miR-142-5p promotes Trichinella spiralis Ts-DNaseⅡ-7-mediated intestinal bar-rier damage by affecting the expression of tight junction protein CLDN1
10.16303/j.cnki.1005-4545.2024.07.15
- VernacularTitle:miR-142-5p通过影响紧密连接蛋白CLDN1的表达介导旋毛虫Ts-DNaseⅡ-7的肠屏障损伤作用
- Author:
Yuting E
1
;
Jing WANG
;
Yicheng SUN
;
Xiaolei LIU
;
Abulaizi WULAMUJIANG
;
Cunzhou WANG
;
Jing DING
Author Information
1. 吉林大学动物医学学院人畜共患传染病重症诊治全国重点实验室/人兽共患病研究所/人兽共患病研究教育部重点实验室,吉林长春 130062
- Keywords:
Trichinella spiralis;
miR-142-5p;
intestinal barrier;
permeability;
CLDN1
- From:
Chinese Journal of Veterinary Science
2024;44(7):1458-1465,1482
- CountryChina
- Language:Chinese
-
Abstract:
Upon penetration into the host organism,the decapsulated larvae(intestinal infective stage 1 larvae)of Trichinella spiralis(T.spiralis)proceed to invade the host's small intestinal epithelial cells to continue their development,which is a critical step for T.spiralis infection and pathogenesis.Based on our prior research,the Ts-DNase Ⅱ-7 protein,which is present in the adult stage of T.spiralis,has a role in promoting the parasite's invasion of the intestinal epithelium.This is achieved by disrupting the integrity of the intestinal barrier,consequently promoting the parasitization of these cells in the host organism.Ts-DNase Ⅱ-7 can induce differential expression of various miRNAs,among which miR-142-5p has been reported to be involved in disrupting the intestinal barrier.Therefore,in this study,we investigated the mechanism by which Ts-DNase Ⅱ-7-induced miR-142-5p regulates intestinal epithelial cell barrier function by affecting target genes.The experiment was divided into Ts-DNase Ⅱ-7-treated group,miR-142-5p overexpression group(OE),miR-142-5p inhibition group(Inhibition),negative control group(NC)and control group(Control),lentiviral vectors for overexpression and suppression of miR-142-5p expression were used to infect human colorectal adenocarcinoma cells Caco-2 at an MOI of 80,and puromycin(8 mg/L)was used to screen cells to obtain cell lines that stably suppressed the expression of miR-142-5p and cell lines that miR-142-5p was overexpressed,cell transfection efficiency was assessed by fluorescence microscopy,and the relative expression of miR-142-5p was detected by RT-qPCR in each group of cells.The target gene Claudin-1(CLDN1)was predicted and validated using miR target prediction database,RT-qPCR,Western blot and dual luciferase assay.HE staining,Western blot and quantitative analysis of monolayer transmembrane electrical resistance(TEER)and FITC permeability were then applied to elucidate the mechanism of action of miR-142-5p.Caco-2 cells were lentivirally infected and successful transfection was verified by fluorescence microscopy and RT-qPCR.Dual luciferase and Western blot results showed that CLDN1 was a direct target gene of miR-142-5p(P＜0.001).Compared to the Ts-DNaseⅡ-7 alone treatment group and the OE+Ts-DNase Ⅱ-7 group,the miR-142-5p inhibited expression group and alleviated Ts-DNase Ⅱ-7-induced down-regulation of CLDN1 mRNA and protein levels(P＜0.01 and P＜0.05,respectively)and re-versed the decrease in TEER and elevated permeability(P＜0.01).Ts-DNase Ⅱ-7 up-regulates miR-142-5p expression,causing reduced CLDN1 expression and impaired intestinal barrier function in Caco-2 monolayer cells,which in turn promotes T.spiralis invasion of the intestinal epithelium to achieve further development.
