Identification of differentially expressed genes in mouse kidney after irradiation using microarray analysis

Irradiation of the kidney induces dose-dependent, progressive renal functional impairment, which is partly mediated by vascular damage. The molecular mechanisms underlying the development of radiation-induced nephropathy are unclear. Given the complexity of radiation-induced responses, microarrays may offer new opportunities to identify a wider range of genes involved in the development of radiation injury. The aim of the present study was to determine whether microarrays are a useful tool for identifying time-related changes in gene expression and potential mechanisms of radiation-induced nephropathy. Microarray experiments were performed using amplified RNA from irradiated mouse kidneys (1 x 16 Gy) and from sham-irradiated control tissue at different intervals (1-30 weeks) after irradiation. After normalization procedures (using information from straight-color, color-reverse and self-self experiments), the differentially expressed genes were identified. Control and repeat experiments were done to confirm that the observations were not artifacts of the array procedure (RNA amplification, probe synthesis, hybridizations and data analysis). To provide independent confirmation of microarray data, semi-quantitative PCR was performed on a selection of genes. At 1 week after irradiation (before the onset of vascular and functional damage), 16 genes were significantly up-regulated and 9 genes were down-regulated. During the period of developing nephropathy (10 to 20 weeks), 31 and 42 genes were up-regulated and 9 and 4 genes were down-regulated. At the later time of 30 weeks, the vast majority of differentially expressed genes (191 out of 203) were down-regulated. Potential genes of interest included TSA-1 (also known as Ly6e) and Jagged 1 (Jag1). Increased expression of TSA-1, a member of the Ly-6 family, has previously been reported in response to proteinuria. Jagged 1, a ligand for the Notch receptor, is known to play a role in angiogenesis, and is particularly interesting in the context of radiation-induced vascular injury. The present study demonstrates the potential of microarrays to identify changing patterns of gene expression in irradiated kidney. Further studies will be required to evaluate functional involvement of these genes in vascular-mediated normal tissue injury.     

Nonsense-mediated mRNA decay (NMD) silences the accumulation of aberrant trypsin proteinase inhibitor mRNA in Nicotiana attenuata

In eukaryotes, genes carrying premature termination codons (PTCs) are often associated with decreased mRNA levels compared with their counterparts without PTCs. PTC-harboring mRNA is rapidly degraded through the nonsense-mediated mRNA decay (NMD) pathway to prevent the accumulation of potentially detrimental truncated proteins. In a native ecotype of Nicotiana attenuata collected from Arizona (AZ), the mRNA levels of a trypsin proteinase inhibitor ( TPI ) gene are substantially lower than in plants collected from Utah (UT). Cloning the AZ TPI gene revealed a 6 bp deletion mutation in exon 2 resulting in a PTC and decreased mRNA levels through NMD. Silencing UPF1 , 2 and 3 in N. attenuata AZ plants by virus-induced gene silencing (VIGS) enhanced the levels of PTC-harboring TPI mRNA, demonstrating a conserved role for UPF genes in plants. Furthermore, using cell suspension cultures that express variants of the TPI construct, we demonstrate that both intron-containing and intronless genes are subject to NMD in plants; unlike PTCs in mammals, PTCs downstream of introns activate NMD in plants. However, when a PTC is only 4 bp upstream of an intron, the NMD surveillance mechanism is abrogated. We also demonstrate that, in an intronless TPI gene, a PTC located at the beginning or the end of the coding sequence triggers NMD less efficiently than do PTCs located at the middle of the coding sequence. Taken together, these results highlight the complexity of the NMD activation mechanisms in plants.     

利用生物信息学研究肥胖与2型糖尿病患者肝组织基因表达变化

目的:利用芯片数据分析工具对GEO基因芯片数据进行数据挖掘,系统分析肥胖与2型糖尿病患者肝组织相关基因表达的变化,探讨肥胖与2型糖尿病的联系及糖尿病早期预防和诊断的新靶点。方法:首先在公共芯片数据库中选择肥胖与2型糖尿病相关芯片数据(GSE15653),利用R等芯片数据分析工具分析肥胖与2型糖尿病患者肝组织基因的表达变化,并预测相关差异表达基因在血中蛋白表达。结果:肥胖患者与正常人肝组织比较发现412个差异表达基因,其中上调表达基因212个,下调表达基因200个,2型糖尿病患者中控制良好者与正常人肝组织比较发现486个差异表达基因,其中上调表达基因253个,下调表达基因233个,而2型糖尿病患者中控制不良者与正常人肝组织比较发现1051个差异表达基因,其中上调表达基因560个,下调表达基因491个;2型糖尿病控制良好者与肥胖患者肝组织有263个相同的表达变化基因,而2型糖尿病控制不良者与肥胖患者肝组织有131个相同的表达变化基因;结合蛋白质组学结果分析肥胖与2型糖尿病相关的差异表达基因中有30个蛋白表达产物是分泌型蛋白。结论:肥胖及2型糖尿病患者肝组织与正常肝组织比较基因表达均发生明显变化,其基因表达变化数目随疾病的严重性增加而增多,而且2型糖尿病的控制情况与肝组织基因表达变化有密切关系。肥胖与2型糖尿病相关的差异表达基因中表达分泌型蛋白的可进一步用于研发监测疾病发生发展的候选靶分子。     

