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| miR-548a-3p调控TLR4抑制脂多糖诱导结肠上皮细胞的凋亡 |
| MiR-548a-3p inhibits apoptosis of colonic epithelial cells induced by lipopolysaccharide by regulating TLR4 |
| 投稿时间：2021-09-09 |
| DOI：10.3969/j.issn.1000-0399.2022.08.008 |
| 中文关键词: 溃疡性结肠炎 细胞凋亡 微小RNA-548a-3p toll样受体4 |
| 英文关键词: Ulcerative colitis Apoptosis MiR-548a-3p Toll-like receptor 4 (TLR4) |
| 基金项目:深圳市宝安区科技计划项目(项目编号:2016CX318) |
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| 中文摘要: |
| 目的 探究微小RNA (miRNA)-548a-3p对脂多糖(LPS)诱导的结肠上皮细胞的凋亡和炎症反应影响及其机制。方法 收集2019年1月至2020年5月于南方医科大学深圳医院就诊,经肠镜及病理学检查确诊为活动期溃疡性结肠炎35例患者的肠黏膜组织及正常肠道黏膜组织。取正常结肠上皮细胞分为6组:对照组不做任何处理;LPS组给予50 μg/mL LPS处理;miR-NC组、miR-548-3p mimics组、miR-548-3p inhibitor组、miR-574-3p mimics+pcDNA3.1-toll样受体4(TLR4)组分别转染相应序列,随后使用等量LPS处理。实时荧光定量聚合酶链反应(PCR)检测miR-548a-3p表达,CCK-8法检测细胞增殖,酶联免疫吸附剂测定(ELISA)检测肿瘤坏死因子α(TNF-α)和白介素8(IL-8)含量,双荧光素酶报告基因实验验证miR-548a-3p与TLR4的结合关系;Western blot实验检测TLR4、B细胞淋巴瘤/白血病-2基因-相关X蛋白(Bax)和半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)蛋白表达。结果 miR-548a-3p在溃疡性结肠炎患者肠黏膜组织中相对表达水平为(1.130±0.568),低于正常肠黏膜组织(1.684±0.733),差异有统计学意义(P<0.05);LPS组中miR-548a-3p表达水平为(0.463±0.042),低于对照组的(1.001±0.090),差异有统计学意义(P<0.05)。转染后,miR-548a-3p inhibitor组中TNF-α、IL-8、细胞凋亡率、BAX表达和Caspase 3表达水平高于miR-NC组,而24小时和48小时细胞增殖水平低于miR-NC组,差异均有统计学意义(P<0.05)。与miR-NC组相比,miR-548a-3pmimics组中含野生型TLR4荧光素酶活性降低(P<0.05)。与miR-548a-3p mimics组相比,miR-574-3p mimics+pcDNA3.1-TLR4组TNF-α、IL-8、细胞凋亡率、BAX表达和Caspase 3表达水平较高,24小时和48小时细胞增殖水平较低,差异有统计学意义(P<0.05)。结论 miR-548a-3p可通过靶向TLR4抑制细胞凋亡和炎症因子分泌,并促进细胞增殖。 |
| 英文摘要: |
| Objective To investigate the effect of microRNA (miRNA)-548a-3p on lipopolysaccharide (LPS)-induced apoptosis and inflammatory response of colonic epithelial cells and its mechanism.Methods Intestinal mucosal tissues and normal intestinal mucosal tissues of patients with active ulcerative colitis who were admitted to Shenzhen Hospital Afflilated to Southern Medical University from January 2019 to May 2020 were collected. Colonic epithelial cells were divided into six groups:Controlgroup was not treated; LPS group was treated with 50 μg/mL LPS; miR-NC group, miR-548-3p mimics group, miR-548-3p inhibitorgroup,miR-574-3p mimics+pcDNA3.1-toll-like receptor 4 (TLR4) group were transfected with corresponding sequence, then treated with LPS. Real-time fluorescent quantitative PCR was used to detect miR-548a-3p expression. CCK-8 assay was used to detect cell proliferation. ELISA was used to detect tumor necrosis factor-α (TNF-α) and interleukin-8 (IL-8) levels. Dual luciferase reporter gene assay was used to verify the combination between miR-548a-3p and TLR4. Western blotting was used to detect the expression of TLR4, B cell lymphoma/leukemia-2 gene-associated X protein (Bax) and cysteine aspartate protease-3(Caspase-3). Results The relative expression of miR-548a-3p in intestinal mucosa of patients with ulcerative colitis (1.130±0.568) was significantly lower than that in normal intestinal mucosa (1.684±0.733), and the difference was statistically significant (P<0.05). The miR-548a-3p expression in LPS group (0.463±0.042) was lower than that in the control group (1.001±0.090), and the difference was statistically significant (P<0.05). After transfection, the TNF-α, IL-8, cell apoptosis, BAX expression and Caspase-3 expression in miR-548a-3p inhibitor group were higher than those in miR-NC group, while the cell proliferation at 24th and 48th were significantly lower, and the difference was statistically significant (P<0.05). Compared with miR-NC group, the luciferase activity of wild type TLR4 in miR-548a-3pmimics group significantly decreased (P<0.05). Compared with the miR-548a-3p mimics group, the TNF-α, IL-8, cell apoptosis, BAX expression and Caspase-3 expression were significantly higher, while the cell proliferation at 24th and 48th were lower, and the difference was statistically significant (P<0.05). Conclusions MiR-548a-3p can inhibit apoptosis and inflammation, as well as promote cell proliferation by targeting TLR4. |
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