated with a poor prognosis, the highest PCDHB2 and PCDHB6 expression, a relatively high abundance of Th1 cells, NKT and class-switched memory B cells, relatively low enrichment of cDC, iDC, and CD4+ memory T cells, and high Edmondson-Steiner grades (III and IV). We also identified Wnt-Hippo signaling pathway-related genes that may influence immune cell infiltration. We developed a panel of 272 Wnt-Hippo signaling pathway-related genes to classify HCC into four groups based on Taiwanese HCC and The Cancer Genome Atlas Liver Hepatocellular Carcinoma datasets. This novel molecular classification system may aid the treatment of HCC.

Mutation Analysis of Second Primary Tumors in Oral Cancer in Taiwanese Patients through Next-Generation Sequencing.

Head and neck cancer has poor overall survival. Patients with head and neck cancer more frequently develop second primary tumors than do patients with other cancers, leading to a poor prognosis. In this study, we used next-generation sequencing to analyze and compare mutations between first tumors and second tumors in oral cancer. We retrieved tumor tissues collected from 13 patients who were diagnosed twice as having cancer. We used driver gene and trunk mutations to distinguish between recurrent cancer and primary cancer in oral cancer. We observed unique driver gene mutations in three patients with an initial clinical diagnosis of recurrent cancer; hence, we believe that the corresponding patients had primary cancer. Four patients with an initial clinical diagnosis of primary cancer were found to actually have recurrent cancer according to our results. Genetic testing can be used to enhance the accuracy of clinical diagnosis.

Long Noncoding RNA NTT Context-Dependently Regulates MYB by Interacting With Activated Complex in Hepatocellular Carcinoma Cells.

BACKGROUND: Long noncoding RNA (lncRNA) mediates the pathogenesis of various diseases, including cancer and cardiovascular, infectious, and metabolic diseases. This study examined the role of lncRNA NTT in the development and progression of cancer. METHODS: The expression of NTT was determined using tissues containing complementary DNA (cDNA) from patients with liver, lung, kidney, oral, and colon cancers. The expression of cis-acting genes adjacent to the NTT locus (CTGF, STX7, MYB, BCLAF1, IFNGR1, TNFAIP3, and HIVEP2) was also assessed. We used knockdown and chromatin immunoprecipitation (ChIP) assays to identify the cis-acting genes that interact with NTT. RESULTS: NTT was most significantly downregulated in hepatocellular carcinoma (HCC), while a higher NTT level correlated with a shorter survival time of patients with HCC. Multivariate analysis indicated NTT was not an independent predictor for overall survival. MYB was significantly upregulated, and its increased expression was associated with dismal survival in HCC patients, similar to the results for NTT. NTT knockdown significantly decreased cellular migration. ChIP of HCC cell lines revealed that NTT is regulated by the transcription factor ATF3 and binds to the MYB promoter via the activated complex. Additionally, when NTT was knocked down, the expression of MYB target genes such as Bcl-xL, cyclinD1, and VEGF was also downregulated. NTT could play a positive or negative regulator for MYB with a context-dependent manner in both HCC tissues and animal model. CONCLUSION: Our study suggests that NTT plays a key role in HCC progression via MYB-regulated target genes and may serve as a novel therapeutic target.

Metatranscriptomic Analysis of Human Lung Metagenomes from Patients with Lung Cancer.

This study was designed to characterize the microbiomes of the lung tissues of lung cancer patients. RNA-sequencing was performed on lung tumor samples from 49 patients with lung cancer. Metatranscriptomics data were analyzed using SAMSA2 and Kraken2 software. 16S rRNA sequencing was also performed. The heterogeneous cellular landscape and immune repertoires of the lung samples were examined using xCell and TRUST4, respectively. We found that nine bacteria were significantly enriched in the lung tissues of cancer patients, and associated with reduced overall survival (OS). We also found that subjects with mutations in the epidermal growth factor receptor gene were less likely to experience the presence of Pseudomonas. aeruginosa. We found that the presence of CD8+ T-cells, CD4+ naive T-cells, dendritic cells, and CD4+ central memory T cells were associated with a good prognosis, while the presence of pro B-cells was associated with a poor prognosis. Furthermore, high clone numbers were associated with a high ImmuneScore for all immune receptor repertoires. Clone numbers and diversity were significantly higher in unpresented subjects compared to presented subjects. Our results provide insight into the microbiota of human lung cancer, and how its composition is linked to the tumor immune microenvironment, immune receptor repertoires, and OS.

