Effects of miR-939 and miR-376A on the pathogenesis of ulcerative colitis by regulating NF-κB and NFAT expression through decoy strategy
Effects of miR-939 and miR-376A on the pathogenesis of ulcerative colitis by regulating NF-κB and NFAT expression through decoy strategy
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miR-939和miR-376A通过诱饵策略调节NF-κB和NFAT表达对溃疡性结肠炎发病机制的影响
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通讯作者：
崔春晖
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作者：
林永伟;谢朗;周志鹏;黄永胜;邱政华;叶丽丽;崔春晖
目的：验证miR-939和miR-376A在UC患者组织中的表达。通过诱骗策略研究miR-939和miR-376A通过调节NF-κB和NFAT的表达对溃疡性结肠炎发病机制的影响，为UC的治疗提供新思路。采用QPCR技术检测正常标本和临床标本UC组织标本静息期和活动期miR-939、miR-376a、NF-κB、NFAT的表达差异。分析正常人和UC组织样本中miR-939、miR-376、NF-κB和NFAT的差异表达。2. 将mirna和抗mirna反义抑制剂的合成、decoyODN的制备、外源性过表达miR-939和miR-376a及其抑制剂引入HT29细胞和UC体外模型细胞。通过miR -376A和miR-939调控荧光素酶基因的转录，测定了荧光素酶的活性。RT-PCR分析评估miRNA与转录因子之间的相互作用。电泳迁移率变化分析（EMSA）证实了mirna结合NF-κB和NFAT的能力。采用WB、QPCR检测UC肠屏障claudin、occludin、Z0-1蛋白的表达，采用免疫荧光技术观察上述紧密连接蛋白在UC的分布。WB、QPCR、免疫荧光检测凋亡相关因子KCNJ2、caspase3、PARP、核小体的变化。3. 建立DSS小鼠UC模型，分为转染人工诱饵（NF-κB、NFAT）组、转染miRNA-decoyODN（-939、-376A）组和空白对照组。治疗后，测定小鼠结肠组织形态变化、血清炎症因子表达变化、尿蛋白或尿白细胞、肝酶、凝血酶原时间变化，检测肠道通透性。通过对比研究，分析miRNAs“窥探”策略在UC炎症控制中的作用。结果：UC患者组织中miR-939和miR-376A的表达明显低于正常人体组织。正常与UC患者miR-939、miR-376A、NF-κB、NFAT的表达呈负相关。由于MiR-939和miR-376A对NF-κ b和NFAT的诱引策略，MiR-939和miR-376A的表达被抑制，转录因子NF-κ b和NFAT的激活被抑制，炎症因子IL-8、IL1B、IL2、G -CSF、GM-CSF、MIP-2α、TNF-a、TLR4的分泌也被抑制，有利于cludins、occludins和Z0-1蛋白的恢复，抑制肠上皮细胞凋亡。动物实验发现，人工诱饵组和转染mirna -诱饵ODN组大鼠血清WBC计数、BUN、Glu、ALT、AST及24种尿蛋白均低于UC模型组。转染后24,48,72小时，
Objective: To verify the expression of miR-939 and miR-376A in the tissues of UC patients. Study the effects of miR-939 and miR-376A on the pathogenesis of ulcerative colitis by regulating the expression of NF-κB and NFAT through decoy strategies to provide new ideas for the treatment of UC..Methods: 1. QPCR technology was used to detect the differences between the expression of miR-939, miR-376a and NF-κB, NFAT in resting and active stages of normal samples and UC tissue samples from clinical samples. Differential expressions of miR-939, miR-376, NF-κB, and NFAT in normal human samples and UC tissue samples were analyzed for their correlation. 2. Synthesis of miRNAs and anti-miRNA antisense inhibitors, preparation of decoyODN, exogenous overexpression of miR-939 and miR-376a and their inhibitors were introduced into HT29 cells and UC in vitro model cells. Luciferase activity was measured in response to miR -376A and miR-939 regulate transcription of the luciferase gene. RT-PCR analysis evaluates the interaction between miRNA and transcription factors. Electrophoretic mobility change analysis (EMSA) validates the ability of miRNAs to bind NF-κB and NFAT. The expression of claudins, occludin, Z0-1 of the intestinal barrier at UC was examined by WB, QPCR, and the distribution of the above-mentioned tight junction proteins at UC was observed by immunofluorescence technology. Changes of apoptosis-related factors (KCNJ2, caspase3, PARP, nucleosomes) were examined by WB, QPCR, and immunofluorescence. 3. Establish DSS mouse UC model, which is divided into transfection artificial decoy (NF-κB, NFAT) group, transfection miRNA-decoyODN (-939, -376A) group and blank control group. After the treatment, the morphological changes of colonic tissues in mice, changes in serum inflammatory factor expression, and changes in urine protein or urine leukocytes, liver enzymes, and prothrombin time were measured to detect intestinal permeability. Through comparative research, analyze the effect of miRNAs "snoop" strategy on controlling UC inflammation..Results: The expressions of miR-939 and miR-376A in UC patient tissues were significantly lower than those in normal human tissues. And the expressions of miR-939, miR-376A, NF-κB, and NFAT were negatively correlated in normal and UC patients. As MiR-939 and miR-376A's decoy strategy for NF-κB and NFAT, the expression of miR-939 and miR-376A were inhibitied, the activation of transcription factors NF-κB and NFAT were inhibited and the the secretion of inflammatory factors IL-8, IL1B, IL2, G - CSF, GM-CSF, MIP-2α, TNF-a, TLR4 were also inhibited which is conducive to the recovery of claudins, occludins and Z0-1 proteins, and inhibits intestinal epithelial cell apoptosis. Animal experiments found that WBC counts, BUN, Glu, ALT, AST and 24 urine proteins in serum of rats in the artificial decoy group and the transfected miRNA-decoy ODN group were lower than those in the UC model group. 24, 48, 72 hours after transfection,