Microarray analysis of human hepatic aging

David W. Boorman

doi:10.17918/etd-2881

Two sets of liver tissue samples of human subjects were investigated by gene expression microarray analysis. One set had normal and disease samples with their telomere lengths; the other set consisted of groups of normal young and old donors, mostly post mortem. The goal was to identify genes related to aging and inflammation and to investigate the hypothesis that telomere length is a biomarker for these processes. The first set of ten surgical liver biopsies were run on the single channel microarray, Codelink Human Bioarray. Because of the liver diseases in the subjects (3 with Hepatitis C, 3 with Primary Sclerosing Cholangitis, 1 with a liver cyst, and 3 controls), the standard preliminary procedure of simple variance filtering was deemed insufficient. Instead, the samples were adjusted for disease using Analysis of Covariance, to identify genes of potential interest. This was conducted in three pairs based on age, telomere length and disease, because of the small sample size. With this procedure, about 1,600 genes had an alpha significance of less than five percent (14.7% total for all comparisons). Hierarchical clustering of these genes grouped the Hepatitis C patients and the young patients, as a partial validation of this model. The second dataset was obtained from the Gene Expression Omnibus (GEO), GSE9588. The samples were run on the Rosetta/Merck Human 44k 1.1 dual channel microarray platform, against a control of 191 pooled human liver samples. The original study analyzed the surgical and post mortem liver samples with respect to disease and single nucleotide polymorphisms. Sixty-seven samples were selected from this dataset, ages 20-29 or ages 70-81. Their telomere lengths are unknown. Simple variance filtering resulted in 1,965 differentially expressed genes. To reduce false positives further, various microarray techniques were employed, including hierarchical clustering, principal component analysis, k-means clustering and Pavlitis Template Modeling. The last technique matches expression to the relative telomere length or age. Resulting genes were then run through the gene ontology programs DAVID and PANTHER, which find gene networks. Networks that were differentially up regulated based on age include ubiquitin, a protein intracellular ransport marker often linked to protein targeted for degradation, as well as cytochrome p450 (CYP), a mono-oxygenase in the endoplasmic reticulum of hepatocytes involved in the breakdown of steroids, fatty acids and xenobiotics. The cytochrome p450 network was up regulated in the older group, but not in elderly who also had steatosis (fatty liver) or who had drug liver risk from the use of a medication. Networks down regulated or mixed based on age included the broad categories of "receptors" and "immunity and defense." IL8, which is involved in inflammation, was one of these hits. Networks that were down regulated with shorter telomere length included genes related to chromatin restructuring and transcription regulation, including the selective down regulation of several Histone H4 isoforms. Hypotheses generated from this study include the identification of six poorly characterized receptors which may be involved in liver regeneration. Additionally, there may be a mechanistic problem between CYP transcription and activated protein, because of the high CYP mRNA levels seen here in elderly, compared to the unchanged or decreased protein levels found in other studies. Additionally, elderly with steatosis or drug liver risk may have a higher hepatocyte turn over and higher Hepatocyte Growth Factor (HGF) expression, since HGF suppresses CYP.

Microarray analysis of human hepatic aging

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Microarray analysis of human hepatic aging

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