The occurrence of obesity, which stands as one of the main risk factors for the development of numerous diseases in the modern world, is significantly influenced not only by lifestyle and environment but also by genetic predisposition. As the most common form of obesity is polygenic and complex, our procedures were carried out on unique obese (FLI) and lean (FHI) mouse lines. In males of these lines, a previous study has shown an association between obesity and increased expression of the Pla2g4e gene, which encodes an enzyme of the phospholipase A2 superfamily. In the first part of the thesis, we wanted to confirm the increased gene expression in females of the obese line. We isolated RNA from the hypothalamus, checked its purity and quality, reverse transcribed it and performed RT-qPCR. We used Ppia and B2M as normalisation control genes. We analysed Pla2g4e expression by the threshold cycle comparison method (2$^{-ΔΔCt}$) and expressed the result as a multiple of gene expression of the obese versus the lean line. We have shown that the gene is upregulated in hypothalamus of females in the obese line compared to the lean line and that the difference in expression is statistically significant.
In another preliminary screening study, several polyadenylation sites were found in some genes expressed in the hypothalamus of the same model mice, that can lead to the formation of differently localised and stable mRNA isoforms and differently functional proteins. For the confirmation of the existence of their long and short isoforms and analysis of their eventual differential expression in female mice of both lines, we selected the Copg1, Pcna and Stx3 genes. Since the short isoform, whose primers also detect the long isoform, cannot be validated by qPCR, we detected the expression of the isoforms by the more specific TaqMan qPCR method. We confirmed the differential expression of the long Pcna isoform, which is upregulated in the obese line, and differential expression of the short Copg1 isoform, although it is only mildly upregulated in the lean line. We also confirmed differential expression of both Stx3 isoforms, with the long isoform being upregulated in the lean line and the short isoform being downregulated in the obese line.
Our results confirm that Pla2g4e is an important genetic factor and therefore a potential therapeutic target for the treatment of obesity. The identification of differentially expressed mRNA isoforms between the obese and lean lines also suggests a potential therapeutic application. By targeting specific isoforms to increase or decrease their expression, it might be possible to modify the expression patterns of the isoforms responsible for the development of obesity.
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