Identification of early salt stress response genes in tomato root by suppression subtractive hybridization and microarray analysis

High salinity is one of the most serious threats to crop production. To understand the molecular basis of plant responses to salt stress better, suppression subtractive hybridization (SSH) and microarray approaches were combined to identify the potential important or novel genes involved in the early stage of tomato responses to severe salt stress. First, SSH libraries were constructed for the root tissue of two cultivated tomato (Solanum lycopersicum) genotypes: LA2711, a salt-tolerant cultivar, and ZS-5, a salt-sensitive cultivar, to compare salt treatment and non-treatment plants. Then a subset of clones from these SSH libraries were used to construct a tomato cDNA array and microarray analysis was carried out to verify the expression changes of this set of clones upon a high concentration of salt treatment at various time points compared to the corresponding non-treatment controls. A total of 201 non-redundant genes that were differentially expressed upon 30 min of severe salt stress either in LA2711 or ZS-5 were identified from microarray analysis; most of these genes have not previously been reported to be associated with salt stress. The diversity of the putative functions of these genes indicated that salt stress resulted in a complex response in tomato plants.     

Identification of genes associated with tumorigenesis of retinoblastoma by microarray analysis

There is no report on the gene expression profile of retinoblastoma (Rb). We analyzed the gene expression profile of Rb by the microarray technique. One thousand four genes were upregulated and 481 genes were downregulated. Microarray data were confirmed by semiquantitative RT-PCR for 5 genes in Rb samples: CDC25A, C17orf75, ERBB3, LATS2, and CHFR. Clusters of differentially expressed genes were identified on chromosomes 1, 16, and 17. Based on the expression profile, we hypothesized that the PI3K/AKT/mTOR (insulin signaling) pathway might be dysregulated in Rb. Our semiquantitative RT-PCR analysis of the PIK3CA, AKT1, FRAP1, and RPS6KB1 genes in Rb samples supported this hypothesis. We suggest that known inhibitors of this pathway could be evaluated for the treatment of Rb.     

Identification of novel universal housekeeping genes by statistical analysis of microarray data

Housekeeping genes are widely used as internal controls in a variety of study types, including real time RT-PCR, microarrays, Northern analysis and RNase protection assays. However, even commonly used housekeeping genes may vary in stability depending on the cell type or disease being studied. Thus, it is necessary to identify additional housekeeping-type genes that show sample-independent stability. Here, we used statistical analysis to examine a large human microarray database, seeking genes that were stably expressed in various tissues, disease states and cell lines. We further selected genes that were expressed at different levels, because reference and target genes should be present in similar copy numbers to achieve reliable quantitative results. Real time RT-PCR amplification of three newly identified reference genes, CGI-119, CTBP1 and GOLGAl, alongside three well-known housekeeping genes, B2M, GAPD, and TUBB, confirmed that the newly identified genes were more stably expressed in individual samples with similar ranges. These results collectively suggest that statistical analysis of microarray data can be used to identify new candidate housekeeping genes showing consistent expression across tissues and diseases. Our analysis identified three novel candidate housekeeping genes (CGI-119, GOLGA1, and CTBP1) that could prove useful for normalization across a variety of RNA-based techniques.     

Identification of novel schizophrenia loci by homozygosity mapping using DNA microarray analysis

The recent development of high-resolution DNA microarrays, in which hundreds of thousands of single nucleotide polymorphisms (SNPs) are genotyped, enables the rapid identification of susceptibility genes for complex diseases. Clusters of these SNPs may show runs of homozygosity (ROHs) that can be analyzed for association with disease. An analysis of patients whose parents were first cousins enables the search for autozygous segments in their offspring. Here, using the Affymetrix® Genome-Wide Human SNP Array 5.0 to determine ROHs, we genotyped 9 individuals with schizophrenia (SCZ) whose parents were first cousins. We identified overlapping ROHs on chromosomes 1, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 16, 17, 19, 20, and 21 in at least 3 individuals. Only the locus on chromosome 5 has been reported previously. The ROHs on chromosome 5q23.3–q31.1 include the candidate genes histidine triad nucleotide binding protein 1 (HINT1) and acyl-CoA synthetase long-chain family member 6 (ACSL6). Other overlapping ROHs may contain novel rare recessive variants that affect SCZ specifically in our samples, given the highly heterozygous nature of SCZ. Analysis of patients whose parents are first cousins may provide new insights for the genetic analysis of psychiatric diseases.     

Identification of genes associated with tumorigenesis of meibomian cell carcinoma by microarray analysis

Meibomian cell carcinoma (MCC) is a malignant tumor of the meibomian glands located in the eyelids. No information exists on the cytogenetic and genetic aspects of MCC. There is no report on the gene expression profile of MCC. Thus there is a need, for both scientific and clinical reasons, to identify genes and pathways that are involved in the development and progression of MCC. We analyzed the gene expression profile of MCC by the microarray technique. Forty-four genes were upregulated and 149 genes were downregulated in MCC. Differential expression data were confirmed for 5 genes by semiquantitative RT-PCR in MCC tumors: GTF2H4, RBM12, UBE2D3, DDX17, and LZTS1. We found dysregulation of two major pathways in MCC: MAPK and JAK/STAT. Clusters of genes on chromosomes 1, 12, and 19 were dysregulated in MCC. The data presented here will facilitate the identification of specific markers and therapeutic targets for the treatment of MCC patients.     

