Biomedical subjects

Sen-Yung Hsieh

Publications and source records attributed to Sen-Yung Hsieh.

7 recordsLinked to original sources

PubMed2006

Differentially profiling the low-expression transcriptomes of human hepatoma using a novel SSH/microarray approach.

BACKGROUND: The main limitation in performing genome-wide gene-expression profiling is the assay of low-expression genes. Approaches with high throughput and high sensitivity for assaying low-expression transcripts are urgently needed for functional genomic studies. Combination of the suppressive subtractive hybridization (SSH) and cDNA microarray techniques using the subtracted cDNA clones as probes printed on chips has greatly improved the efficiency for fishing out the differentially expressed clones and has been used before. However, it remains tedious and inefficient sequencing works for identifying genes including the great number of redundancy in the subtracted amplicons, and sacrifices the original advantages of high sensitivity of SSH in profiling low-expression transcriptomes. RESULTS: We modified the previous combination of SSH and microarray methods by directly using the subtracted amplicons as targets to hybridize the pre-made cDNA microarrays (named as "SSH/microarray"). mRNA prepared from three pairs of hepatoma and non-hepatoma liver tissues was subjected to the SSH/microarray assays, as well as directly to regular cDNA microarray assays for comparison. As compared to the original SSH and microarray combination assays, the modified SSH/microarray assays allowed for much easier inspection of the subtraction efficiency and identification of genes in the subtracted amplicons without tedious and inefficient sequencing work. On the other hand, 5015 of the 9376 genes originally filtered out by the regular cDNA microarray assays because of low expression became analyzable by the SSH/microarray assays. Moreover, the SSH/microarray assays detected about ten times more (701 vs. 69) HCC differentially expressed genes (at least a two-fold difference and P < 0.01), particularly for those with rare transcripts, than did the regular cDNA microarray assays. The differential expression was validated in 9 randomly selected genes in 18 pairs of hepatoma/non-hepatoma liver tissues using quantitative RT-PCR. The SSH/microarray approaches resulted in identifying many differentially expressed genes implicated in the regulation of cell cycle, cell death, signal transduction and cell morphogenesis, suggesting the involvement of multi-biological processes in hepato-carcinogenesis. CONCLUSION: The modified SSH/microarray approach is a simple but high-sensitive and high-efficient tool for differentially profiling the low-expression transcriptomes. It is most adequate for applying to functional genomic studies.

Carcinoma, Hepatocellular↗

PubMed2006

Systematical evaluation of the effects of sample collection procedures on low-molecular-weight serum/plasma proteome profiling.

Blood is an ideal source for biomarker discovery. However, little has been done to address the effects of sampling, handling and storage procedures on serum/plasma proteomes. We used magnetic bead-based MALDI-TOF MS to systematically evaluate the influence of each procedure on low-molecular-weight serum/plasma proteome profiling on the basis of the whole spectra. We found that sampling procedures, including the selection of blood collection tubes and anticoagulants, variations in clotting time or time lag before centrifugation, and hemolysis, displayed significant effects on the proteomes. Moreover, serum and plasma were mutually incompatible for proteome comparison. By contrast, overnight fasting, handling procedures, including centrifugation speeds (1500 x g vs. 3000 x g) or time (15 min vs. 30 min), and storage conditions, such as at 4 degrees C or 25 degrees C for up to 24 h or at -80 degrees C for up to 3 months, and repeated freeze/thaw of up to ten cycles, had relatively minor effects on the proteomes based upon our analysis of about 100 peaks. We concluded that low-molecular-weight serum/plasma proteomes were diversely affected by sampling, handling and storage with most change from variations of sampling procedures. We therefore suggest the necessity of standardizing sampling procedure for proteome comparison and biomarker discovery.

Adult↗

PubMed2006

Comparative proteomic studies on the pathogenesis of human ulcerative colitis.

Ulcerative colitis (UC) is a chronic inflammatory disorder primarily affecting the colon mucosa. Its etiology and pathogenesis remain unclear. We used 2-DE and MS to identify differentially expressed proteins among the UC active, UC inactive, nonspecific colitis, and normal colon mucosa. Thirteen down-regulated and six up-regulated proteins were identified. Of the down-expressed proteins, eight (heat-shock protein 90 (HSPA9B), heat-shock protein 60 (HSPD1), H+-transporting two-sector ATPase (ATP5B), prohibitin (PHB), mitochondrial malate dehydrogenase (MDH2), voltage-dependent anion-selective channel protein 1 (VDAC1), thioredoxin peroxidase (PRDX1), and thiol-specific antioxidant (PRDX2)) were mitochondrial proteins, three (ATP5B, MDH2, triosephosphate isomerase) were involved in energy generation, three (PRDX1, PRDX2, SELENBP1) were cellular antioxidants, and six (HSPD1, HSPA9B, PRDX1, PRDX2, PHB, VDAC1) were stress-response proteins. Transmission electron microscopy revealed pathological alterations of mitochondrial ultrastructures even before the global colonocyte changes in the UC colon mucosa. PHB, an essential mitochondrial component protein, was down-expressed in the disease active as well as inactive colon mucosa from the patients of UC, indicative of an early event of mitochondrial changes during UC development. In contrast, aberrant activation of NFAT and ectopic expression of potential immunogenic proteins (tumor rejection antigen 1 and poliovirus receptor related protein 1) were found in the UC-diseased colon mucosa. Our findings suggest the implications of colonocyte mitochondrial dysfunction and perturbed mucosa immune regulation in the pathogenesis of UC and provide potential targets for the development of a new therapy.