The Fusion Gene Landscape in Taiwanese Patients with Non-Small Cell Lung Cancer.

BACKGROUND: Analyzing fusion gene transcripts may yield an effective approach for selecting cancer treatments. However, few comprehensive analyses of fusions in non-small cell lung cancer (NSCLC) patients have been performed. METHODS: We enrolled 54 patients with NSCLC, and performed RNA-sequencing (RNA-Seq). STAR (Spliced Transcripts Alignment to a Reference)-Fusion was used to identify fusions. RESULTS: Of the 218 fusions discovered, 24 had been reported and the rest were novel. Three fusions had the highest occurrence rates. After integrating our gene expression and fusion data, we found that samples harboring fusions containing ASXL1, CACNA1A, EEF1A1, and RET also exhibited increased expression of these genes. We then searched for mutations and fusions in cancer driver genes in each sample and found that nine patients carried both mutations and fusions in cancer driver genes. Furthermore, we found a trend for mutual exclusivity between gene fusions and mutations in the same gene, with the exception of DMD, and we found that EGFR mutations are associated with the number of fusion genes. Finally, we identified kinase gene fusions, and potentially druggable fusions, which may play roles in lung cancer therapy. CONCLUSION: The clinical use of RNA-Seq for detecting driver fusion genes may play an important role in the treatment of lung cancer.

Mutation profile of non-small cell lung cancer revealed by next generation sequencing.

BACKGROUND: Precision therapy for lung cancer requires comprehensive genomic analyses. Specific effects of targeted therapies have been reported in Asia populations, including Taiwanese, but genomic studies have rarely been performed in these populations. METHOD: We enrolled 72 patients with non-small cell lung cancer, of whom 61 had adenocarcinoma, 10 had squamous cell carcinoma, and 1 had combined adenocarcinoma and squamous cell carcinoma. Whole-exome or targeted gene sequencing was performed. To identify trunk mutations, we performed whole-exome sequencing in two tumor regions in four patients. RESULTS: Nineteen known driver mutations in EGFR, PIK3CA, KRAS, CTNNB1, and MET were identified in 34 of the 72 tumors evaluated (47.22%). A comparison with the Cancer Genome Atlas dataset showed that EGFR was mutated at a much higher frequency in our cohort than in Caucasians, whereas KRAS and TP53 mutations were found in only 5.56% and 25% of our Taiwanese patients, respectively. We also identified new mutations in ARID1A, ARID2, CDK12, CHEK2, GNAS, H3F3A, KDM6A, KMT2C, NOTCH1, RB1, RBM10, RUNX1, SETD2, SF3B1, SMARCA4, THRAP3, TP53, and ZMYM2. Moreover, all ClinVar pathogenic variants were trunk mutations present in two regions of a tumor. RNA sequencing revealed that the trunk or branch genes were expressed at similar levels among different tumor regions. CONCLUSIONS: We identified novel variants potentially associated with lung cancer tumorigenesis. The specific mutation pattern in Taiwanese patients with non-small cell lung cancer may influence targeted therapies.

Genome-Wide Analysis of Prognostic Alternative Splicing Signature and Splicing Factors in Lung Adenocarcinoma.

