Scientific Research Center, The First Affiliated Hospital/School of Clinical Medicine of Guangdong Pharmaceutical University, Guangzhou, Guangdong 510080, PR China.
Scientific Research Center, The First Affiliated Hospital/School of Clinical Medicine of Guangdong Pharmaceutical University, Guangzhou, Guangdong 510080, PR China; Dongguan bioshine biotechnology Co. Ltd..
Cell Signal. 2021 Jun;82:109957. doi: 10.1016/j.cellsig.2021.109957. Epub 2021 Feb 17.
To investigate the influence of HuR on the apoptosis rate of epithelial cells in rats with ulcerative colitis (UC) and its mechanism.
UC cell models were established in LPS induced Caco-2 cells. After transfection of si-HuR, pcDNA3.1-HuR, pcDNA3.1-HMGB1, miR-29a-3p mimic or miR-29a-3p inhibitor and their negative controls, apoptosis rate and apoptosis-related proteins (Bcl-2, Bax and cleaved-caspase-3) were tested by flow cytometry, qRT-PCR and Western blot. Actinomycin D treatment was applied to verify the effect of HuR in Caco-2 cells. The binding of HMGB1 to HuR/miR-29a-3p was measured by RIP and dual luciferase reporter gene assays. Experimental UC rat models were established by rectum administration of TNBS/ethanol. The colonic weight/length ratio was calculated at the day 15. The morphology of colon tissues and the apoptosis of tissues were separately detected by H&E staining and TUNEL staining. qRT-PCR and Western blot were conducted to determine the levels of HuR, miR-29a-3p and HMGB1 in colon tissues.
The apoptosis of LPS-treated Caco-2 cells was inhibited following transfection of si-HuR or miR-29a-3p mimic while facilitated following transfection of pcDNA3.1-HMGB1 or miR-29a-3p inhibitor. RIP and dual luciferase reporter gene assays showed that both HuR and miR-29a-3p can bind HMGB1. Overexpression of HuR in Caco-2 cells results in less HMGB1 that can be bind to miR-29a-3p. The degradation rate of HMGB1 mRNA was increased after transfection of si-HuR in Caco-2 cells. Additionally, miR-29a-3p overexpression can abolish the increases of HMGB1 mRNA induced by HuR, therefore consequently suppress the HMGB1 mRNA that can be bind to HuR. Knockdown of HuR can alleviate TNBS-induced UC in rats and inhibit the apoptosis of colon tissues.
HuR competitively binds HMGB1 with miR-29a-3p to promote apoptosis of colonic epithelia in rats with UC.
研究 HuR 对溃疡性结肠炎（UC）大鼠上皮细胞凋亡率的影响及其机制。
采用脂多糖诱导 Caco-2 细胞建立 UC 细胞模型。转染 si-HuR、pcDNA3.1-HuR、pcDNA3.1-HMGB1、miR-29a-3p 模拟物或 miR-29a-3p 抑制剂及其阴性对照后，通过流式细胞术、qRT-PCR 和 Western blot 检测细胞凋亡率和凋亡相关蛋白（Bcl-2、Bax 和 cleaved-caspase-3）。应用放线菌素 D 处理验证 HuR 在 Caco-2 细胞中的作用。通过 RIP 和双荧光素酶报告基因实验测定 HMGB1 与 HuR/miR-29a-3p 的结合。通过直肠给予 TNBS/乙醇建立实验性 UC 大鼠模型。在第 15 天计算结肠重量/长度比。通过 H&E 染色和 TUNEL 染色分别检测结肠组织形态和组织细胞凋亡。通过 qRT-PCR 和 Western blot 检测结肠组织中 HuR、miR-29a-3p 和 HMGB1 的水平。
转染 si-HuR 或 miR-29a-3p 模拟物可抑制 LPS 处理的 Caco-2 细胞凋亡，而转染 pcDNA3.1-HMGB1 或 miR-29a-3p 抑制剂则促进其凋亡。RIP 和双荧光素酶报告基因实验表明，HuR 和 miR-29a-3p 均可与 HMGB1 结合。在 Caco-2 细胞中转染 HuR 可导致与 miR-29a-3p 结合的 HMGB1 减少。转染 si-HuR 可增加 Caco-2 细胞中 HMGB1 mRNA 的降解率。此外，miR-29a-3p 过表达可消除 HuR 诱导的 HMGB1 mRNA 增加，从而抑制与 HuR 结合的 HMGB1 mRNA。HuR 敲低可减轻 TNBS 诱导的 UC 大鼠模型中结肠组织的凋亡。
HuR 与 miR-29a-3p 竞争性结合 HMGB1，促进 UC 大鼠结肠上皮细胞凋亡。