Standardization strategy for quantitative PCR in human seminoma and normal testis

Housekeeping genes are commonly used as endogenous references in quantitative RT-PCR. Ideally these genes are constitutionally expressed by all cell types and do not vary under experimental conditions. Tissues of 9 normal testes and 22 classical pure seminoma were obtained for RNA-extraction. Real-time RT-PCR was used to examine the mRNA-expression of ubiquitin C, beta-actin, GAPDH, 18S ribosomal RNA (18S rRNA) and porphobilinogen-deaminase (PBGD). Additionally, 3 normal testicular tissues and 39 seminoma, including 1 normal testis and 17 seminoma of the RT-PCR group, were utilized for microarray analyses. Ubiquitin C (protein degradation) was down-regulated, GAPDH (carbohydrate metabolism), beta-actin (cytoskeleton), 18S rRNA (ribosome) and PBGD (porphyrin metabolism) were up-regulated in seminoma. A normalization of the target gene data with up-regulated housekeeping genes would equalize or underestimate up-regulated data and overestimate down-regulated data. We demonstrate that none of the investigated housekeeping genes is suitable for normalization of the target gene RT-PCR data, but may be essential for tumor metabolism in human seminoma. Further, we developed a standardization strategy, which is applicable to many experimental investigations.     

利用GenMAPP筛查鼻咽癌差异表达基因

利用GenMAPP软件对鼻咽癌和正常鼻咽上皮基因微阵列表达谱结果进行分析,筛查鼻咽癌差异表达基因. 结果显示：在17 000个基因中,与正常鼻咽上皮相比,在鼻咽癌中发生2倍以上差异表达的基因共有339个,其中有160个基因在鼻咽癌中表达上调,179个表达下调. 这些基因分别与细胞增殖、基因转录、凋亡、信号转导、DNA损伤修复、肿瘤分化和浸润转移及细胞周期调节等相关. 鼻咽癌的发生发展存在多基因表达调控的改变,对其差异表达基因的研究有助于阐明鼻咽癌发生发展机制.     

LDHA induces EMT gene transcription and regulates autophagy to promote the metastasis and tumorigenesis of papillary thyroid carcinoma

Papillary thyroid carcinoma (PTC) is one of the most common kinds of endocrine-related cancer and has a heterogeneous prognosis. Metabolic reprogramming is one of the hallmarks of cancers. Aberrant glucose metabolism is associated with malignant biological behavior. However, the functions and mechanisms of glucose metabolism genes in PTC are not fully understood. Thus, data from The Cancer Genome Atlas database were analyzed, and lactate dehydrogenase A (LDHA) was determined to be a potential novel diagnostic and therapeutic target for PTCs. The research objective was to investigate the expression of LDHA in PTCs and to explore the main functions and relative mechanisms of LDHA in PTCs. Higher expression levels of LDHA were found in PTC tissues than in normal thyroid tissues at both the mRNA and protein levels. Higher expression levels of LDHA were correlated with aggressive clinicopathological features and poor prognosis. Moreover, we found that LDHA not only promoted PTC migration and invasion but also enhanced tumor growth both in vitro and in vivo. In addition, we revealed that the metabolic products of LDHA catalyzed induced the epithelial–mesenchymal transition process by increasing the relative gene H3K27 acetylation. Moreover, LDHA knockdown activated the AMPK pathway and induced protective autophagy. An autophagy inhibitor significantly enhanced the antitumor effect of FX11. These results suggested that LDHA enhanced the cell metastasis and proliferation of PTCs and may therefore become a potential therapeutic target for PTCs.Subject terms: Thyroid cancer, Target validation     

Monitoring expression profiles of Arabidopsis genes during cold acclimation and deacclimation using DNA microarrays

