Differential methylation of G-protein coupled receptor signaling genes in gastrointestinal neuroendocrine tumors.

Neuroendocrine tumors (NETs) of the small intestine undergo large chromosomal and methylation changes. The objective of this study was to identify methylation differences in NETs and consider how the differentially methylated genes may impact patient survival. Genome-wide methylation and chromosomal copy number variation (CNV) of NETs from the small intestine and appendix were measured. Tumors were divided into three molecular subtypes according to CNV results: chromosome 18 loss (18LOH), Multiple CNV, and No CNV. Comparison of 18LOH tumors with MultiCNV and NoCNV tumors identified 901 differentially methylated genes. Genes from the G-protein coupled receptor (GPCR) pathways are statistically overrepresented in the differentially methylated genes. One of the highlighted genes from the GPCR pathway is somatostatin (SST), a clinical target for NETs. Patient survival based on low versus high methylation in all samples identified four significant genes (p < 0.05) OR2S2, SMILR, RNU6-653P, and AC010543.1. Within the 18LOH molecular subtype tumors, survival differences were identified in high versus low methylation of 24 genes. The most significant is TRHR (p < 0.01), a GPCR with multiple FDA-approved drugs. By separating NETs into different molecular subtypes based on chromosomal changes, we find that multiple GPCRs and their ligands appear to be regulated through methylation and correlated with survival. These results suggest opportunities for better treatment strategies for NETs based on molecular features.

Differential alternative splicing between hepatocellular carcinoma with normal and elevated serum alpha-fetoprotein.

BACKGROUND: Serum alpha-fetoprotein (AFP) is the approved serum marker for hepatocellular carcinoma (HCC) screening. However, not all HCC patients show high (≥ 20 ng/mL) serum AFP, and the molecular mechanisms of HCCs with normal (< 20 ng/mL) serum AFP remain to be elucidated. Therefore, we aimed to identify biological features of HCCs with normal serum AFP by investigating differential alternative splicing (AS) between HCCs with normal and high serum AFP. METHODS: We performed a genome-wide survey of AS events in 249 HCCs with normal (n = 131) and high (n = 118) serum AFP levels using RNA-sequencing data obtained from The Cancer Genome Atlas. RESULTS: In group comparisons of RNA-seq profiles from HCCs with normal and high serum AFP levels, 161 differential AS events (125 genes; ΔPSI > 0.05, FDR < 0.05) were identified to be alternatively spliced between the two groups. Those genes were enriched in cell migration or proliferation terms such as "the cell migration and growth-cone collapse" and "regulation of insulin-like growth factor (IGF) transport and uptake by IGF binding proteins". Most of all, two AS genes (FN1 and FAM20A) directly interact with AFP; these relate to the regulation of IGF transport and post-translational protein phosphorylation. Interestingly, 42 genes and 27 genes were associated with gender and vascular invasion (VI), respectively, but only eighteen genes were significant in survival analysis. We especially highlight that FN1 exhibited increased differential expression of AS events (ΔPSI > 0.05), in which exons 25 and 33 were more frequently skipped in HCCs with normal (low) serum AFP compared to those with high serum AFP. Moreover, these events were gender and VI dependent. CONCLUSION: We found that AS may influence the regulation of transcriptional differences inherent in the occurrence of HCC maintaining normal rather than elevated serum AFP levels.

The landscape of alternative splicing in HIV-1 infected CD4 T-cells.

BACKGROUND: Elucidating molecular mechanisms that are altered during HIV-1 infection may provide a better understanding of the HIV-1 life cycle and how it interacts with infected T-cells. One such mechanism is alternative splicing (AS), which has been studied for HIV-1 itself, but no systematic analysis has yet been performed on infected T-cells. We hypothesized that AS patterns in infected T-cells may illuminate the molecular mechanisms underlying HIV-1 infection and identify candidate molecular markers for specifically targeting infected T-cells. METHODS: We downloaded previously published raw RNA-seq data obtained from HIV-1 infected and non-infected T-cells. We estimated percent spliced in (PSI) levels for each AS exon, then identified differential AS events in the infected cells (FDR < 0.05, PSI difference > 0.1). We performed functional gene set enrichment analysis on the genes with differentially expressed AS exons to identify their functional roles. In addition, we used RT-PCR to validate differential alternative splicing events in cyclin T1 (CCNT1) as a case study. RESULTS: We identified 427 candidate genes with differentially expressed AS exons in infected T-cells, including 20 genes related to cell surface, 35 to kinases, and 121 to immune-related genes. In addition, protein-protein interaction analysis identified six essential subnetworks related to the viral life cycle, including Transcriptional regulation by TP53, Class I MHC mediated antigen, G2/M transition, and late phase of HIV life cycle. CCNT1 exon 7 was more frequently skipped in infected T-cells, leading to loss of the key Cyclin_N motif and affecting HIV-1 transcriptional elongation. CONCLUSIONS: Our findings may provide new insight into systemic host AS regulation under HIV-1 infection and may provide useful initial candidates for the discovery of new markers for specifically targeting infected T-cells.

Differential alternative splicing regulation among hepatocellular carcinoma with different risk factors.

