基于ERK1/2通路研究CCL21对HT29细胞紧密连接蛋白损伤的作用及机制

Effect of CCL21 on Tight Junction Protein Injury in HT29 Cells Based on ERK1/2 Pathway and Its Mechanism

本研究旨在探讨趋化因子配体21(C-C chemokine ligand 21,CCL21)对肠上皮屏障紧密连接蛋白的影响及其机制。在GEO数据库中使用115例溃疡性结肠炎(ulcerative colitis,UC)样本表达谱数据筛选验证CCL21在UC中的差异表达,构建蛋白质-蛋白质相互作用(protein-protein interaction,PPI)网络;体外培养人结肠癌细胞HT29,将其分为对照组、CCL21组、CCL21+ERK1/2抑制剂组(CCL21+U0126组)、CCL21+ERK1/2激活剂组(CCL21+C6 Ceramide组);使用Western blot检测各组细胞紧密连接蛋白ZO-1、occluding、claudin-1及ERK1/2、p-ERK1/2的蛋白表达;用实时定量PCR(real time quantitative PCR,RT-qPCR)检测ZO-1、occluding和claudin-1的mRNA表达;用透射电镜观察对照组与CCL21组紧密连接结构。结果发现,CCL21在UC中显著高表达,且与多个分子存在相互作用关系;CCL21可导致ZO-1、occluding和claudin-1的蛋白表达量及mRNA表达量下降(P<0.05),同时可上调HT29细胞ERK1/2通路的蛋白磷酸化水平(P<0.05);CCL21可导致HT29细胞紧密连接结构间隙增宽,连续性中断;使用ERK1/2抑制剂U0126后可阻断CCL21导致的ZO-1、occluding和claudin-1的蛋白表达量及其mRNA表达量的下调(P<0.05),而使用ERK1/2激活剂C6 Ceramide则进一步下调ZO-1、occluding和claudin-1的蛋白表达量及mRNA表达量(P<0.05)。以上结果表明,CCL21与UC发病密切相关,ERK1/2通路参与CCL21下调HT29细胞紧密连接蛋白ZO-1、occluding和claudin-1的表达。

The aim of this study was to investigate the effect of C-C Chemokine ligand 21(CCL21)on intestinal epithelial barrier tight junction protein and its mechanism.The expression profile data of 115 cases of ulcerative colitis(UC)colon tissue samples were screened in GEO database to verify the differential expression of CCL21 in UC and the protein-protein interaction(PPI)network was constructed.Human colon cancer cells HT29 were culturedin vitro and divided into a control group,CCL21 group,CCL21+ERK1/2 pathway inhibitor group(CCL21+U0126 group)and CCL21+ERK1/2 pathway activator group(CCL21+C6 Ceramide group).Western blot was used to detect the protein expression of ZO-1,occluding,claudin-1,ERK1/2 and p-ERK1/2.Real time quantitative PCR(RT-qPCR)was used to detect the mRNA expression of ZO-1,occluding and claudin-1.The tight junction structures of the control group and CCL21 group were observed by transmission electron microscopy.The results showed that CCL21 was significantly overexpressed in UC by bioinformatics analysis,and there was an interaction relationship between CCL21 and several molecules.CCL21 induced the decrease of the protein expression level and mRNA expression level of ZO-1,occluding and claudin-1(P<0.05),and upregulated the phosphorylation level of ERK1/2 pathway in HT29 cells(P<0.05).CCL21 destroyed the tight junction structure of HT29 cells,widened the gap and interrupted the continuity.U0126,an ERK1/2 inhibitor,blocked the down-regulation of the protein expression and mRNA expression of ZO-1,occluding and claudin-1 induced by CCL21(P<0.05).The protein and mRNA expression levels of ZO-1,occluding and claudin-1 were further down-regulated after using C6 Ceramide,an ERK1/2 activator(P<0.05).These results indicate that CCL21 is closely related to the pathogenesis of UC,and the ERK1/2 pathway is involved in CCL21 down-regulating the expression of tight junction proteins ZO-1,occluding and claudin-1 in HT29 cells.