Identification of conserved pathways of DNA-damage response and radiation protection by genome-wide RNAi

Ionizing radiation is extremely harmful for human cells, and DNA double-strand breaks (DSBs) are considered to be the main cytotoxic lesions induced. Improper processing of DSBs contributes to tumorigenesis, and mutations in DSB response genes underlie several inherited disorders characterized by cancer predisposition. Here, we performed a comprehensive screen for genes that protect animal cells against ionizing radiation. A total of 45 C. elegans genes were identified in a genome-wide RNA interference screen for increased sensitivity to ionizing radiation in germ cells. These genes include orthologs of well-known human cancer predisposition genes as well as novel genes, including human disease genes not previously linked to defective DNA-damage responses. Knockdown of eleven genes also impaired radiation-induced cell-cycle arrest, and seven genes were essential for apoptosis upon exposure to irradiation. The gene set was further clustered on the basis of increased sensitivity to DNA-damaging cancer drugs cisplatin and camptothecin. Almost all genes are conserved across animal phylogeny, and their relevance for humans was directly demonstrated by showing that their knockdown in human cells results in radiation sensitivity, indicating that this set of genes is important for future cancer profiling and drug development.     

Biochemical pathways analysis of microarray results: regulation of myogenesis in pigs

Background  

Combining microarray results and biological pathway information will add insight into biological processes. Pathway information is widely available in databases through the internet.     

Identification of estrogen-responsive genes in the GH3 cell line by cDNA microarray analysis

To identify estrogen-responsive genes in somatolactotrophic cells of the pituitary gland, a rat pituitary cell line GH3 was subjected to cDNA microarray analysis. GH3 cells respond to estrogen by growth as well as prolactin synthesis. RNAs extracted from GH3 cells treated with 17beta-estradiol (E2) at 10(-9) M for 24 h were compared with the control samples. The effect of an antiestrogen ICI182780 was also examined. The array analysis indicated 26 genes to be up-regulated and only seven genes down-regulated by E2. Fourteen genes were further examined by real-time RT-PCR quantification and 10 were confirmed to be regulated by the hormone in a dose-dependent manner. Expression and regulation of these genes were then examined in the anterior pituitary glands of female F344 rats ovariectomized and/or treated with E2 and 8 out of 10 were again found to be up-regulated. Interestingly, two of the most estrogen-responsive genes in GH3 cells were strongly dependent on E2 in vivo. #1 was identified as calbindin-D9k mRNA, with 80- and 118-fold induction over the ovariectomized controls at 3 and 24 h, respectively, after E2 administration. #2 was found to be parvalbumin mRNA, with 30-fold increase at 24 h. Third was c-myc mRNA, with 4.5 times induction at 24 h. The levels were maintained after one month of chronic E2 treatment. Identification of these estrogen-responsive genes should contribute to understating of estrogen actions in the pituitary gland.     

Identification of hepatocellular-carcinoma-associated antigens and autoantibodies by serological proteome analysis combined with protein microarray

To comprehensively study autoantibodies in patients with hepatocellular carcinoma (HCC), we used an approach-based serology and proteomics technologies. Total proteins extracted from HepG2 cells and HepG2.2.15 cells were separated by two-dimensional gel electrophoresis (2DE) and then transferred onto polyvinylidene difluoride (PVDF) membranes, which were subsequently incubated with sera from HCC patients or from normal controls. As a result, 13 HCC-associated antigens were identified. Antigenicity of eight proteins was further confirmed using recombinant proteins by Western blotting (WB) and protein microarray. The results of antigen microarray analysis showed strong signals of keratin 8 and lamin A/C in chronic hepatitis controls; therefore, the autoantibodies to keratin 8 and lamin A/C may not be HCC-specific. These two antigens were removed from subsequent analyses. The frequencies of positive reactions to DEAD (Asp-Glu-Ala-Asp) box polypeptide 3, eukaryotic translation elongation factor 2 (eEF2), apoptosis-inducing factor (AIF), heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2), prostatic binding protein, and triosephosphate isomerase (TIM) were significantly higher in HCC than in chronic hepatitis and normal individuals. Positive reactions to DEAD box polypeptide 3, eEF2, AIF, and prostatic binding protein were significantly more frequent in HCC than in any other cancer. The sensitivity of any individual antigen in HCC at stage I ranged from 50 to 85%. When the combinations of six antigens were analyzed, the sensitivity increased to 90%. We conclude that the detection of autoantibodies against the six antigens may have value on early diagnosis of HCC.     

Identification of potential genes/proteins regulated by Tiam1 in colorectal cancer by microarray analysis and proteome analysis

Tiam1 (T-cell lymphoma invasion and metastasis-inducing protein 1), a guanine nucleotide exchange factor that activates Rac, is a colorectal cancer metastasis-related gene. In this study, we aimed to better understand the mechanism underlying Tiam1-mediated metastasis. We applied gene microarray and proteome analysis and compared expression of genes and proteins in a stable Tiam1-silencing colorectal cancer cell line and in a control cell line. Our analysis identified three genes, high-mobility group box1 (HMGB1), annexin IV (ANXA4) and phosphoglycerate mutase 1 (PGAM1) that were associated with Tiam1. Analysis of these proteins, which may be directly or indirectly regulated by Tiam1, may provide insight into the role and mechanism of Tiam1 in colorectal cancer metastasis.