Lactobacillus casei LC01 Regulates Intestinal Epithelial Permeability through miR-144 Targeting of OCLN and ZO1
1Department of Gastroenterology, The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510176, P.R. China
2School of Chinese Medicine, Hong Kong Baptist University, Hong Kong, China
3Department of Orthopedics, The Second Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510176, P.R. China
4Department of Preventive Medicine and Health Statistics, Guangzhou University of Chinese Medicine, Guangzhou 510176, Guangdong, P.R. China
J. Microbiol. Biotechnol. 2020; 30(10): 1480-1487
Published October 28, 2020
Copyright © The Korean Society for Microbiology and Biotechnology.
Irritable bowel syndrome  is a chronic, functional bowel disorder that is characterized by visceral pain and bloating and altered bowel habits in the absence of anatomical or biochemical abnormalities . Up to 30% of IBS patients have been reported to develop acute gastroenteritis, and most are diagnosed with irritable bowel syndrome with diarrhea (IBS-D), which is a major subtype and correlates with changes in the intestinal microflora after infection . Brain-gut axis dysfunction , visceral hypersensitivity , gastrointestinal dysmotility , and alterations in psychosomatic  or psychosocial behavior  have been implicated in the pathophysiology of IBS-D, but the exact mechanisms remain largely undefined.
Probiotics are beneficial bacteria that can improve health and maintain the micro-ecological balance of the host intestine. Screening probiotic strains with excellent probiotic characteristics has become a research hotspot. Studies have shown that the probiotic
MicroRNAs (miRNAs) are a type of non-coding small-molecule RNA endogenously expressed in eukaryotic cell organisms and can act on the 3'-untranslated region (3'-UTR) of target gene mRNA . If the miRNAs are completely complementary to the 3'-UTR, it may cause the target gene mRNA to degrade; if not completely complementary, it may inhibit the target gene mRNA translation . Current research reports that miRNAs play an important role in regulating IBS-D intestinal epithelial permeability [15, 16]. We also found in our previous studies that the downregulation of miR-144 can target OCLN and ZO1 to reverse the intestinal epithelial permeability of IBS-D rats , but little is known about whether
In this study, we aimed to evaluate whether and how
Materials and Methods
Human IEC Lines
Human IEC lines (HCoEpiC, C1388) were purchased from the Chinese Academy of Sciences Shanghai Cell Bank and cultured in DMEM containing 100 ml/l fetal bovine serum in a CO2 incubator with saturated humidity at 37°C. Fibroblasts were removed using phase difference digestion and adherence. When 80%-90% of the IECs were adherent to culture plates, the IECs were passaged by trypsin digestion.
L. casei LC01 Preparation
Paracellular Flux Measurements
Intestinal epithelial permeability as a surrogate marker of layer integrity was measured by using fluorescein isothiocyanate-dextran 4 kDa (FD4; Sigma-Aldrich, USA). The IECs were treated with or without
miRNA Microarray Analysis
IECs treated with or without
Enzyme-Linked Immunosorbent Assays (ELISA)
The method was as described previously . Briefly, the IECs were treated with or without
RNA Oligoribonucleotide Synthesis and Transfection
The method was as described previously . Briefly, miR-144 mimic and inhibitor, mimic control and inhibitor control and the short interference RNAs (siRNA) were used to repress ZO1 or OCLN (siZO1 or siOCLN), and siRNA negative control was prepared for transfection (RiboBio, China). IECs treated with or without
Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
The method was as described previously . Briefly, after total RNA was extracted and complementary DNA (cDNA) was synthesized, qRT-PCR was performed using an ABI Prism 7500 PCR system (Applied Biosystems, USA). β-Actin was used as an internal reference, and the 2−ΔCt method was used to calculate the relative mRNA expression level. The measurements were repeated in triplicate and averaged. The primers were designed as follows: OCLN: 5’-AAGACGATGAGGTGCAGAAG-3’ (forward), 5’-GTGAAGAGAGCCTGACCAAA-3’ (reverse); ZO1: 5’-GGAGAGGTGTTTCGTGTTGT-3’ (forward), 5’-ACTGCTCAGCCCTGTTCTTA-3’(reverse); β-actin: 5’-CGTGACATTAAGGAGAAGCTG-3’(forward), 5’-CTAGAAGCATTTGCGGTGGAC-3’ (reverse); miR-144: 5'-GCGCGCTACAGTATAGATGATG-3' (forward), 5'-GCTGTCAACGATACGCTACG-3' (reverse).
The method was as described previously . Briefly, after proteins were extracted from the IECs, protein concentrations were determined using the BCA Protein Assay Kit (Beyotime, Beijing, China). Equal amounts of protein were loaded onto sodium dodecyl sulphate-polyacrylamide gels (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were incubated with primary antibodies (1:300 dilution) overnight at 4°C, incubated with a secondary antibody (1:3,000 dilution) at 37°C for 1 h, and examined using a chemiluminescence detection kit. β-Actin was used as the internal reference.