A comparative analysis of gene expression profiles during cold acclimation and deacclimation is necessary to elucidate the molecular mechanisms of cold stress responses in higher plants. We analyzed gene expression profiles in the process of cold acclimation and deacclimation (recovery from cold stress) using two microarray systems, the 7K RAFL cDNA microarray and the Agilent 22K oligonucleotide array. By both microarray analyses, we identified 292 genes up-regulated and 320 genes down-regulated during deacclimation, and 445 cold up-regulated genes and 341 cold down-regulated genes during cold acclimation. Many genes up-regulated during deacclimation were found to be down-regulated during cold acclimation, and vice versa. The genes up-regulated during deacclimation were classified into (1) regulatory proteins involved in further regulation of signal transduction and gene expression and (2) functional proteins involved in the recovery process from cold-stress-induced damages and plant growth. We also applied expression profiling studies to identify the key genes involved in the biosynthesis of carbohydrates and amino acids that are known to play important roles in cold acclimation. We compared genes that are regulated during deacclimation with those regulated during rehydration after dehydration to discuss the similarity and difference of each recovery process.Electronic Supplementary Material Supplementary materials are available for this article at     

Effects of acetaminophen on hepatic gene expression in mice

Acetaminophen (APAP) is one of the most commonly used drugs for the safe and effective treatment of fever and pain. However, it is a well-established hepatotoxin. The objective of this study was to identify alternation in various genes in liver of mice after administration of low and high doses of APAP. Male C57BL/6J mice received APAP (30 or 300 mg/kg, i.p.). They were sacrificed after 6 hr and 24 hr for assessment of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), total RNA isolation, cDNA microarray analysis and histopathological analysis of liver injury. Low dose of APAP did not cause hepatotoxicity in mice. However, it was toxic at a high dose. Using microarray technology, we selected changed genes more than 1.5 fold. Gene expression changes were recorded even at a low dose treatment with APAP. Six (6) hr after APAP treatment at low dose, 6 genes were up-regulated and 25 genes were down-regulated. However, 24 hr after treatment at low dose 8 genes were up-regulated and 34 genes were down-regulated. 6 hr after of high dose treatment 29 genes were down-regulated and none was up-regulated. A 24 hr treatment with high dose up-regulated 6 genes and down-regulated 18 genes. These expression patterns provide information on high versus low dose mechanisms of APAP toxicity. Gene expression signatures recorded after a nontoxic dose of APAP strongly support the validity of gene expression changes as meaningful markers of hepatotoxicity.     

Gene Expression in the Cyanobacterium <Emphasis Type="Italic">Anabaena</Emphasis> sp. PCC7120 under Desiccation

The N₂-fixing cyanobacterium Anabaena sp. PCC7120 showed an inherent capacity for desiccation tolerance. A DNA microarray covering almost the entire genome of Anabaena was used to determine the genome-wide gene expression under desiccation. RNA was extracted from cells at intervals starting from early to late desiccation. The pattern of gene expression in DNA fragments was categorized into seven types, which include four types of up-regulated and three types of down-regulated fragments. Validation of the data was carried out by RT-PCR on selected up-regulated DNA fragments and was consistent with the changes in mRNA levels. Our conclusions regarding desiccation tolerance for Anabaena sp. PCC7120 are as follows: (i) Genes for osmoprotectant metabolisms and the K⁺ transporting system are up-regulated from early to mid-desiccation; (ii) genes induced by osmotic, salt, and low-temperature stress are up-regulated under desiccation; (iii) genes for heat shock proteins are up-regulated after mid-desiccation; (iv) genes for photosynthesis and the nitrogen-transporting system are down-regulated during early desiccation; and (v) genes for RNA polymerase and ribosomal protein are down-regulated between the early and the middle phase of desiccation. Profiles of gene expression are discussed in relation to desiccation acclimation.     

Evaluation of Genome-Wide Expression Profiles of Blood and Sputum Neutrophils in Cystic Fibrosis Patients Before and After Antibiotic Therapy

