| ATP-binding cassette transporters (ABC) sub-family G member 4 (ABCG4) is an integrated membrane protein. ABC members transport various biomolecules, including ion, amino acids, carbohydrate, phospholipids and drug etc. across the cell membrane, using the energy derived from ATP hydrolysis. It was shown that the main function of ABCG4 was to promote cell cholesterol efflux. In 2008, Uehara Y et al, using in situ hybridization, found that the expression of abcg4 in microglial cells near senile plaques in the brain of AD patients was significantly higher than those in none-AD patients. As abcg4 may play an important role in the occurrence and development of AD, some experiments regarding this gene were carried out in the lab.Firstly, we cloned and sequenced abcg4 gene in the KM mouse strain. There was three-bases difference of the cloned gene from the one shown in GenBank (accession number NM138955. 3); The expression of abcg4 in different tissues of KM mouse was measured using semi-quantitative RT-PCR, and it showed that abcg4 gene was mainly expressed in the brain, eyes and spleen, with higher expression in the first two tissues.Secondly, the effects of RNA interference on abcg4 expression were investigated. Three small interfering RNAs (siRNAs) targeting to abcg4 gene were designed: siRNA-001, siRNA-002, siRNA-003. siRNAs were injected into mice lateral ventricle using stereotaxic apparatus. The result showed that only siRNA-003 could reduce abcg4 mRNA expression remarkably; and its interference effect was best at 48h after injection; We also found that the inhibitory effect to abcg4 gene was dependent upon the dose of siRNA: it reached best when injected at the dose 6ug per mouse into the brain.Finally, the expressions of the genes amyloid precursor protein (APP)-app, apolipoprotein E (APOE)-ApoE and 3-Hydroxy-3-methylglutaryl CoA synthase (HMGS)-hmgs were measured by means of RT-PCR, when abcg4 was successfully silenced by siRNA. As a result, hmgs gene, which has a key role in cholesterol synthesis, was down-regulated in the abcg4 knockdown mice; the expression of ApoE gene had no obvious change; the app mRNA level was significantly higher. Accordingly, the expression of APP ( Phospho-Thr668 ) protein, as shown by western blot, was down-regulated, which was different from the change in the level of app mRNA. In 2007, Feyt C et al reported that phosphorylation of Amyloid Precursor Protein (APP) at Thr668 would decrease Aβproduction. In our case, when abcg4 was successfully silenced by specific siRNA, APP (Phospho-Thr668 ) was down-regulated in the abcg4 knockdown mice, we could deduce that Aβwould increase accordingly. The study provides a direct evidence for the association between ABCG4 and AD by means of siRNA interference, which has not been reported at home and abroad.In this paper, by abcg4 gene cloning and sequencing, its expression profile analysis, specific gene interference in vivo, and the expressional change of the other related genes such as APP(Phospho-Thr668)induced by the interference, we had a better understanding of the function of abcg4 gene and its possible role in AD pathogenesis. |
