Unknown,Transcriptomics,Genomics,Proteomics

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Microarray analysis of aristolochic acid in normal human cells

ABSTRACT: Aim： Use microarray analysis to understand the molecular mechanism underlying the effect of aristolochic acid (AA), a major active component of plants from the Aristolochiaceae family, in normal human kidney (HK-2) cells. Methods： HK-2 cells were treated with AA for 24 hours and cell viability was measured by a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide assay. Complementary DNA microarrays were used to investigate the gene expression pattern of HK-2 cells exposed to AA and the results of this study were in triplicate. Quantitative real-time RT-PCR assay was used to verify the microarray data for selected nuclear factor kappa B (NF-κB)-regulated genes. Furthermore, subcellular localization of NF-κB p65 was visualized by immunofluorescence confocal microscopy in HK-2 cells. NF-κB activity was examined by luciferase reporter assay in HK-2/NF-κB transgenic cells. Results： AA exhibited a dose-dependent cytotoxic effect in HK-2 cells and induced alterations in gene expression profiles related to DNA damage response, stress response, etc. In addition, 9 biological pathways associated with immunomodulatory functions were down-regulated in AA-treated HK-2 cells. Network analysis revealed that NF-κB played a central role in the network topology. Among NF-kB-regulated genes, 8 differentially expressed genes were verified by real-time RT-PCR. The inhibition of NF-κB activity by AA was further confirmed by immunofluorescence confocal microscopy and by NF-κB luciferase reporter assay. Conclusion： Our data revealed that AA could suppress NF-κB activity in normal human cells, perhaps partially accounting for the reported anti-inflammatory effects of some plants from the genus Aristolochia. HK-2 cells were grown in keratinocyte serum-free basal medium (Gibco) supplemented with 5 ng/ml of recombinant epidermal growth factor and 50 μg/ml of bovine pituitary extract without antibiotics in 5 % CO2 at 370C. HK-2 cells were seeded in 25-T flasks and incubated for 24 h before aristolochic acid treatment. Aristolochic acid (10 90 μM) were added to HK-2 cells for 24 h. The control cells received equal amounts of water only.

ORGANISM(S): Homo sapiens

SUBMITTER: Chien-Yun Hsiang

PROVIDER: E-GEOD-18243 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:MicroRNAs (miRNAs) play an important role in carcinogenesis by regulating the protein translation from their targeted messenger RNAs (mRNA). Aristolochic Acid (AA) is a potent carcinogen that can induce kidney tumors in both human and experimental animals. Although the expression and roles of miRNAs have been extensively studied in cancer tissues or tissues treated by carcinogens, it is still unclear how the three levels of gene expression, including miRNA, mRNA and protein, are regulated and coordinated during the early stage of carcinogenesis. Here, we treated rats with 10 mg/kg AA or vehicle control for 12 weeks and the kidney tissues were quickly isolated and frozen immediately after the treatment. Eight kidney samples (4 for the treatment and 4 for the control) were used for analyzing miRNA and mRNA expressions with deep sequencing, and protein expressions with proteomics. MiRNA expressions were significantly changed by AA treatment. The treated samples were well separated from the control samples in both principal component analysis (PCA) and hierarchical clustering analysis (HCA). Quantitative measurements of six miRNAs using TaqMan real-time PCR were consistent with the deep sequencing results in terms of both direction and magnitude of gene expression change. Sixty-three miRNAs (adjusted p value < 0.05 and fold change > 1.5), 6,794 mRNAs (adjusted p value < 0.05 and fold change > 2.0), and 800 proteins (fold change > 2.0) were significantly altered by AA treatment. By combining the results of a computational target predicting algorithm, and the mRNA and protein expression data, 143 genes were found by the differentially expressed miRNAs (DEMs). The DEM-targeted genes are mainly related to cancer, cell growth, which reflects the carcinogenic processes in the AA-treated rat kidneys. Groups of four 6 week-old male Big Blue rats were treated with AA as its sodium salt at 10.0 mg/kg body weight by gavage (4 ml/kg body weight) 5 times a week for 12 weeks. The rats that were treated with 0.9% sodium chloride on the same schedule were used as control. microRNAs were isolated from rat kidneys and subject to next-generation sequencing to determine the expression profile.

Project description:Aristolochic acid (AA) is the causative agent of urothelial tumours associated with aristolochic acid nephropathy and is also implicated in the development of Balkan endemic nephropathy-associated urothelial tumours. These tumours contain AA-characteristic TP53 mutations. We examined gene expression changes in Hupki (human TP53 knock-in) mice after treatment with aristolochic acid I (AAI) by gavage (5 mg/kg body weight). After 3, 12 and 21 days of treatment gene expression profiles were investigated using Agilent Whole Mouse 44K Genome Oligo Array. Expression profiles were significantly altered by AAI treatment in both target (kidney) and non-target (liver) tissue. Renal pathology and DNA adduct analysis confirmed kidney as the target tissue of AAI-induced toxicity. Gene ontology for functional analysis revealed that processes related to apoptosis, cell cycle, stress response, immune system, inflammatory response and kidney development were altered in kidney. Canonical pathway analysis indicated Nfkb, aryl hydrocarbon receptor, Tp53 and cell cycle signalling as the most important pathways modulated in kidney. Expression of Nfkb1 and other Nfkb-target genes was confirmed by quantitative real-time PCR (qRT-PCR) and was consistent with the induction of Nfkb1 protein. Myc oncogene, frequently over-expressed in urothelial tumours, was up-regulated by AAI on the microarrays and confirmed by qRT-PCR and protein induction. Collectively we found that microarray gene expression analysis is a useful tool to define tissue-specific responses in AAI-induced toxicity. Several genes identified such as TP53, Rb1, Mdm2, Cdkn2a and Myc are frequently affected in human urothelial cancer, and may be valuable prognostic markers in future clinical studies. Keywords: Carcinogen treatment Two-color Agilent array. A reference design was chosen that all samples were hybridised to universal mouse reference RNA (UMRR). 12-condition experiment (2 mouse organs: kidney and liver; 2 treatments: AAI and water; 3 time points: 3, 12 and 21 days). Three biological replicates for each condition. One replicate per array.

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Microarray analysis of aristolochic acid in normal human cells

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