Analysis of The Cancer Genome Atlas data revealed that alternative splicing (AS) events could serve as prognostic biomarkers in various cancer types. This study examined lung adenocarcinoma (LUAD) tissues for AS and assessed AS events as potential indicators of prognosis in our cohort. RNA sequencing and bioinformatics analysis were performed. We used SUPPA2 to analyze the AS profiles. Using univariate Cox regression analysis, overall survival (OS)-related AS events were identified. Genes relating to the OS-related AS events were imported into Cytoscape, and the CytoHubba application was run. OS-related splicing factors (SFs) were explored using the log-rank test. The relationship between the percent spliced-in value of the OS-related AS events and SF expression was identified by Spearman correlation analysis. We found 1957 OS-related AS events in 1151 genes, and most were protective factors. Alternative first exon splicing was the most frequent type of splicing event. The hub genes in the gene network of the OS-related AS events were FBXW11, FBXL5, KCTD7, UBB and CDC27. The area under the curve of the MIX prediction model was 0.847 for 5-year survival based on seven OS-related AS events. Overexpression of SFs CELF2 and SRSF5 was associated with better OS. We constructed a correlation network between SFs and OS-related AS events. In conclusion, we identified prognostic predictors using AS events that stratified LUAD patients into high- and low-risk groups. The discovery of the splicing networks in this study provides an insight into the underlying mechanisms.

Male-Specific Long Noncoding RNA TTTY15 Inhibits Non-Small Cell Lung Cancer Proliferation and Metastasis via TBX4.

Gender affects cancer susceptibility. Currently, there are only a few studies on Y chromosome-linked long noncoding RNAs (lncRNAs), and the potential association between lncRNAs and cancers in males has not been fully elucidated. Here, we examined the expression of testis-specific transcript Y-linked 15 (TTTY15) in 37 males with non-small cell lung cancer (NSCLC), and performed circular chromosome conformation capture with next-generation sequencing to determine the genomic interaction regions of the TTTY15 gene. Our results showed that the expression levels of TTTY15 were lower in NSCLC tissues. Lower TTTY15 expression levels were associated with Tumor-Node-Metastasis (TNM) stage. A TTTY15 knockdown promoted malignant transformation of NSCLC cells. Based on the bioinformatics analysis of circular chromosome conformation capture data, we found that T-box transcription factor 4 (TBX4) may be a potential target gene of TTTY15. The RNA immunoprecipitation and chromatin immunoprecipitation results showed that TTTY15 may interact with DNA (cytosine-5)-methyltransferase 3A (DNMT3A), and the TTTY15 knockdown increased the binding of DNMT3A to the TBX4 promoter. We concluded that low TTTY15 expression correlates with worse prognosis among patients with NSCLC. TTTY15 promotes TBX4 expression via DNMT3A-mediated regulation. The identification of lncRNAs encoded by male-specific genes may help to identify potential targets for NSCLC therapy.

lncRNA NTT/PBOV1 Axis Promotes Monocyte Differentiation and Is Elevated in Rheumatoid Arthritis.

Monocytes/macrophages are important in orchestrating inflammatory responses. However, knowledge of the long noncoding RNA (lncRNA) regulation of monocytic cell differentiation and diseases remains limited. We aimed to elucidate the role of the 17 kb lncRNA noncoding transcript in T cells (NTT) in monocyte functions. Knockdown and chromatin immunoprecipitation (ChIP) assays in THP-1 cells (human monocytic leukemia cell line) revealed that NTT is regulated by the monocyte key transcription factor C/EBPß and that it binds to the promoter of nearby gene PBOV1 via hnRNP-U. Overexpression of PBOV1 in THP-1 cells resulted in cell cycle G1 arrest, differentiation into macrophages, a marked increase in IL-10 and CXCL10 mRNA levels, and upregulation of the costimulatory molecules. In contrast to the downregulated NTT observed in lipopolysaccharide (LPS)-treated THP-1 cells, the C/EBPß/NTT/PBOV1 axis was found to be hyperactivated in peripheral blood mononuclear cells (PBMCs) of first-time diagnosed untreated early rheumatoid arthritis (RA) patients, and their gene expression levels decreased markedly after treatment. Higher initial C/EBPß/NTT/PBOV1 expression levels were associated with a trend of higher disease activity DAS28 scores. In conclusion, our study suggests that the lncRNA NTT is a regulator of inflammation in monocytes, and its activation participates in monocyte/macrophage differentiation and the pathogenesis of RA.

Prognostic Value of RNASEH2A-, CDK1-, and CD151-Related Pathway Gene Profiling for Kidney Cancers.

