AUF-1 knock down in mice overarches butyrate driven hypo-cholesteraemia by conjuring AUF-1-Dicer-1-miR122 hierarchy

Abstract

This discourse probes the mechanistic molecular details of butyrate action in maintaining host- cholesterol balance. Hepatic miR122 being the most indispensable regulator of cholesterol metabolic enzymes, we studied upstream players of miR122 biogenesis in the presence and absence of butyrate in Huh7 cells and mice model. We showed that butyrate treatment caused upregulation of RNA-binding protein, AUF-1 resulting in RNase-III nuclease, Dicer-1 instability, and significant diminution of miR122. We proved its importance of AUF-1 and sequential downstream players in AUF-1-knock-down mice. We synthesized unique self-transfecting GMO (guanidinium-morpholino- oligonucleotides) linked PMO (Phosphorodiamidate-Morpholino Oligonucleotides)-based antisense reagent and injection of which in mouse caused near absence of AUF-1 coupled with increased Dicer- 1 and miR122, and reduced serum cholesterol regardless of butyrate treatment indicating that butyrate acts though AUF-1. The roster of intracellular players was as follows: AUF-1-Dicer-1-miR122 for triggering butyrate driven hypocholesterlaemia. To our knowledge this is the first report linking AUF-1 with cholesterol biogenesis.

Butyrate ameliorates inflammation in colon biopsy samples of IBD patients and experimental colitis in mice involving RNA binding protein, AUF1-IL-27 axis and accelerating B1a to B10 polarization

“…To further establish the link between AUF-1 and IL-10 production in B1a cells, we knocked down AUF1 by deploying morpholino oligomer. Morpholino oligomers (MO) which are short single-stranded DNA analogues that are built upon a backbone of morpholine rings and are resistant to host enzymes present, a characteristic that makes them highly suitable for application in primary cells and in vivo (65), (26). Earlier we have used the AUF1 knock down mice model using MO to establish the role of AUF1 in cholesterol synthesis (26).…”

“…Morpholino oligomers (MO) which are short single-stranded DNA analogues that are built upon a backbone of morpholine rings and are resistant to host enzymes present, a characteristic that makes them highly suitable for application in primary cells and in vivo (65), (26). Earlier we have used the AUF1 knock down mice model using MO to establish the role of AUF1 in cholesterol synthesis (26). Unlike a knockout, AUF1-MO mediated knock down causes partial knock down of AUF1 in organs like in kidney, heart (26), but nearly 100 % knock down in liver (26), colon and spleen.…”

“…Earlier we have used the AUF1 knock down mice model using MO to establish the role of AUF1 in cholesterol synthesis (26). Unlike a knockout, AUF1-MO mediated knock down causes partial knock down of AUF1 in organs like in kidney, heart (26), but nearly 100 % knock down in liver (26), colon and spleen. Therefore, warranting morpholino administration in mice a unique knockdown (KD) model to explore the role of AUF1 in B10 generation.…”

“…Overproduction of TNF-, in AUF1 knockout mice accounts for higher susceptibility to septicaemia (25). Previously we showed that AUF-1 is increased due to butyrate treatment (26) in mice liver and AUF-1 knock down using a cell penetrating morpholino in mice, inhibits butyrate mediated hypo-cholesterolemic effects in mice (26).…”

“…Butyrate activates RNA binding protein, AUF1 (26), is known to regulate cytokine mRNA stability (27), (28), (29). It is recently reported that B-1a cells (30) and Treg produce IL-27 (31).…”