In seeking more specific biomarkers of the cystic fibrosis (CF) lung inflammatory disease that would be sensitive to antibiotic therapy, we sought to evaluate the gene expression profiles of neutrophils in CF patients before treatment in comparison with non-CF healthy individuals and after antibiotic treatment. Genes involved in neutrophil-mediated inflammation, i.e. chemotaxis, respiratory burst, apoptosis, and granule exocytosis, were the targets of this study. Microarray analysis was carried out in blood and airway neutrophils from CF patients and in control subjects. A fold change (log) threshold of 1.4 and a cut-off of p<0.05 were utilized to identify significant genes. Community networks and principal component analysis were used to distinguish the groups of controls, pre- and post-therapy patients. Control subjects and CF patients before therapy were readily separated, whereas a clear distinction between patients before and after antibiotic therapy was not possible. Blood neutrophils before therapy presented 269 genes down-regulated and 56 up-regulated as compared with control subjects. Comparison between the same patients before and after therapy showed instead 44 genes down-regulated and 72 up-regulated. Three genes appeared to be sensitive to therapy and returned to “healthy” condition: phorbol-12-myristate-13-acetate-induced protein 1 (PMAIP1), hydrogen voltage-gated channel 1 (HVCN1), and β-arrestin 1 (ARRB1). The up-regulation of these genes after therapy were confirmed by real time PCR. In airway neutrophils, 1029 genes were differentially expressed post- vs pre-therapy. Of these, 30 genes were up-regulated and 75 down-regulated following antibiotic treatment. However, biological plausibility determined that only down-regulated genes belonged to the gene classes studied for blood neutrophils. Finally, it was observed that commonly expressed genes showed a greater variability in airway neutrophils than that found in blood neutrophils, both before and after therapy. These results indicate more specific targets for future interventions in CF patients involving respiratory burst, apoptosis, and granule exocytosis.     

Metabolic genes in cancer: Their roles in tumor progression and clinical implications

Re-programming of metabolic pathways is a hallmark of physiological changes in cancer cells. The expression of certain genes that directly control the rate of key metabolic pathways including glycolysis, lipogenesis and nucleotide synthesis are drastically altered at different stages of tumor progression. These alterations are generally considered as an adaptation of tumor cells; however, they also contribute to the progression of tumor cells to become more aggressive phenotypes. This review summarizes the recent information about the mechanistic link of these genes to oncogenesis and their potential utility as diagnostic markers as well as for therapeutic targets. We particularly focus on three groups of genes; GLUT1, G6PD, TKTL1 and PGI/AMF in glycolytic pathway, ACLY, ACC1 and FAS in lipogenesis and RRM2, p53R2 and TYMS for nucleotide synthesis. All these genes are highly up-regulated in a variety of tumor cells in cancer patients, and they play active roles in tumor progression rather than expressing merely as a consequence of phenotypic change of the cancer cells. Molecular dissection of their orchestrated networks and understanding the exact mechanism of their expression will provide a window of opportunity to target these genes for specific cancer therapy. We also reviewed existing database of gene microarray to validate the utility of these genes for cancer diagnosis.     

Copy number alteration and uniparental disomy analysis categorizes Japanese papillary thyroid carcinomas into distinct groups

The aim of the present study was to investigate chromosomal aberrations in sporadic Japanese papillary thyroid carcinomas (PTCs), concomitant with the analysis of oncogene mutational status. Twenty-five PTCs (11 with BRAF(V600E), 4 with RET/PTC1, and 10 without mutation in HRAS, KRAS, NRAS, BRAF, RET/PTC1, or RET/PTC3) were analyzed using Genome-Wide Human SNP Array 6.0 which allows us to detect copy number alteration (CNA) and uniparental disomy (UPD), also referred to as copy neutral loss of heterozygosity, in a single experiment. The Japanese PTCs showed relatively stable karyotypes. Seven cases (28%) showed CNA(s), and 6 (24%) showed UPD(s). Interestingly, CNA and UPD were rarely overlapped in the same tumor; the only one advanced case showed both CNA and UPD with a highly complex karyotype. Thirteen (52%) showed neither CNA nor UPD. Regarding CNA, deletions tended to be more frequent than amplifications. The most frequent and recurrent region was the deletion in chromosome 22; however, it was found in only 4 cases (16%). The degree of genomic instability did not depend on the oncogene status. However, in oncogene-positive cases (BRAF(V600E) and RET/PTC1), tumors with CNA/UPD were less frequent (5/15, 33%), whereas tumors with CNA/UPD were more frequent in oncogene-negative cases (7/10, 70%), suggesting that chromosomal aberrations may play a role in the development of PTC, especially in oncogene-negative tumors. These data suggest that Japanese PTCs may be classified into three distinct groups: CNA(+), UPD(+), and no chromosomal aberrations. BRAF(V600E) mutational status did not correlate with any parameters of chromosomal defects.     

