Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptional profiling by array of mouse hearts from animals with heterozygous truncation of Igf1 gene (HZ), or HZ treated with low doses of IGF-1 (substitutive therapy) against wild-type control

ABSTRACT: 'This study investigates the effects produced by IGF-1 partial deficiency and if those effects were restored with the hormone''s substitutive therapy. Partial IGF-1 deficiency is achieved by heterozygous animals for a truncated gene of IGF-1 (Hz Igf1+/-), supplied by The Jackson Laboratories, which show a 60-75% reduction in circulating IGF-1 levels. The mice were a cross of 129SV Igf1+/- mice with non-consanguineous MF-1 mice to avoid consanguinity. Animals are treated for 10 days 2ug/100g bw/day with rhIGF-1 in succinate vehicle. After the 10 days of treatment, the animals were sacrificed, hearts were extracted, stored in RNAlater and frozen. On the day of the experiment, hearts were thawed, homogenised and subsequently processed as the RNA extraction kit instructions indicate (RNAqueousH-Micro Kit, Ambion, USA).'

ORGANISM(S): Mus musculus

SUBMITTER: Gabriel Aguirre

PROVIDER: E-MTAB-5791 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In this study we used the cre-loxP recombinase system. All mice were genetically modified in as much as all mice had exon 4 of the IGF-I gene flanked by loxP sites. In the knockouts, Mx-Cre had also been introduced, which results in inactivation of the IGF-I gene specifically in the liver. RNA from liver, skeletal muscle, heart, fat (retroperitoneal fat) and bone (vertebrae) was extracted from 6 control and 7 mice deficient of liver-derived insulin-like growth factor-I (LI-IGF-I-/- mice) by Tri Reagent (Sigma, St. Louis, MO, USA) and purified using spin columns from Rneasy Total RNA Isolation Kit (Qiagen, Chatsworth, CA, USA). For microarray analysis, the RNA samples were pooled three or two, resulting in three pools per group while for the confirmatory RT-PCR analyses individual mice (n=6-7) were analyzed. For each organ, there were three pooled samples from the mice with deficiency of liver-derived IGF-I and 3 pooled samples from the wildtype mice i.e. intact IGF-I gene. The pooled RNA was reverse transcribed into cDNA, labeled, and analyzed by DNA microarray (MG-U74Av2 Array; Affymetrix, Santa Clara, CA, USA). Scanned output files were analyzed using Affymetrix Micro Array Suite version 4.0.1 software (Affymetrix). To allow comparison of gene expression, the gene chips were globally scaled to an average intensity of 500. Each of the three LI-IGF-I-/- chips was compared with the three control chips, generating a total of nine comparison files. Only the genes that were regarded as M-^SchangedM-^T according to the Affymetrix algorithm in four to nine of the comparisons were selected for further analysis. We then calculated an average-fold change for the nine comparisons of the selected genes. For a gene to be regarded as consistently regulated in the LI-IGF-I-/- mice, the average-fold increase or decrease of the nine comparisons was set as at least 1.5 fold in at least 4 of the 5 analyzed organs. Microarray showed that heat shock protein 1A (Hspa1a), heat shock protein 1B (Hspa1b), and connective tissue growth factor (Ctgf) were decreased in all organs that expressed these genes. CCAAT/enhancer protein delta (Cebpd) decreased in 4 of the 5 analyzed organs. The RT-PCR analyses that were performed later confirmed that mRNA levels of Hspa1a, Hspa1b, and Ctgf decreased in LI-IGF-I-/- mice in skeletal muscle tissue while mRNA levels of Cebpd were unchanged.

Project description:Background: Studies suggested that mesenchymal stem cells (MSCs) have intrinsic neurogenic potential and can be differentiated into neural stem cell/ neural progenitor cells (NPCs) under specific microenvironment. Manipulation of growth factors is one of the popular method to achieve trans-lineage differentiation of MSCs. Synergistic effect of epidermal growth factor (EGF) and fibroblast growth factor 2 (bFGF) have been widely identified as basic requirement for neural differentiation to take place. Insulin-like growth factor 1 (IGF-1) also known as somatomedin C is an important growth promoting protein during embryonic development which control numerous cellular responses and biological systems. Our recent study has found that the combination of EGF, bFGF and IGF-1 could significantly improved the growth and survivability of MSCs-derived NPCs. Therefore, to understand the genomic mechanism underlying the differentiation in vitro, we have studied the miRNAs profile of MSCs-derived NPCs under IGF-1 influenced conditioned microenvironments. Objectives: To evaluate the effects of IGF-1 in trans-lineage differentiation of MSCs, we have induced MSCs into neural lineage in 3 groups; Group A (positive control) - EGF+bFGF, Group B (Treatment) - EGF+bFGF+IGF-1, and Group C (negative control/ untreated). To unravel the role of regulatory miRNAs involved in the early differentiation, we have performed detailed miRNA profiling for MSCs-derived NPCs at three time intervals (day 1, day 3 and day 5). The data has explored crucial miRNAs involved in early differentiation of MSCs into NPCs. Stage specific MSCs-derived NPCs at Passage 1 were collected for total RNA extraction at three time-points (D1, D3 and D5) and hybridization on Affymetrix miRNA geneChip 2.0 arrays. Each experiment were repeated three times independently (Exp 1, Exp 2 and Exp 3). Group A served as positive control (EGF+bFGF), Group B as treatment (EGF+bFGF+IGF-1) and Group C without growth factor as negative control.

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Transcriptional profiling by array of mouse hearts from animals with heterozygous truncation of Igf1 gene (HZ), or HZ treated with low doses of IGF-1 (substitutive therapy) against wild-type control

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