Meng Shuang, Li Yingjie, Zang Xiaozhen, Jiang Zheng, Ning Huahan, Li Jing
1Department of Gastroenterology, The First Affiliated Hospital of Jinzhou Medical University, No. 2, Section 5 Renmin Street, Guta District, Jinzhou City, 121001 Liaoning Province China.
2Jinzhou Medical University, No. 40, Section 3, Songpo Road, Linghe District, Jinzhou City, 121001 Liaoning Province China.
Cancer Cell Int. 2020 Mar 30;20:95. doi: 10.1186/s12935-020-01184-0. eCollection 2020.
Colitis-associated cancer (CAC) is a complication of inflammatory bowel disease (IBD) with a poor prognosis because it is often diagnosed in advanced stages with local progression or metastasis. Compared with the more common polyp-induced sporadic colorectal cancer (sCRC), CAC has different molecular mechanisms. Toll-like receptor 2 (TLR2) expression is not limited to cells related to inflammation and immune function. High levels of TLR2 expression in tumor tissues of colorectal cancer (CRC) patients have been reported. This report is to investigate the effects of knockout and knockdown of the TLR2 gene on the proliferation of CAC and sCRC.
Twelve C57BL/6 J wild-type mice (WT) and 12 TLR2 knockout mice (TLR2-/-) were used to rapidly establish a colitis-associated cancer (CAC) model via the 1,2-dimethylhydrazine-dextran sodium sulfate (DMH-DSS) method and were divided into the normal WT control group (NC), TLR2 knockout control group (KC), normal wild-type tumor modeling group (NT), and TLR2 knockout tumor modeling group (KT), with 6 mice in each group. The general performance of the mice during modeling, the gross changes of the colon and the rectum, and the pathological score of HE staining were used to observe tumor growth. The expression of TLR2 was detected by immunohistochemistry, and tumor proliferation was detected by Ki67 labeling. Lentivirus carrying TLR2-RNAi was used to stably infect colorectal cancer cells (HCT116 and HT29) to knock down TLR2 gene expression. The experimental groups included the uninfected control group, negative control group, and gene knockdown group. After infection, the expression of TLR2 protein was detected by Western blot, and cell proliferation and the cell cycle were detected by the CCK-8 method and fluorescence-activated cell sorting. Western blot was used to detect the expression levels of p- NF-κβ, cyclin D1 and cyclin D3 protein in each group of cells.
TLR2 knockout in the CAC model resulted in greater changes in body weight and more severe diarrhea and colorectal hemorrhage. However, knocking out the TLR2 gene reduced the shortening of colorectal length, the number of tumors, and the total tumor volume and inhibited the growth of CAC. Knocking out the TLR2 gene also reduced the pathological score and tumor severity. TLR2 was localized in the cell membrane of the colorectal epithelium of the NC group and of the colorectal tumors of the NT group and was highly expressed in the NT group, while antigen Ki67 was localized in the nucleus of the colorectal tumor cells of the NT group and the KT group, and its expression was reduced in the KT group. In an in vitro sporadic colorectal cancer cell experiment, TLR2 protein in the TLR2 knockdown group was significantly downregulated, and TLR2 knockdown significantly inhibited the proliferation of HCT116 and HT29 colorectal cancer cells, resulting in G1 phase arrest. The expression levels of p-NF-κβ, cyclin D1 and cyclin D3 proteins in TLR2 gene knockdown group cells were significantly reduced.
Knockout and knockdown of TLR2 can inhibit the proliferation of inflammation-related colorectal cancer and sporadic colorectal cancer.
结肠炎相关癌（CAC）是炎症性肠病（IBD）的一种并发症，预后较差，因为它常于疾病晚期伴局部进展或转移时才被诊断出来。与更常见的息肉诱导的散发性结直肠癌（sCRC）相比，CAC具有不同的分子机制。Toll样受体2（TLR2）的表达并不局限于与炎症和免疫功能相关的细胞。已有报道称结直肠癌（CRC）患者肿瘤组织中TLR2表达水平较高。本报告旨在研究TLR2基因敲除和敲低对CAC和sCRC增殖的影响。
采用12只C57BL/6 J野生型小鼠（WT）和12只TLR2基因敲除小鼠（TLR2-/-），通过1,2-二甲基肼-葡聚糖硫酸钠（DMH-DSS）法快速建立结肠炎相关癌（CAC）模型，并分为正常WT对照组（NC）、TLR2基因敲除对照组（KC）、正常野生型肿瘤建模组（NT）和TLR2基因敲除肿瘤建模组（KT），每组6只小鼠。通过建模过程中小鼠的一般表现、结肠和直肠的大体变化以及HE染色的病理评分来观察肿瘤生长情况。采用免疫组化法检测TLR2的表达，采用Ki67标记法检测肿瘤增殖情况。使用携带TLR2-RNAi的慢病毒稳定感染结肠癌细胞（HCT116和HT29）以敲低TLR2基因表达。实验组包括未感染对照组、阴性对照组和基因敲低组。感染后，采用蛋白质免疫印迹法检测TLR2蛋白的表达，采用CCK-8法和荧光激活细胞分选法检测细胞增殖和细胞周期。采用蛋白质免疫印迹法检测每组细胞中p-NF-κβ、细胞周期蛋白D1和细胞周期蛋白D3蛋白的表达水平。
在CAC模型中敲除TLR2导致体重变化更大、腹泻和结直肠出血更严重。然而，敲除TLR2基因可减少结直肠长度缩短、肿瘤数量和肿瘤总体积，并抑制CAC的生长。敲除TLR2基因还可降低病理评分和肿瘤严重程度。TLR2定位于NC组结直肠上皮细胞膜以及NT组结直肠肿瘤细胞膜，且在NT组中高表达，而抗原Ki67定位于NT组和KT组结直肠肿瘤细胞核，其在KT组中的表达降低。在体外散发性结肠癌细胞实验中，TLR2基因敲低组中TLR2蛋白显著下调，TLR2基因敲低显著抑制HCT116和HT29结肠癌细胞的增殖，导致细胞停滞于G1期。TLR2基因敲低组细胞中p-NF-κβ、细胞周期蛋白D1和细胞周期蛋白D3蛋白的表达水平显著降低。
TLR2基因的敲除和敲低均可抑制炎症相关结直肠癌和散发性结直肠癌的增殖。
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