Lactobacillus casei LC01 Regulates Intestinal Epithelial Permeability through miR-144 Targeting of OCLN and ZO1

Abstract

Our previous report determined that miR-144 is a key regulator of intestinal epithelial permeability in irritable bowel syndrome with diarrhea (IBS-D) rats. Recent evidence has shown that lactobacilli play an important role in the relief of IBS-D symptoms. However, few studies have addressed the mechanisms by which microRNAs and lactobacilli exert their beneficial effects on intestinal epithelial permeability. Hence, to elucidate whether miRNAs and lactobacilli play roles in intestinal epithelial barrier regulation, we compared miRNA expression levels in intestinal epithelial cells (IECs) under Lactobacillus casei (L. casei LC01) treatment. IECs and L. casei LC01 were co-cultured and then subjected to microRNA microarray assay. qRT-PCR, western blot and ELISA were used to detect the expression of occludin (OCLN) and zonula occludens 1 (ZO1/TJP1). The interaction between miRNAs and L. casei LC01 acting in IECs was investigated through transfection of RNA oligoribonucleotides and pcDNA 3.1 plasmid. The results are as follows: 1) L. casei LC01 decreased the expression of miR-144 and FD4 and promoted OCLN and ZO1 expression in IECs; 2) L. casei LC01 enhanced the barrier function of IECs via downregulation of miR-144 and upregulation of OCLN and ZO1; 3) Under L. casei LC01 treatment, OCLN and ZO1 overexpression could partially eliminate the promoting effect of miR-144 on intestinal permeability in IECs. Our results demonstrate that L. casei LC01 regulates intestinal permeability of IECs through miR-144 targeting of OCLN and ZO1. L. casei LC01 can be a possible therapeutic target for managing dysfunction of the intestinal epithelial barrier.

Keywords: intestinal epithelial permeability; lactobacilli; miR-144; occludin (OCLN); zonula occludens 1 (ZO1/TJP1).

Fig. 1. L. casei LC01 significantly inhibited intestinal permeability of IECs.

(A) ELISA showed that the expression levels of OCLN and ZO1 were significantly increased in IECs under L. casei LC01 treatment compared to IECs that did not receive L. casei LC01 treatment for 3 days. (B & C) Protein and mRNA expression levels of OCLN and ZO1 were significantly upregulated in the experimental group compared with the control group. (D) FD4 was significantly downregulated in the experimental group compared with the control group. *p < 0.05.

Fig. 2. L. casei LC01 decreased the expression of miR-144 in IECs.

(A) Heatmap showing that 26 miRNAs were differentially expressed in IECs under L. casei LC01 treatment, of which 10 were upregulated and 16 were downregulated. (B) Six miRNAs (including miR-144) were randomly selected to verify the reliability of the microarray analysis through qRT-PCR.(C) OCLN and ZO1 share a matching 3′ UTR sequence that targets the seed region of miR-144. *p < 0.05 (Tukey’s test).

Fig. 3. L. casei LC01 regulated intestinal permeability of IECs via miR-144.

(A) qRT-PCR results showed that the expression levels of OCLN, ZO1 were significantly decreased in the miR-144 mimic group but were enhanced in the miR-144 inhibitor and miR control group compared with NC group. (B) The western blot results were consistent with the qRT-PCR results. *p < 0.05, difference between all groups (ANOVA & LSD).

Fig. 4. miR-144 strongly promoted intestinal permeability of IECs compared to si-OCLN and si-ZO1 during L. casei LC01 treatment.

(A) qRT-PCR demonstrated successful transfection of miR-144, si-OCLN and si-ZO1. (B and C) Western blot and qRT-PCR results both showed that OCLN and ZO1 were significantly decreased in the miR-144, si-OCLN, and si-ZO1 groups compared to the NC and si-control group. In addition, OCLN and ZO1 were the most decreased in the miR-144 group compared with all other groups. *p < 0.05, difference between all groups; ^NSp > 0.05, difference between the si-OCLN and si-ZO1 groups (ANOVA & LSD).

Fig. 5. Overexpression of OCLN/ZO1 partially rescued the promoting effect of miR-144 on intestinal permeability in IECs during L. casei LC01 treatment.

(A) qRT-PCR showed that pcDNA3.1-OCLN/ZO1 remarkably increased the expression of OCLN/ZO1, but miR-144 suppressed OCLN/ZO1 expression in IECs during L. casei LC01 treatment. Moreover, pcDNA3.1-OCLN/ZO1 also markedly increased OCLN/ZO1 expression, even in the presence of miR-144. (B) The western blot results were consistent with the qRT-PCR results.