Small RNAs as molecular tools to dissect function of Glutathione S-Transferase Mu type 1

Graham, Laura (2010) Small RNAs as molecular tools to dissect function of Glutathione S-Transferase Mu type 1. PhD thesis, University of Glasgow.

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Human essential hypertension is a substantial public health problem with greater than 25% of the adult population affected worldwide. It is a complex disorder and a major risk factor for various cardiovascular diseases such as heart disease, renal disease and stroke. The stroke-prone spontaneously hypertensive rat (SHRSP) is a commonly used model of human essential hypertension. Previous studies identified a quantitative trait locus (QTL) for blood pressure regulation on rat chromosome 2. To further enhance the investigation of this QTL, rat chromosome 2 congenic strains were generated by transferring regions of chromosome 2 from the normotensive WKY onto the genetic background of the SHRSP. Congenic strains exhibited significantly lower blood pressure than the SHRSP parental strain indicating that genes within the congenic region are important for blood pressure regulation. Renal microarray analysis of congenic 2c* and parental strains led to the identification of glutathione s-transferase mu type 1 (Gstm1) as a positional and possible functional candidate gene for hypertension in the SHRSP. Expression of Gstm1 was significantly lower in the SHRSP kidney than in 2c* and WKY and renal oxidative stress was increased in SHRSP. Glutathione s-transferase mu type 1 is part of a large family of antioxidant enzymes and may play a role in hypertension by modulating levels of oxidative stress.

This study has utilised small RNAs to examine the role of Gstm1 in cellular oxidative stress and also investigated microRNA (miRNA) expression in the rat congenic strains. RNA interference (RNAi) was utilised to knock-down expression of Gstm1 in a rat kidney tubular epithelial cell line (NRK-52E). Three different short interfering RNA (siRNA) sequences designed to target Gstm1 were evaluated. Each sequence significantly reduced expression of Gstm1 and was confirmed at both mRNA and protein level. Off-target effects on other Gstm isoforms and the interferon response gene, oligoadenylate synthetase 1 were prevented by reducing the concentration of siRNA used. To take knock-down of Gstm1 expression into an in vivo setting, local delivery to the kidney via the renal artery was assessed but was found to cause significant kidney damage. Instead, kidney targeted vectors that can be delivered systemically were evaluated in the SHRSP. Immunohistochemistry confirmed specific targeting of kidney tubules. Plasmid vectors were then generated that express Gstm1 specific short-hairpin RNA (shRNA) molecules based on the sequences that successfully knocked down Gstm1 expression in vitro. Transfection of these plasmids into NRK-52E cells was poor and knock-down of Gstm1 could not be confirmed.

The role of Gstm1 in protection against cellular oxidative stress was evaluated in NRK-52E cells by measuring markers of oxidative stress following knock-down of Gstm1 expression. Total GST activity was not reduced in cells transfected with Gstm1 specific siRNA however activity was increased following over-expression of Gstm1. No change in the levels of reduced glutathione was observed in cells following knock-down of Gstm1. Oxidative stress was determined by measuring 8-isoprostane (a marker of lipid peroxidation), 8-hydroxy-2-deoxyguanosine (8-OH-dG) (a marker of oxidative DNA damage) and by the comet assay (DNA damage). No significant difference in the levels of 8-isoprostane or 8-OH-dG was observed in cells treated with Gstm1 specific siRNA compared to control siRNA. However a small but significant increase in ‘comet’ tail length was observed in cells with reduced Gstm1 expression indicating greater DNA damage in these cells.

Two miRNAs that map to the chromosome 2 congenic regions were investigated (miR-137 and miR-9-1). The expression of each miRNA was investigated in kidney from SHRSP, WKY and congenic rat strains at 5, 16 and 21 weeks of age. Expression of miR-9-1 was unchanged in the SHRSP but was significantly reduced in 16 week 2c* and WKY compared to 5 week. Expression of miR-137 was unchanged in both WKY and 2k but was significantly increased in 21 week old salt-loaded SHRSP compared to 21 week old SHRSP without salt. Successful over-expression of each miRNA was demonstrated in NRK-52E cells but inhibition of each miRNA could not be confirmed. MicroRNA target prediction methods were employed to identify potential gene targets for each miRNA. A list of predicted targets for each miRNA was generated and combined with gene expression datasets generated from 5 week, 16 week, 21 week and 21 week plus salt kidney microarray analysis carried out previously. Using Ingenuity Pathway Analysis software, predicted target genes were identified according to patterns of expression. Analysis of predicted miR-137 targets failed to find any genes that followed the correct pattern of expression (down-regulated in SHRSP but unchanged in both WKY and 2k). Of the targets predicted for miR-9-1, seven were identified that were up-regulated in both WKY and 2c* but not SHRSP. The expression levels of each of the seven genes were assessed in kidney and NRK-52E cells over-expressing miR-9-1 however none of the predicted targets could be validated.

In summary, a wide range of techniques have been employed in an attempt to investigate the function of Gstm1 and the possible role of miRNA that map to the congenic regions implicated in blood pressure regulation. Although a specific role for Gstm1 in protection against oxidative stress has yet to be fully determined, the integration of targeted vectors and modulation of Gstm1 expression will allow the role of Gstm1 to be investigated more fully in vivo.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Subjects: R Medicine > RC Internal medicine
Q Science > QP Physiology
Colleges/Schools: College of Medical Veterinary and Life Sciences > School of Cardiovascular & Metabolic Health
Supervisor's Name: Dominiczak, Prof. Anna F.
Date of Award: 2010
Depositing User: Miss Laura Graham
Unique ID: glathesis:2010-1883
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 08 Jun 2010
Last Modified: 10 Dec 2012 13:47

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