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
miR-142-5p promotes Trichinella spiralis Ts-DNaseⅡ-7-mediated intestinal bar-rier damage by affecting the expression of tight junction protein CLDN1
鄂玉婷 1王静 1孙奕成 1刘晓雷 1吾拉木江·阿布来孜 2王存洲 2丁静1
作者信息
- 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
- 2. 新疆维吾尔自治区喀什地区伽师县维吾尔医医院,新疆 伽师 844300
- 折叠
摘要
旋毛虫进入宿主后,脱囊幼虫(肠道感染性第1期幼虫)进入宿主的小肠上皮细胞内继续发育,是旋毛虫感染并致病的关键步骤.本课题组前期研究结果显示,旋毛虫成虫期Ts-DNaseⅡ-7蛋白通过影响肠屏障,促进旋毛虫侵入肠上皮细胞,从而有利于其在宿主体内寄生.并且,Ts-DNase Ⅱ-7能够诱导多种miRNA出现差异化表达,有文献报道其中的miR-142-5p能够参与破坏肠屏障,因此本研究对Ts-DNaseⅡ-7诱导的miR-142-5p通过影响靶基因调控肠上皮细胞屏障功能的机制进行了探讨.试验分为Ts-DNase Ⅱ-7处理组、miR-142-5p过表达组(OE)、miR-142-5p抑制组(Inhibition)、阴性对照组(NC)和空白对照组(Control),将 miR-142-5p过表达及抑制表达的慢病毒载体,在MOI为80的条件下感染人结直肠腺癌细胞Caco-2,嘌呤霉素(8 mg/L)筛选获得稳定抑制miR-142-5p表达及过表达miR-142-5p的细胞系,通过荧光显微镜评估细胞转染情况,采用RT-qPCR检测各组细胞miR-142-5p相对表达量;利用miRNA靶标预测数据库、RT-qPCR、Western blot和双荧光素酶试验对目的基因进行预测和验证;再应用HE染色、Western blot和单层跨膜电阻(TEER)与FITC通透性定量分析阐明miR-142-5p的作用机制.结果显示:Caco-2细胞经慢病毒感染后,经荧光显微镜及RT-qPCR验证转染成功.此外,双荧光素酶和 Western blot结果显示CLDN1是miR-142-5p的直接靶基因(P＜0.001),与Ts-DNase Ⅱ-7单独处理组和OE+Ts-DNase Ⅱ-7组相比,抑制miR-142-5p表达组可缓解Tss-DNaseⅡ-7引起的CLDN1 mRNA和蛋白水平下调(分别为P＜0.01和P＜0.05),并逆转TEER下降和通透性升高(P＜0.01).结果表明,Ts-DNase Ⅱ-7可上调miR-142-5p的表达,引起Caco-2单层细胞CLDN1表达减少和肠屏障功能受损,进而促进旋毛虫入侵肠上皮,实现进一步发育.
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.
关键词
旋毛虫/miR-142-5p/肠道屏障/通透性/CLDN1Key words
Trichinella spiralis/miR-142-5p/intestinal barrier/permeability/CLDN1引用本文复制引用
基金项目
吉林省自然科学基金(20220508052RC)
吉林省自然科学基金(20220101294JC)
出版年
2024
NETL
NSTL
万方数据