The nucleotide degrading enzyme gene RNASEH2A (ribonuclease H2 subunit A) has been found to be overexpressed in cancers. Our aim was to understand the role of RNASEH2A in cancer prognostication and to establish a scoring system based on the expressions of genes interacting with RNASEH2A. We screened the nucleotide degrading enzyme gene expression in RNAseq data of 14 cancer types derived from The Cancer Genome Atlas (TCGA) and found that RNASEH2A overexpression was associated with poor patient survival only in renal cell carcinomas (RCCs). Further cluster analyses of samples with poor outcomes revealed that cluster of differentiation 151 (CD151) upregulation correlated with low cyclin dependent kinase 1 (CDK1) and high RNASEH2A expression. The combination of low CD151 expression and high RNASEH2A expression resulted in impaired proliferation in four kidney cancer cell lines, suggesting potential synthetic dosage lethality (SDL) interactions between the two genes. A prognostication scoring system was established based on the expression levels of RNASEH2A-, CDK1-, and CD151-related genes, which could effectively predict the overall survival in a TCGA clear cell RCC cohort (n = 533, 995.3 versus 2242.2 days, p < 0.0001), in another clear cell renal cell carcinoma (ccRCC) cohort E-GEOD-22541 (n = 44, 390.0 versus 1889.2 days, p = 0.0007), and in a TCGA papillary RCC (pRCC) cohort (n = 287, 741.6 versus 1623.7 days, p < 0.0001). Our results provide a clinically applicable prognostication scoring system for renal cancers.

Long noncoding RNA MIAT promotes non-small cell lung cancer proliferation and metastasis through MMP9 activation.

Long noncoding RNAs (lncRNAs) play crucial roles in carcinogenesis. Myocardial infarction-associated transcript (MIAT), originally isolated as a candidate gene for myocardial infarction, has been found to act as an oncogene in chronic lymphocytic leukaemias and neuroendocrine prostate cancer (NEPC); however, little is known about its expression pattern, biological function, and underlying mechanism in non-small cell lung cancer (NSCLC). In this study, we observed that MIAT expression was upregulated in NSCLC, and its overexpression was associated with advanced tumor stage. Moreover, MIAT knockdown decreased cell proliferation, migration, invasion, and cell cycle arrested in G1 phase. Mechanistic investigation revealed that MIAT could interact with histone methyltransferase mixed-lineage leukemia (MLL). MIAT silencing impeded the binding of MLL on the matrix metalloproteinase 9 (MMP9) promoter region and epigenetically reduced MMP9 transcriptional activity. Overall, our findings suggest that MIAT expression is associated with NSCLC and may be one of the critical targets in progression and metastasis in NSCLC.

Long noncoding RNA TUG1 is downregulated in non-small cell lung cancer and can regulate CELF1 on binding to PRC2.

BACKGROUND: Long noncoding RNAs (lncRNAs) play crucial roles in tumorigenesis, and lncRNA taurine-upregulated gene 1 (TUG1) has been proven to be associated with several human cancers. However, the mechanisms of TUG1-involved regulation remain largely unknown. METHODS: We examined the expressions of TUG1 in a cohort of 89 patients with non-small cell lung cancer (NSCLC) to determine the association between TUG1 expression and clinical parameters. We used circular chromosome conformation capture (4C) coupled with next-generation sequencing to explore the genome regions that interact with TUG1 and the TUG1-mediated regulation. RESULTS: TUG1 was significantly downregulated, and the TUG1 downregulation correlated with sex (p = 0.006), smoking status (p = 0.016), and tumor differentiation grade (p = 0.001). Knockdown of TUG1 significantly promoted the proliferation of NSCLC cells. According to the bioinformatic analysis result of TUG1 4C sequencing data, 83 candidate genes and their interaction regions were identified. Among these candidate genes, CUGBP and Elav-like family member 1 (CELF1) are potential targets of TUG1 in-trans regulation. To confirm the interaction between TUG1 and CELF1, relative expressions of CELF1 were examined in TUG1 knockdown H520 cells; results showed that CELF1 was significantly upregulated in TUG1 knockdown H520 cells. RNA immunoprecipitation was then performed to examine whether TUG1 RNA was bound to PRC2, a TUG1-involved regulation mechanism reported in previous studies. The results demonstrated that TUG1 RNA was bound to enhancer of zeste protein 2/embryonic ectoderm development (EZH2/EED), which is essential for PRC2. Finally, our designed ChIP assay revealed that the EZH2/EED was bound to the promotor region of CELF1 within 992 bp upstream of the transcript start site. CONCLUSION: TUG1 is downregulated in NSCLC. Using TUG1 4C sequencing and bioinformatic analysis, we found CELF1 to be a potential target of TUG1 RNA in in-trans regulation. Moreover, subsequent experiments showed that TUG1 RNA could bind to PRC2 in the promotor region of CELF1 and negatively regulate CELF1 expressions in H520 cells. Our results may facilitate developing new treatment modalities targeting TUG1/PRC2/CELF1 interactions in patients with NSCLC.