BACKGROUND: Hepatitis B virus (HBV), hepatitis C virus (HCV), and alcohol consumption are predominant causes of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying how differently these causes are implicated in HCC development are not fully understood. Therefore, we investigated differential alternative splicing (AS) regulation among HCC patients with these risk factors. METHODS: We conducted a genome-wide survey of AS events associated with HCCs among HBV (n = 95), HCV (n = 47), or alcohol (n = 76) using RNA-sequencing data obtained from The Cancer Genome Atlas. RESULTS: In three group comparisons of HBV vs. HCV, HBV vs. alcohol, and HCV vs. alcohol for RNA seq (ΔPSI> 0.05, FDR < 0.05), 133, 93, and 29 differential AS events (143 genes) were identified, respectively. Of 143 AS genes, eight and one gene were alternatively spliced specific to HBV and HCV, respectively. Through functional analysis over the canonical pathways and gene ontologies, we identified significantly enriched pathways in 143 AS genes including immune system, mRNA splicing-major pathway, and nonsense-mediated decay, which may be important to carcinogenesis in HCC risk factors. Among eight genes with HBV-specific splicing events, HLA-A, HLA-C, and IP6K2 exhibited more differential expression of AS events (ΔPSI> 0.1). Intron retention of HLA-A was observed more frequently in HBV-associated HCC than HCV- or alcohol-associated HCC, and intron retention of HLA-C showed vice versa. Exon 3 (based on ENST00000432678) of IP6K2 was less skipped in HBV-associated in HCC compared to HCV- or alcohol-associated HCC. CONCLUSION: AS may play an important role in regulating transcription differences implicated in HBV-, HCV-, and alcohol-related HCC development.

Effective therapeutic options for elderly patients with hepatocellular carcinoma: A nationwide cohort study.

We evaluated the post-treatment overall survival (OS) of elderly hepatocellular carcinoma (HCC) patients.The archived records of 10,578 HCC patients registered at the Korean Central Cancer Registry from 2008 through 2014 were retrospectively analyzed. In this registry, we selected Barcelona Clinic Liver Cancer (BCLC) 0, A, or B staged HCC patients (n = 4744) treated by surgical resection (SR), local ablation therapy (LAT), or locoregional therapy (LRT). OSs in nonelderly (<70 years) and elderly (≥70 years) patients were compared after propensity score matching (PSM).In BCLC 0-A staged HCC, the cumulative OS rates of elderly patients were poorer than those of nonelderly patients after PSM (P < .001), but not in those with BCLC stage B (P > .05). In BCLC 0-A staged elderly patients, OS after SR was significantly better than after LAT (P = .005) or LRT (P < .001). In BCLC B staged elderly patients, SR achieved better OS than LRT (P = .006). Multivariable analysis showed that LAT (hazard ratio [HR] 1.52, P = .048) or LRT (HR, 2.01, P < .001) as compared with SR, and large (>3 cm) tumor size (HR1.49, P = .018) were poor predictors of OS for elderly patients with BCLC stage 0-A, and that LRT (HR, 2.64, P = .042) was a poor predictor for those with BCLC stage B.SR provided a better OS rate than LAT or LRT in elderly HCC patients with BCLC stage 0-A, than LRT in those with BCLC stage B. SR should be considered the first therapeutic option even in elderly HCC patients with these stages.

Identification of exon skipping events associated with Alzheimer's disease in the human hippocampus.

BACKGROUND: At least 90% of human genes are alternatively spliced. Alternative splicing has an important function regulating gene expression and miss-splicing can contribute to risk for human diseases, including Alzheimer's disease (AD). METHODS: We developed a splicing decision model as a molecular mechanism to identify functional exon skipping events and genetic variation affecting alternative splicing on a genome-wide scale by integrating genomics, transcriptomics, and neuroimaging data in a systems biology approach. In this study, we analyzed RNA-Seq data of hippocampus brain tissue from Alzheimer's disease (AD; n = 24) and cognitively normal elderly controls (CN; n = 50) and identified three exon skipping events in two genes (RELN and NOS1) as significantly associated with AD (corrected p-value < 0.05 and fold change > 1.5). Next, we identified single-nucleotide polymorphisms (SNPs) affecting exon skipping events using the splicing decision model and then performed an association analysis of SNPs potentially affecting three exon skipping events with a global cortical measure of amyloid-ß deposition measured by [18F] Florbetapir position emission tomography (PET) scan as an AD-related quantitative phenotype. A whole-brain voxel-based analysis was also performed. RESULTS: Two exons in RELN and one exon in NOS1 showed significantly lower expression levels in the AD participants compared to CN participants, suggesting that the exons tend to be skipped more in AD. We also showed the loss of the core protein structure due to the skipped exons using the protein 3D structure analysis. The targeted SNP-based association analysis identified one intronic SNP (rs362771) adjacent to the skipped exon 24 in RELN as significantly associated with cortical amyloid-ß levels (corrected p-value < 0.05). This SNP is within the splicing regulatory element, i.e., intronic splicing enhancer. The minor allele of rs362771 conferred decreases in cortical amyloid-ß levels in the right temporal and bilateral parietal lobes. CONCLUSIONS: Our results suggest that exon skipping events and splicing-affecting SNPs in the human hippocampus may contribute to AD pathogenesis. Integration of multiple omics and neuroimaging data provides insights into possible mechanisms underlying AD pathophysiology through exon skipping and may help identify novel therapeutic targets.