Fig 1: 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 2: 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 3: 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 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.
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