Aflatoxin M1 and ochratoxin A induce a competitive endogenous RNA regulatory network of intestinal immunosuppression by whole-transcriptome analysis

Abstract

Aflatoxin M1 (AFM1) and ochratoxin A (OTA) are common mycotoxins in cereal foods and milk products, and may cause serious negative impacts on human health. The intestine is crucial for immune regulation as it protects host homeostatic health from external contaminants; however, the underlying mechanisms of AFM1 and OTA mediated intestinal immunotoxicity remain unclear. In this study, whole transcriptome analysis was used to characterize BALB/c mouse intestines exposed to individual and combined AFM1 and OTA [3.0 mg/kg body weight (BW)] for 28 days to screen for key intestinal immunotoxicity-related differentially expressed mRNAs (DEmRNAs), differentially expressed microRNAs (DEmiRNAs), differentially expressed long non-coding RNAs (DElncRNAs), and associated enriched signaling pathways. Functional validation was then conducted in intestinal differentiated Caco-2 cells using different inhibitor assays to verify the accuracy of transcriptome and the importance of the key screened regulatory factors. In vivo data revealed that AFM1 and OTA exposure disrupted the intestines and exerted intestinal immunosuppression effects. When compared with AFM1, OTA had stronger intestinal toxicity in combined treatments. Further analyses of competitive endogenous RNA (ceRNA) regulatory networks in mice showed that AFM1 and OTA mediated-intestinal immunosuppression was putatively explained as follows: (i) toxins affected DEmRNAs regarding transfer and transduction mechanisms between cells (Csf1, Csf1r, Cxcl10, Cx3cr1, and Irf1), which were regulated by key DEmiRNAs (miR-106-x, miR-107-y, and miR-124-y) and the DElncRNA Rian, and (ii) toxins inhibited transforming growth factor-β-activated kinase 1 (TAK1)/I-kappaB kinase (IKK)/inhibitor of kappa Bα (IκBα)/p65 nuclear factor-κB (NF-κB) signaling phosphorylation levels, which was validated in differentiated Caco-2 cells using the TAK1 inhibitor (5Z-7-oxozeaenol). In conclusion, we evaluated the risk of co-exposure to AFM1 and OTA and associated health hazards from a whole transcriptome perspective.

Keywords: Aflatoxin M1; Differentially expressed RNA; Immunotoxicity; Intestinal toxicity; Ochratoxin A; Whole transcriptome.