The regulation of Niemann-Pick C1-Like 1 (NPC1L1) gene expression in opposite direction by Bacteroides spp. and related outer membrane vesicles in Caco-2 cell line

Abstract

Purpose: The intestine has substantial role in cholesterol homeostasis due to the presence of various cholesterol transporters and gut microbiota. Bacteroides spp. are important members of gut microbiota that employ outer membrane vesicles (OMVs) to interact with host. In this regard, we evaluated the effect of Bacteroides fragilis, Bacteroides thetaiotaomicron and related OMVs on the gene expression of important cholesterol transporters, Niemann-Pick C1-Like 1 (NPC1L1), ATP-binding cassette (ABCA1), and liver X receptors (LXRs) in Caco-2 cells.

Methods: OMVs were isolated from overnight brain heart infusion (BHI) broth of bacterial standard strains using deoxycholate and assessed by Scanning electron microscopy (SEM). The relative change in genes expression was assessed by Quantitative reverse transcription PCR (RT-qPCR) based on SYBR Green and 2^-∆∆ct method in Caco-2 cells that were treated with bacteria and OMVs. Data were statistically analyzed with GraphPad Prism software. Finally, pathway enrichment based on the studied genes was performed using Cytoscape plugin ClueGO.

Results: B. fragilis (P value = 0.002) and B. thetaiotaomicron (P value = 0.001) significantly reduced NPC1L1 gene expression in Caco-2 cells. Interestingly, NPC1L1 transcripts were significantly increased by both OMVs(P value = 0.04) (P value = 0.01). Also, LXRβ was significantly down regulated by B. thetaiotaomicron (P value = 0.02). ClueGO analysis on the studied genes demonstrated several functional groups which involve in lipid and cholesterol metabolism.

Conclusion: The opposite effect of B. fragilis, B. thetaiotaomicron and related OMVs on the NPC1L1 gene expression was observed in Caco-2 cells. Interestingly, these effects partially were in line with the alternation of LXRs expression. However, based on pathway enrichment analysis, further molecular investigations are required to elaborate in details the specific association between Bacteroides spp. and OMVs with regulation of cholesterol signaling pathways including cholesterol transport, lipid storage, lipid homeostasis and cholesterol homeostasis.

Keywords: Bacteroides spp.; Cholesterol homeostasis; Gut microbiota; Niemann-pick C1-like 1; Outer membrane vesicles (OMVs).

Fig. 1

The relative gene expression of NR1H3, NR1H2, ABCA1 and NPC1L1 based on RT-qPCR experiments in Caco-2 cells treated with B. fragilis (a), B. thetaiotaomicron (b) at multiplicity of infection, MOI = 10 and PBS as control group for overnight. Values of triplicate experiments are demonstrated as mean ± SD. Statistical Significant are presented as * (P < 0.05) and^,** (P < 0.01)

Fig. 2

The relative mRNA expression of NR1H3, NR1H2, ABCA1 and NPC1L1 based on RT-qPCR experiments in Caco-2 cells treated with B. fragilis OMVs (a), B. thetaiotaomicron OMVs (b) at 50 μg/ml and sucrose as control group overnight. Values of triplicate experiments are demonstrated as mean ± SD. Statistical Significant are presented as * (P < 0.05) and^,** (P < 0.01)

Fig. 3

The GO terms were classified into several functional groups (different node colors) according to the kappa value. Each node represented a GO term, and the node size was proportional to the significance of the term. The most significant GO terms were labeled with a highlighted color. The edge in the nodes indicated that they shared common genes, and the width of the edge was proportional to the number of common genes