Adult↗

PubMed2005

High-frequency Alu-mediated genomic recombination/deletion within the caspase-activated DNase gene in human hepatoma.

The Alu repetitive elements, which constitute 10% of human genome, may serve as bridges for genomic recombination. However, their roles in tumorigenesis remain to be elucidated. Caspase-activated DNase (CAD), whose gene (hCAD) is mapped at chromosome 1p36, a region frequently displaying hemizygote deletion in many human cancers, is the key enzyme for nucleosome fragmentation during apoptosis. Recently, we detected many aberrant mRNAs for hCAD in many human hepatoma cells. To elucidate the genetic basis leading to the mRNA aberration, we used PCR-based chromosome walking to clone the corresponding genomic DNA identifying a novel Alu/Alu homologous recombination/deletion within hCAD in HepG2 and Hep3B cells. We then detected similar recombination events in 13 out of the 20 hepatoma tissues and in eight of the para-cancerous cirrhotic livers. The recombination was inclined to occur in males (P=0.031) and had marginal association with high-grade hepatoma (P=0.070) and tumor recurrence (P=0.070). The recombination caused exon-3 deletion, which in turn led to exon-3 skipping or replacement with a partial Alu-sequence, and consequential C-truncation of CAD. Our findings of high frequency of Alu-mediated hCAD deletion in human hepatoma not only underscore the implication of hCAD in hepatocarcinogenesis, but also highlight the potential roles of human repetitive sequences in mediating genome instability in human cancers.

Alu Elements↗

PubMed2005

Dys-regulation of clusterin in human hepatoma is not associated with tumorigenesis but is secondary to cell response to external tresses.

It becomes feasible to perform genome-wide differential gene or protein expression in the post genome era. However, little has been addressed on the effects of external stresses and microenvironment alterations on the outcomes of gene and protein expression. To identify downregulated genes during hepatoma development, we combined the cDNA representational difference analysis (RDA) and reverse Northern blot analysis identifying eight genes. Of interest, the expression of the clusterin gene was either down or upregulated in 8 and 7 out of the 20 hepatoma tissues, respectively. Further analysis revealed that its expression was independent of patients' age, gender, causes of liver disease, tumor size, tumor histological stage, or clinical outcome, but was strongly associated with the methods of hepatectomy procedures. In vitro studies disclosed that the clusterin mRNA was increased twofold in early exponential phase of cell proliferation followed by downregulation in the subsequent quiescence phase, whereas it was rapidly increased up to twelvefold upon UV-induced apoptosis. These results suggest that dys-regulation of the clusterin gene in human hepatoma was most likely due to cellular responses to external stresses especially during the procedures for sample collection rather than any correlation to hepatoma development or progression. Our findings that external stresses or microenvironmental changes could greatly affect gene or protein expression offer a general caution to all the studies conducted via genomic and proteomic approaches.

Adult↗

PubMed2005

Fishing for biomarkers: analyzing mass spectrometry data with the new ClinProTools software.

Recently, applications of mass spectrometry in the field of clinical proteomics have gained tremendous visibility in the scientific and clinical community. One major objective is the search for potential biomarkers in complex body fluids like serum, plasma, urine, saliva, or cerebral spinal fluid. For this purpose, efficient visualization of large data sets derived from patient cohorts is crucial to provide clinical experts an interactive impression of the data quality. Additionally, it is necessary to apply statistical analysis and pattern matching algorithms to attain validated signal patterns that may allow for later applications in sample classification. We introduce the new ClinProTools bioinformatics software, which performs all major steps of profiling, screening, and monitoring applications in clinical proteomics. ClinProTools is the data interpretation software of the mass spectrometry-based ClinProt solutions for biomarker analysis. ClinProTools performs data pretreatment, visualization, statistics, pattern determination, pattern evaluation, and classification of spectra. This article will focus on ClinProTool's powerful and intuitive visualization options for clinical proteomics applications.

Algorithms↗

PubMed2004

Dickkopf-3/REIC functions as a suppressor gene of tumor growth.

To identify putative tumor suppressor genes in hepatocarcinogenesis, we combined the representational difference analysis and reverse northern blot identifying downregulated genes in human hepatoma tissues. One of them was Dkk-3/REIC. Dkk-3/REIC was downregulated in 11 out of the 20 human hepatoma tissues as compared to their counterparts of noncancerous liver tissues by northern blot analysis. It was also downregulated in 29 out of 48 human cancer samples including the kidney, urinary bladder, prostate, pancreas and lung cancers. Its gene product, Dkk-3/REIC, was found to be N-glycosylated and have two isoforms, the 55 kDa in the cytosol and 50 kDa secreted in the medium. Ectopic expression of Dkk-3/REIC in HeLa, Hep3B and Huh 7 cells led to suppression of cell growth, which was primarily attributable to induction of cell apoptosis. The suppression phenomenon was found to be cell-type related (most prominent in HeLa and least in Hep3B cells) and cell-density dependent (attenuated as the cell density increased). Transduction of Dkk-3/REIC into HeLa and Hep3B cells caused suppression on colony formation in vitro and reduced tumor growth rate in inoculated athymic nude mice. In conclusion, these data indicate that Dkk-3/REIC functions as a suppressor for human tumor growth.

Adaptor Proteins, Signal Transducing↗