Mechanisms of mutagenesis in mammalian cells. Application to human thyroid tumours

Mutations are defined as stable and irreversible modifications of the normal genetic message due to small changes in the number or type of bases, or to large modifications of the genome such as deletions, insertions or chromosome rearrangements. These lesions are due to either polymerase errors during normal DNA replication or unrepaired DNA lesions, which will give rise to mutations through a mutagenic pathway. The molecular process leading to mutagenesis depends largely on the type of DNA lesions. Base modifications, such as 8-oxo-guanine or thymine glycol, both induced by ionizing radiations (IR), are readily replicated leading to direct mutations, usually base-pair substitutions. The 8-oxo-G gives rise predominantly to G to T transversions, the type of mutations found in ras or p53 gene from IR-induced tumors. Bulky adducts produced by chemical carcinogens or UV-irradiation are usually repaired by the nucleotide excision repair (NER) pathway which is able to detect structural distortion in the normal double-strand DNA backbone. These lesions represent a blockage to DNA and RNA polymerases as well as some signal for p53 accumulation in the damaged cell. In the absence of repair, these lesions could be eventually replicated owing to the induction of specific proteins at least in bacteria during the SOS process. The precise nature of the error-prone replication across an unexcised DNA lesion in the template is not fully understood in detailed biochemical terms, in mammalian cells. IR basically produce a very large number of DNA lesions from unique base modifications to single- or double-strand breaks and even complex DNA lesions due to the passage of very high energy particles or to a local re-emission of numerous radicals. The breakage of the double-helix is a difficult lesion to repair. Either it will result in cell death or, after an incorrect recombinational pathway, it will induce frameshifts, large deletions or chromosomal rearrangements. Most of the IR-induced mutations are recessive ones, requiring therefore a second genetic event in order to exhibit any harmful effect and a long latency period before the development of a radiation-induced tumor. The fact that IR essentially induced deletions and chromosomal translocations renders very difficult the use of the p53 gene as a marker for mutation analysis. In agreement with the type of lesions induced by IR, it is interesting to point out that the presence has been observed, in a vast majority of radiation-induced papillary thyroid carcinomas (PTC), of an activated ret proto-oncogene originated by the fusion of the tyrosine kinase 3' domain of this gene with the 5' domain of four different genes. These ret chimeric genes which are due to intra- or inter-chromosomal translocations, were called RET/PTC1 to PTC5. The RET/PTC rearrangements were found in PTC from children contaminated by the Chernobyl fall-out as well as in tumours from patients with a history of therapeutic external radiation, with a frequency of 60-84%. This frequency was only 15% in 'spontaneous' PTC. The type of ret chimeric gene predominantly originated by the accidental or therapeutic IR was different. Indeed, PTC1 was present in 75% of the tumours linked to a therapeutic radiation and PTC3 in 75% of the Chernobyl ones. The other forms of RET/PTC were observed in only a minority of the post-Chernobyl PTC (< 20%). The difference in the frequency of PTC1 and PTC3 in both types of PTC, is statistically significant (P < 10(-5), Fischer's exact test). In two of the post-therapeutic radiation PTC, RET/PTC1 and PTC3 were simultaneously present. A PTC1 gene was also observed in 45% of the adenomas appearing after therapeutic radiation. The long-period of latency between exposure to IR and the appearance of thyroid tumours is probably due to the conversion of a heterozygote genotype of IR-induced mutations to a homozygote one. It will be interesting to use this time lag in accidental or therapeutic-irradiated p     

Molecular cytogenetic characterization of chromosome 9-derived material in a human thyroid cancer cell line

The incidence of papillary thyroid carcinoma (PTC) increases significantly after exposure of the head and neck region to ionizing radiation, yet we know neither the steps involved in malignant transformation of thyroid epithelium nor the specific carcinogenic mode of action of radiation. Such increased tumor frequency became most evident in children after the 1986 nuclear accident in Chernobyl, Ukraine. In the eight years following the accident, the average incidence of childhood PTCs (chPTC) increased 70-fold in Belarus, 200-fold in Gomel, 10-fold in the Ukraine and 50-fold in Tschnigov, Kiev, Rovno, Shitomyr and Tscherkassy compared to the rate of about 1 tumor incidence per 106 children per year prior to 1986 (Likhtarev et al., 1995; Sobolev et al., 1997; Jacob et al., 1998). To study the etiology of radiation-induced thyroid cancer, we formed an international consortium to investigate chromosomal changes and altered gene expression in cases of post-Chernobyl chPTC. Our approach is based on karyotyping of primary cultures established from chPTC specimens, establishment of cell lines and studies of genotype-phenotype relationships through high resolution chromosome analysis, DNA/cDNA micro-array studies, and mouse xenografts that test for tumorigenicity. Here, we report the application of fluorescence in situ hybridization (FISH)-based techniques for the molecular cytogenetic characterization of a highly tumorigenic chPTC cell line, S48TK, and its subclones. Using chromosome 9 rearrangements as an example, we describe a new approach termed 'BAC-FISH' to rapidly delineate chromosomal breakpoints, an important step towards a better understanding of the formation of translocations and their functional consequences.