The method was as described previously . Briefly, IECs were transfected with the pcDNA 3.1 plasmid (pcDNA3.1-OCLN/ZO1), or the miR-144 mimic, or co-transfected with the overexpression vector and the miR-144 mimic. Western blot and qRT-PCR were then used to detect the expression of OCLN/ZO1.
SPSS 17.0 (SPSS Inc., USA) was used for statistical analyses. The measurement data that conformed to a normal distribution were expressed as the mean ± standard deviation. One-way analysis of variance (ANOVA) and least significant difference (LSD) multiple comparison tests were used to compare the differences between groups. Parameters with abnormal distribution were expressed as the median (quartile). Non-parametric (Mann-Whitney U) tests were used to compare the differences between groups. Differences were regarded as statistically significant when
L. casei LC01 Significantly Inhibited Intestinal Permeability of IECs
IECs treated with or without
( L. caseiLC01 significantly inhibited intestinal permeability of IECs. A) ELISA showed that the expression levels of OCLN and ZO1 were significantly increased in IECs under L. caseiLC01 treatment compared to IECs that did not receive L. caseiLC01 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.
L. casei LC01 Suppressed the Expression of miR-144 in IECs
To study the differential expression of miRNAs in IECs in response to
( L. caseiLC01 decreased the expression of miR-144 in IECs. A) Heatmap showing that 26 miRNAs were differentially expressed in IECs under L. caseiLC01 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).
L. casei LC01 Regulated Intestinal Permeability of IECs Via miR-144
To evaluate the role of miR-144 in IECs during
( L. caseiLC01 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).
miR-144 Promoted Intestinal Permeability of IECs More Strongly than si-OCLN and si-ZO1 during
L. casei LC01 Treatment
First, we transfected IECs with miR-144, si-OCLN or si-ZO1, and verified the transfection efficiency of miR-144, si-OCLN, and si-ZO1 in IECs by qRT-PCR (Fig. 4A). Next, we examined the expression of OCLN and ZO1 by western blot (Fig. 4B) and qRT-PCR (Fig. 4C) during
miR-144 strongly promoted intestinal permeability of IECs compared to si-OCLN and si-ZO1 during( L. caseiLC01 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; NS p> 0.05, difference between the si-OCLN and si-ZO1 groups (ANOVA & LSD).
Overexpression of OCLN/ZO1 Partially Rescued the Promoting Effect of miR-144 on Intestinal Permeability in IECs during
L. casei LC01 Treatment
To further understand the interaction between miR-144, OCLN/ZO1 and
Overexpression of OCLN/ZO1 partially rescued the promoting effect of miR-144 on intestinal permeability in IECs during( L. caseiLC01 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. caseiLC01 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.
In a previous study, intestinal microflora were reported to play an important role in the maintenance of the host intestinal epithelial barrier function . Some probiotics can act through molecular and cellular mechanisms that prevent pathogenic bacterial adhesion, enhance innate immunity, decrease pathogen-induced inflammation and promote intestinal epithelial cell survival, barrier function, and protective responses . It has been reported that
The aim of this study was to further identify the molecular mechanisms underlying the mutual effect of
Recent evidence indicates that the communication between the gut microbiome and the host may be partially carried out by miRNAs, which are differentially expressed under specific microflora states . However, the role of miRNAs in the microbial-host communication of intestinal diseases is not yet fully understood. Interestingly, an important finding of the present study is that miR-144 was downregulated in IECs during
IECs are key mediators of intestinal homeostasis and are able to establish an immune environment that allows symbiotic bacterial colonization. IECs provide physical and biochemical barriers that sequester host tissues and commensal bacteria to maintain intestinal homeostasis. IECs have a mechanism enabling them to respond to microbial signals, which confers tolerance against continuous exposure to symbiotic bacteria . In this study, IECs were used to investigate intestinal epithelial permeability stimulated by
It has also been reported that OCLN and ZO1 are not only capable of controlling paracellular macromolecule permeability but also interacting with intracellular signaling pathways that regulate and maintain intestinal epithelial permeability, subsequently enhancing intestinal epithelial barrier function [25-29]. To further understand the interaction between miR-144, OCLN/ZO1 and
In addition, the results of this study have several important clinical implications. First, we conducted miRNA microarray analysis in this study, which provides examples of identifying differentially expressed miRNAs and their target genes after intervention with beneficial bacteria. Second, we provide the basis for further research and characterization of the beneficial bacteria-miRNA-OCLN/ZO1 relationship, which not only helps to better understand the key molecular switches regarding the permeability of the intestinal mucosa but also to characterize the miRNAs related to intestinal epithelial permeability-related diseases. Third,
In summary, regulation of intestinal epithelial permeability through tight junction proteins is an important mechanism for enhancing mucosal defense functions and maintaining intestinal homeostasis.
This study was supported by the National Natural Science Foundation (No. 81804047), Guangdong Provincial Traditional Chinese Medicine Research Project (No. 20181095), Hong Kong Scholar Program (XJ2018059), Innovative Research Team Project of “Innovative Strong Institute”, the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine (2017TD05) and Guangzhou University of Traditional Chinese Medicine's first-class discipline research key project (A1-AFD018191A16).
Conflict of Interest
The authors have no financial conflicts of interest to declare.
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