Project description:The CCA-adding enzyme adds CCA to the 3' ends of transfer RNAs (tRNAs), a critical step in tRNA biogenesis that generates the amino acid attachment site. We found that the CCA-adding enzyme plays a key role in tRNA quality control by selectively marking unstable tRNAs and tRNA-like small RNAs for degradation. Instead of adding CCA to the 3' ends of these transcripts, CCA-adding enzymes from all three kingdoms of life add CCACCA. Here, we report deep sequencing analysis of the 3' ends of tRNA-Ser-CGA and tRNA-Ser-UGA from S. cerevisiae strains and show that hypomodified mature tRNAs are subjected to CCACCA (or poly(A) addition) as part of a rapid tRNA decay pathway in vivo. We conjecture that CCACCA addtion is a universal mechanism for controlling tRNA levels and preventing errors in translation.
Project description:Introduction: Accurate diagnosis of distal cholangiocarcinoma (CCA) and pancreatic ductal adenocarcinoma (PDAC) is a challenge with clinical consequences. Both are lethal malignancies with distinct therapeutic options. This study aimed to identify a circulating microRNA (miRNA) signature to diagnose and discriminate distal CCA from PDAC. Methods: In the discovery phase, microarray profiling of 752 miRNAs was performed on plasma samples of seven patients with distal CCA and seven age- and sex-matched healthy controls. Significant candidate miRNAs were selected for validation based on predefined selection criteria. In the validation phase, these miRNAs were analyzed by RT-qPCR in an independent cohort of healthy controls (n=32), benign periampullary disease (n=20), distal CCA (n=24), and age- sex- and stage-matched PDAC (n=24). Data were normalized to a combination of two reference genes. The optimal diagnostic combination of miRNAs was determined by logistic regression. Sensitivity and specificity were evaluated by ROC curves and AUC values. Results: In the discovery phase, 19 miRNAs were significantly deregulated in patients with distal CCA compared to healthy controls. In the validation phase, 12 candidate miRNAs were selected for validation by RT-qPCR based on pre-defined selection criteria. A two-miRNA panel of downregulated miR-16 and upregulated miR-877 was able accurately distinguish distal CCA from benign disease as well as from PDAC. Conclusion: This is the first study to identify a combined panel of plasma miRNAs which shows promising diagnostic capability to serve as distal CCA signature with the potential to discriminate distal CCA from PDAC.
Project description:Cholangiocarcinoma (CCA) is a type of highly aggressive cancer arising from the biliary system. Through serum exosome miRNA sequencing, we screened out the differentially expressed miRNA in patients with cholangiocarcinoma(CCA) and common bile duct stones(CBDS).
Project description:We used Affymetrix SNP6.0 microarray profiling to analyze Copy Number Variation in tumor and paired non-tumor tissue of HCC and CCA patients. We used copy number data to confirm molecular subgroups of HCC and CCA patients from Thailand
Project description:The goal of this study is to provide a global transcriptome profiling (RNA-seq) of stem-like subset in human cholangiocarcinoma (CCA). Functional enrichment of CCA stem-like subset was performed by 3D sphere culture (SPH) in CCA cell lines. Comparison to parental CCA cells grown as 2D monolayer is provided.
Project description:Expression profiling of 2555 miRNAs was performed using microarray. Of these, 451 miRNAs expressed in CCA cells. Microarray identified 17 miRNAs that were increased by > 1.5-fold and 56 miRNAs that were decreased by < 0.67-fold in CCA cells treated with TGF-β.
Project description:Cholangiocarcinoma (CCA) is an epithelial malignancy with a dismal prognosis owing to limited treatment options. Here, we identified several compound candidates against CCA using a high-throughput drug screen with approved or emerging oncology drugs, among which kinesin spindle protein (KSP) inhibitors showed potent cytotoxic effects on CCA cells. Treatment with KSP inhibitors SB743921 and ARRY520 caused significant tumor suppression in CCA xenograft models in vivo. Mechanistically, KSP inhibitors led to the formation of abnormal monopolar spindles, which further resulted in the mitotic arrest and cell death of CCA cells both in vivo and in vitro. KEGG pathway analysis of transcriptional data confirmed this finding. Moreover, our clinical data as well as the TCGA database showed KIF11 expression was abundant in most CCA tumor specimens and associated with poor outcomes of CCA patients. Our results demonstrate that the therapeutic regimen of KSP inhibitors could be a promising treatment strategy in CCA.
