YAP蛋白通过激活STAT3调控肠上皮细胞增殖在DSS诱导肠炎及相关肠癌中的作用

YAP regulates intestinal epithelial cell proliferation through activation of STAT3 in DSS-induced colitis and associated cancer

目的:溃疡性结肠炎(ulcerative colitis,UC)是一种肠道慢性、易复发的非特异性炎症,肠上皮修复障碍是其重要的生物学特点,促进肠上皮愈合达到内镜下黏膜愈合是UC的最终治疗目标。Yes相关蛋白(Yes-associated protein,YAP)是影响器官发育、组织生长及肿瘤发生的重要辅助转录因子,近年来越来越多的研究关注YAP在肠黏膜修复中的作用,这对于靶向治疗UC及预防相关并发症的发生具有重要意义。本研究探究YAP调控肠黏膜上皮细胞增殖、修复及肠炎相关肿瘤(colitis associated cancer,CAC)发生的分子机制。方法:使用3%葡聚糖硫酸钠盐(dextran sulfate sodium salt,DSS)连续诱导5 d构建小鼠急性肠炎模型。构建YAP过表达(YAP^(WT))及阴性对照(NC)的慢病毒载体,分别腹腔注射入DSS小鼠体内,于DSS诱导5 d及撤除DSS后正常饮水的5 d(5+5 d)脊椎脱臼处死小鼠,取结肠测量其长度,选择近肛管1~2 cm肠管行免疫组织化学、蛋白质印迹法分析。构建YAP过表达的肠上皮细胞系及信号转导与转录激活因子3(signal transducer and activator of transcription 3,STAT3)小干扰RNA(si-STAT3),用蛋白质印迹法探究YAP对STAT3的调控方式;用功能学细胞计数试剂盒-8(cell counting kit-8,CCK-8)和划痕实验研究YAP调控STAT3在肠上皮细胞增殖中的作用;通过蛋白质免疫共沉淀探究YAP与STAT3的相互作用。结果:DSS炎症诱导导致YAP在肠上皮细胞中表达显著降低,相对于NC小鼠,过表达YAP促进了小鼠在DSS炎症损伤后肠黏膜的修复,表现为肠上皮的结构更为完整,黏膜中炎性细胞的数量减少。蛋白质印迹法、功能学实验及免疫共沉淀结果表明:细胞核中的YAP在DSS撤除后的2 h显著高表达,同时伴随STAT3及磷酸化STAT3(phosphorylated-STAT3,p-STAT3)高表达;YAP过表达上调STAT3、p-STAT3及其转录靶基因细胞-骨髓细胞瘤病毒癌基因(cellularmyelocytomatosis viral oncogene,c-Myc)和G1/S-特异性周期蛋白-D1(cell cycle protein D1,Cyclin D1)的表达,且促进肠上皮细胞的增殖及伤口“愈合”。然而,在过表达YAP的基础上沉默STAT3则c-Myc及Cyclin D1表达降低,细胞增殖能力被逆转。蛋白质免疫共沉淀结果表明YAP和STAT3可以在细胞核内直接结合,促进STAT3的转录表达。在CAC发生过程中,过表达YAP细胞质磷酸化突变体下调STAT3的表达,抑制CAC的发生。结论:YAP通过激活STAT3调控肠上皮细胞增殖,促进DSS急性炎症小鼠的肠黏膜修复,YAP可作为UC肠黏膜修复的重要治疗靶点。然而,长期、过度的YAP激活可导致CAC的发生。

Objective:Ulcerative colitis(UC)is a chronic,relapsing inflammation of the colon.Impaired epithelial repair is an important biological features of UC.Accelerating intestinal epithelial repair to achieve endoscopic mucosal healing has become a key goal in UC.Yesassociated protein(YAP)is a key transcriptional coactivator that regulates organ size,tissue growth and tumorigenesis.Growing studies have focused on the role of YAP in intestinal epithelial regeneration.This study explore the molecular mechanism for the role YAP in modulating colonic epithelial proliferation,repair,and the development of colitis associated cancer.Methods:We constructed the acute colitis mouse model through successive 5 days of 3%dextran sulfate sodium salt(DSS)induction.Then YAP-overexpressed mouse model was constructed by intraperitoneal injection the YAP overexpressed and negative control lentivirus into DSS mice.On the 5th day of DSS induction and the 5th day of normal drinking water after removing DSS(5+5 d),the mice were killed by spinal dislocation.The colon was taken to measure the length,and the bowel 1−2 cm near the anal canal was selected for immunohistochemical and Western blotting.We used YAP over-expressed colonic epithelial cells and small interfering signal transducer and activator of transcription 3(STAT3)RNA to probe the regulation of YAP on STAT3,using cell counting kit-8 and scratch assays to explore the role of YAP on colonic epithelial cell proliferation.Finally,we conducted co-immunoprecipitation to test the relationship between YAP and STAT3.Results:After DSS treatment,the expression of YAP was dramatically diminished in crypts.Compared with the empty control mice,overexpression of YAP drastically accelerated epithelial regeneration after DSS induced colitis,presenting with more intact of structural integrity in intestinal epithelium and a reduction in the number of inflammatory cells in the mucosa.Further Western blotting,functional experiment and coimmunoprecipitation analyses showed that the expression of YAP in nucleus was significantly increased by 2 h post DSS cessation,accompanied with up-regulated total protein levels of STAT3 and phosphorylated-STAT3(p-STAT3).Overexpression of YAP enhanced the expression of STAT3,p-STAT3,and their transcriptional targets including c-Myc and Cyclin D1.In addition,it promoted the proliferation and the“wound healing”of colonic cells.However,these effects were reversed when silencing STAT3 on YAPoverexpressed FHC cells.Moreover,protein immunoprecipitation indicated that YAP could directly interact with STAT3 in the nucleus,up-regulatvng the expressvon of STAT3.Finally,during the process of CAC,overexpression of YAP mutant caused the downregulated expression of STAT3 and inhibited the development and progress of CAC.Conclusion:YAP activates STAT3 signaling in regulation of epithelial cell proliferation and promotes mucosal regeneration after DSS induced colitis,which may serve as a potential therapeutic target in UC.However,persistent and excessive YAP activation may promote CAC development.