Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Expression data from cytoplasmic hybrid (cybrid) and rho0 cells

ABSTRACT: Mitochondria have been implicated in insulin resistance and beta cell dysfunction, both of which comprise the core pathophysiology of type 2 diabetes mellitus (T2DM). It has also recently been found that mtDNA haplogroups are distinctively associated with susceptibility to T2DM at least in Koreans and Japanese. To investigate the functional consequences of different mtDNA, we compared gene expression profiles between cybrid clones harboring three different mtDNA haplogroups (D5, F, and N9a). To produce hybrid clones, we fused mtDNA-depleted osteosarcoma cell line (143B TK- rho0) with nucleus-lacking platelets from twelve donors harboring the three haplogroups. A total of twelve cybrid clones from the three mtDNA haplogroups were obtained: D5 (n=3), F (n=5), and N9a (n=4). For each clone, four technical replicates were obtained and hybridized to the array. For rho0 cell, six technical replicates were obtained and hybridized to the array.

ORGANISM(S): Homo sapiens

SUBMITTER: Seungwoo Hwang

PROVIDER: E-GEOD-26244 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Gene expression pattern in transmitochondrial cytoplasmic hybrid cells harboring type 2 diabetes-associated mitochondrial DNA haplogroups.

Hwang Seungwoo S Kwak Soo Heon SH Bhak Jong J Kang Hae Sun HS Lee You Ri YR Koo Bo Kyung BK Park Kyong Soo KS Lee Hong Kyu HK Cho Young Min YM

PloS one 20110713 7

Decreased mitochondrial function plays a pivotal role in the pathogenesis of type 2 diabetes mellitus (T2DM). Recently, it was reported that mitochondrial DNA (mtDNA) haplogroups confer genetic susceptibility to T2DM in Koreans and Japanese. Particularly, mtDNA haplogroup N9a is associated with a decreased risk of T2DM, whereas haplogroups D5 and F are associated with an increased risk. To examine functional consequences of these haplogroups without being confounded by the heterogeneous nuclear ...[more]

PMID: 21765942

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Project description:The mitochondrial and nuclear genomes contribute to mitochondrial function, and when mitochondrial function is compromised, mitochondrial retrograde signaling alters nuclear gene expression. We performed gene expression profiling of engineered cells that had mitochondria containing a disease-associated mutation that causes mitochondrial dysfunction. By generating networks of transcription factors that targeted these genes, the authors revealed putative mitochondrial retrograde signaling pathways. One such pathway involved retinoic acid receptor alpha (RXRA), the mRNA for which was reduced in the mutant cells. Network analysis and experiments in cells suggested that mitochondrial dysfunction caused by the mutation initiated a positive feedback loop that aggravated mitochondrial dysfunction: Reduced RXRA abundance further compromised expression of genes encoding products involved in mitochondrial function and translation. This gene-transcription factor mapping-network approach may reveal targets for therapeutic intervention of diseases associated with mitochondrial dysfunction. To investigate retrograde signaling pathways induced by the mitochondrial dysfunction caused by the mt3243 mutation, we generated the three types of cybrid cells using a mitochondria-mediated transformation method. Cells lacking mtDNA (rho0) were fused with mtDNAs with the mt3243 mutation isolated from platelets of a diabetic patient with sensorineural hearing loss. We then isolated three types of cybrid cells: W cells had wild-type mtDNA (3243A homoplasmy), H cells had both the mutant and wild-type mtDNA (3243A/G heteroplasmy) with 70% of the mtDNA containing 3243G, and M cells with only mutant mtDNA (3243G homoplasmy). Gene expression profiles of cybrid cells were generated using Illumina HumanHT-12-v3-BeadChip (Illumina, San Diego, CA), which includes 49,896 probes corresponding to 25,202 annotated genes. According to the Illumina protocols, three biological triplicates of each type of cybrid cells were analyzed. Total RNA (500ng) was isolated from cybrid cells using RNeasy Mini Kit (Qiagen, GmbH, Germany). RNA integrity number (RIN) was in the range of RIN = 9.2 and 10 when measured with an Agilent 2100 Bioanalyzer. RNA was reversely transcribed and amplified using IlluminaTotalPrep RNA amplification kit (Ambion, Austin, TX). In vitro transcription was then carried out to prepare cRNA. The cRNAs were hybridized to the array and then labeled with Cy3-streptavidin (Amersham Bioscience, Little Chalfont, UK). The fluorescent signal on the array was measured with a BeadStation 500 System (Illumina, San Diego, CA).

Project description:Mitochondrial DNA (mtDNA) mutations may contribute to aging and age-related disorders. Previously, we created mice expressing a proofreading-deficient version of the mtDNA polymerase gamma (Polg) which accumulate age-related mtDNA mutations and display premature aging. Here we performed microarray gene expression profiling to identify mtDNA mutation-responsive genes in the cochlea of aged mitochondrial mutator mice. Age-related accumulation of mtDNA mutations was associated with transcriptional alternations consistent with reduced inner ear function, mitochondrial dysfunction, neurodegeneration, and reduced cell structural modulation. Hearing assessment and histopathological results confirmed that aged PolgD257A/D257A (D257A) mice exhibited moderate hearing loss and severe cochlear degenerations. Age-related accumulation of mtDNA mutations also resulted in alternations in gene expression consistent with induction of apoptosis, proteolysis, stress response, and reduced DNA repair. TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assay confirmed that the cochleae from aged D257A mice showed significantly more TUNEL positive cells compared to wild-type (WT) mice. The levels of cleaved caspase-3 were also found to increase in the cochleae of aged D257A mice. These observations provide evidence that age-related accumulation of mtDNA mutations is associated with apoptotic cell death in aged cochlea. Our results provide the first global view of molecular events associated with mtDNA mutations in postmitotic tissue, and suggest that apoptosis is the major mechanism of mtDNA mediated cell death in the development of age-related hearing disorder. Experiment Overall Design: To determine the effects of age-related accumulation of mtDNA mutations, each WT sample (n = 5) was compared to each D257A sample (n = 5), generating a total of twenty-five pairwise comparisons. Genes with significantly altered expression levels were sorted into gene ontology biological process categories. Experiment Overall Design: Gene expression change was called statistically significant when at least one gene was called present in a group, the P value was < 0.0500, FC was > 1.1, and FDR was > 30.00 for identification of mtDNA mutations-induced genes. Experiment Overall Design: Affymetrix standard spike controls were used in all experiments (eukaryotic hybridization control kit). Quality control measures were not used. No replicates were done. Dye swap was not used.

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Dataset Information

Expression data from cytoplasmic hybrid (cybrid) and rho0 cells

Publications

Gene expression pattern in transmitochondrial cytoplasmic hybrid cells harboring type 2 diabetes-associated mitochondrial DNA haplogroups.

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