【摘要】 棕榈酸（Palmitic acid, PA）会损伤人体肠道上皮细胞紧密连接，导致肠道屏障功能紊乱。我们前研究发现，6-姜烯酚可通过调控miR-216a-5p靶向抑制Toll样受体4(Toll-like receptor 4,TLR4)进而抑制炎症因子，保护肠道细胞免受PA诱导的损伤。然而，6-姜烯酚调控miR-216a-5p表达的具体机制不清楚。本研究采用PA处理人结直肠腺癌Caco-2细胞建立肠道紧密连接损伤模型，通过DNA pulldown实验和双荧光素酶报告基因实验，初步确定尾型同源框转录因子2(Caudal-related homeobox transcription factor 2,CDX2)是miR-216a-5p的候选调控因子。通过染色质免疫共沉淀-聚合酶链反应（Chromatin immunoprecipitation followed by polymerase chain reaction,ChIP-PCR）实验和电泳迁移率实验（Electrophoretic mobility shift assay,EMSA）实验相结合，证实转录因子CDX2可以与miR-216a-5p启动子相结合并促进其转录。最后评估了转录因子CDX2在促炎因子和紧密连接相关蛋白表达中的作用，证明了CDX2是6-姜烯酚调节miR-216a-5p表达的靶标。本研究首次揭示CDX2是miR-216a-5p的直接转录激活因子，为揭示生姜的健康调节作用和相关功能食品的研究提供了参考和依据。更多还原

【Abstract】 Palmitic acid(PA) can damage the tight junction of human intestinal epithelial cells, leading to intestinal barrier dysfunction. Our previous study demonstrates that 6-shogaol can modulate the expression of microRNA miR-216a-5p, leading to the targeted inhibition of the Toll-like receptor 4(TLR4) signaling pathway. This, in turn, reduces the production of inflammatory factors and protects intestinal cells from PA-induced damage. However, the specific mechanism by which 6-shogaol regulates miR-216a-5p expression remains unclear. This study establishes a model of intestinal tight junction injury by treating human colorectal adenocarcinoma Caco-2 cells with PA. Through DNA pulldown assays and dual-luciferase reporter gene experiments, this study preliminarily identifies Caudal-related homeobox transcription factor 2(CDX2) as a candidate regulator of miR-216a-5p. By combining chromatin immunoprecipitation followed by polymerase chain reaction(ChIP-PCR) and electrophoretic mobility shift assay(EMSA), this study confirms that the transcription factor CDX2 can bind to the miR-216a-5p promoter and promotes its transcription. Finally, this study evaluates the role of transcription factor CDX2 in regulating the expression of pro-inflammatory factors and tight junction-related proteins, proving that CDX2 is the target through which 6-shogaol regulates miR-216a-5p expression. This study is the first to show that CDX2 is a direct transcriptional activator of miR-216a-5p, providing a reference and basis for revealing the health-regulation effects of ginger and related functional food research.更多还原