Project description:Genetic abnormalities of cholangiocarcinoma (CCA) have been widely studied; however, epigenomic changes related to cholangiocarcinogenesis have been less well characterized. We have profiled the DNA methylomes of 28 primary CCA and six matched adjacent normal tissues using Infinium’s HumanMethylation27 BeadChips with the aim of identifying gene sets aberrantly epigenetically regulated in CCA. Using a linear model for microarray data we identified 1610 differentially methylated autosomal CpG sites with 809 CpG sites (representing 603 genes) being hypermethylated and 801 CpG sites (representing 712 genes) being hypomethylated in CCA versus adjacent normal (false discovery rate ? 0.05). Gene Ontology and Gene Set Enrichment analyses identified gene sets significantly associated with hypermethylation at linked CpG sites in CCA including homeobox genes and target genes of PRC2, EED, SUZ12 and Histone H3 trimethylation at lysine 27. We confirmed frequent hypermethylation at the homeobox genes HOXA9 and HOXD9 by bisulfite pyrosequencing in a larger cohort of CCA (n = 103). Our findings indicate a key role for hypermethylation of multiple CpG sites at genes associated with a stem cell-like phenotype as a common molecular aberration in CCA. These data have implications for CCA carcinogenesis, as well as possible novel treatment options using histone methyltransferase inhibitors. Methods: Thirty-two primary CCA, 6 matched adjacent normal, 5 CCA cell lines and an immortal biliary cell line were analyzed for DNA methylation profiles using the Infinium HumanMethylation27 BeadChip. Differential methylation was statistically analyzed using LIMMA. Specific functional terms and Gene Set Enrichment Analysis were performed to identify specific function of aberrantly methylated genes. Results: We identified 711 and 590 autosomal CpG sites as being hypermethylated and hypomethylated representing 527 and 521 genes, respectively, in CCA compared to adjacent normal (false discovery rate 0.05). A number of gene sets were significantly associated with hypermethylation at linked CpG sites in CCA including homeobox genes and the target genes of PRC2, EED, SUZ12, and H3K27me3, whereas target genes of NOS and OCT4 were hypomethylated at linked CpG sites in tumors. Frequent aberrant DNA methylation at HOXA9 and HOXD9 was confirmed by bisulfite pyrosequencing in a larger cohort of CCA (n = 107). Conclusions: This is the first report of a DNA methylation profile of multiple CpG sites at genes associated with a stem cell-like phenotype as a common molecular aberration in CCA. These data have implications for CCA carcinogenesis, as well as possible novel treatment options using histone methyltransferase inhibitors. Array-based methylation profiling was performed using the Infinium HumanMethylation27 BeadChip in 28 primary cholangiocarcinomas and six matched adjacent normal tissues. The reproducibility of the Infinium HumanMethylation27 BeadChips was evaluated using five technical replicates of the ovarian cancer cell line PEO1. Differential methylation cutoff was estimated from five technical replicates by bootstrap resampling and set at ?? ? 0.15 corresponding to a FDR ? 0.05.
Project description:We explore whether a low-energy diet intervention for Metabolic dysfunction-associated steatohepatitis (MASH) improves liver disease by means of modulating the gut microbiome. 16 individuals were given a low-energy diet (880 kcal, consisting of bars, soups, and shakes) for 12 weeks, followed by a stepped re-introduction to whole for an additional 12 weeks. Stool samples were obtained at 0, 12, and 24 weeks for microbiome analysis. Fecal microbiome were measured using 16S rRNA gene sequencing. Positive control (Zymo DNA standard D6305) and negative control (PBS extraction) were included in the sequencing. We found that low-energy diet improved MASH disease without lasting alterations to the gut microbiome.