Circulating exosomal microRNA as potential biomarkers of hepatic injury and inflammation in glycogen storage disease type 1a

Most patients affected by glycogen storage disease type 1a (GSD-1a), an inherited metabolic disorder caused by mutations in the enzyme glucose-6-phosphatase-alpha (G6Pase- α), develop renal and liver complications, including development of hepatocellular adenoma/carcinoma. The purpose of this study was to identify potential biomarkers of the pathophysiology of the GSD1a affected liver. To this end, we utilized the plasma exosomes of a murine model of GSD-1a, LS-G6pc-/- mice, to uncover microRNA expression modulation associated with the disease. Differentially expressed microRNA between LS-G6pc-/- and wild type mice, LS-G6pc-/- mice with hepatocellular adenoma and LS-G6pc-/- mice without adenoma, and LS-G6pc-/- mice with amyloidosis and LS-G6pc-/- mice without amyloidosis were identified. Pathway analysis demonstrated that the target genes of the differentially expressed miRNAs were significantly enriched for insulin signaling pathway, glucose and lipid metabolism, Wnt-beta catenin, telomere maintenance and hepatocellular carcinoma, and chemokine and immune regulation signaling pathways. While some microRNA were common to the different pathologic conditions, others were unique to cancerous or inflammatory status of the animals. Therefore, the altered expression of several microRNA correlates with various pathologic liver statuses and may help discriminate during the progression of the disease and the development of late GSD1-associated complications. Exo-miR were analyzed by the TaqMan Array Card Technology. Exo-miR were reverse transcribed with the TaqMan microRNA Reverse Transcription Kit, using the MegaplexTM RT primers Rodent Pool A (Thermo Fisher Scientific, Monza, MB, Italy). Pre-amplification of cDNA was performed with TaqMan PreAmp Master Mix and MegaplexTM Pre-Amp primers Rodent Pool A. The pre-amplification product was diluted according to the manufacturer’s instructions and used to perform microRNA profiling on the ViiATM 7 Real-Time PCR System. The Exo-miR modulation of expression in LS-G6pc-/- mice was validated for five microRNA by qRT-PCR on the ViiATM 7 Real-Time PCR System.