Down-regulated and Commonly mutated ALPK1 in Lung and Colorectal Cancers.

The ALPK1 gene located in the 4q25 region encodes a newly explored protein kinase which could phosphorylate the amino acid of a domain full of α-helices. Recently, several studies have indicated that the expression of ALPK1 is related to inflammation and various diseases; therefore, the purpose of this investigation was to determine whether the expression of ALPK1 has an influence on tumorigenesis and to further scrutinize its gene polymorphism in order to better understand its clinical importance. In lung and colorectal cancer tissues, the ALPK1 RNA level of the normal part was higher than that of the tumor part using the RT-qPCR analysis. Moreover, differences in HRM melting curves could effectively separate the known mutation sites and be used to identify the two novel variants that might cause the bio-dysfunctions of ALPK1 found in silico predictions. Additionally, in both Lovo colorectal and A549 lung cancer cells with enhanced and depleted expression of ALPK1, the encoded ALPK1 could exert its activity on cell migration without interfering with cell viability. Taken together, these findings suggested that ALPK1 might play a vital role in cancer development and that the newly explored SNPs are found in a Taiwanese cohort.

Ancient migration routes of Austronesian-speaking populations in oceanic Southeast Asia and Melanesia might mimic the spread of nasopharyngeal carcinoma.

Mitochondrial DNA (mtDNA) and non-recombining Y chromosome (NRY) are inherited uni-parentally from mother to daughter or from father to son respectively. Their polymorphism has initially been studied throughout populations of the world to demonstrate the "Out of Africa" hypothesis. Here, to correlate the distribution of nasopharyngeal carcinoma (NPC) in different populations of insular Asia, we analyze the mtDNA information (lineages) obtained from genotyping of the hyper variable region (HVS I & II) among 1400 individuals from island Southeast Asia (ISEA), Taiwan and Fujian and supplemented with the analysis of relevant coding region polymorphisms. Lineages that best represented a clade (a branch of the genetic tree) in the phylogeny were further analyzed using complete genomic mtDNA sequencing. Finally, these complete mtDNA sequences were used to construct a most parsimonious tree which now constitutes the most up-to-date mtDNA dataset available on ISEA and Taiwan. This analysis has exposed new insights of the evolutionary history of insular Asia and has strong implications in assessing possible correlations with linguistic, archaeology, demography and the NPC distribution in populations within these regions. To obtain a more objective and balanced genetic point of view, slowly evolving biallelic Y single nucleotide polymorphism (Y-SNP) was also analyzed. As in the first step above, the technique was first applied to determine affinities (macro analysis) between populations of insular Asia. Secondly, sixteen Y short tandem repeats (Y-STR) were used as they allow deeper insight (micro analysis) into the relationship between individuals of a same region. Together, mtDNA and NRY allowed a better definition of the relational, demographic, cultural and genetic components that constitute the make up of the present day peoples of ISEA. Outstanding findings were obtained on the routes of migration that occurred along with the spread of NPC during the settlement of insular Asia. The results of this analysis will be discussed using a conceptual approach.