Xu Yan-Jie, Zhao Jie-Min, Gao Cao, Ni Xue-Feng, Wang Wei, Hu Wen-Wei, Wu Chang-Ping
Department of Oncology, The Third Affiliated Hospital of Soochow University, Changzhou 213000, Jiangsu Province, PR China.
Department of Anesthesiology, The Third Affiliated Hospital of Soochow University, Changzhou 213000, Jiangsu Province, PR China.
Cell Signal. 2021 Oct;86:110095. doi: 10.1016/j.cellsig.2021.110095. Epub 2021 Jul 25.
In the rankings of cancer mortality and incidence worldwide, colorectal cancer ranks fourth and the third, respectively. Circular RNA hsa_circ_0136666 (hsa_circ_0136666) is reported to participate in the growth of colorectal cancer. However, the mechanism by which hsa_circ_0136666 regulates the tumorigenesis of colorectal cancer needs to be further explored. In this study, we report here the role of hsa_circ_0136666 in the aberrant activation of Treg cells and immune evasion of tumor cells, providing a new strategy for the treatment of colorectal cancer.
Western blotting assay and qRT-PCR assay were used to determine protein and mRNA expression levels. Dual-luciferase reporter assay was used to evaluate the targeted regulatory relationship. RNA immunoprecipitation was used to detect RNA binding. Colony formation assay was utilized to measure the cell proliferation. Flow cytometry was used to assess cell apoptosis. Xenograft model was setup to evaluate tumor growth.
The results showed that hsa_circ_0136666 and PD-L1 was increased in colorectal cancer cells while miR-497 was decreased in colorectal cancer cells when compared with normal colon epithelial cell line. Hsa_circ_0136666 was demonstrated to directly target miR-497, which also regulated PD-L1 by binding to its 3'UTR. Further mechanistic studies identified that hsa_circ_0136666 controlled cell proliferation and apoptosis via targeting miR-497 and regulating PD-L1 expression. Of note, hsa_circ_0136666 stimulated Treg cells mediated by miR-497/PD-L1 axis and its downstream signal pathway in Treg cells. Finally, hsa_circ_0136666 was found to accelerate the tumor growth in vivo.
Our findings demonstrated that hsa_circ_0136666 promoted the expression of PD-L1 by inhibiting miR-497 level in colorectal cancer, thus inducing the activation of Treg cells and leading to the immune escape of tumor, providing a novel mechanistic insight into the pathogenesis of colorectal cancer.
在全球癌症死亡率和发病率排名中，结直肠癌分别位列第四和第三。据报道，环状RNA hsa_circ_0136666（hsa_circ_0136666）参与结直肠癌的生长。然而，hsa_circ_0136666调节结直肠癌肿瘤发生的机制尚需进一步探索。在本研究中，我们报告了hsa_circ_0136666在调节性T细胞（Treg细胞）异常激活和肿瘤细胞免疫逃逸中的作用，为结直肠癌的治疗提供了新策略。
采用蛋白质免疫印迹法和qRT-PCR法测定蛋白质和mRNA表达水平。采用双荧光素酶报告基因检测法评估靶向调控关系。采用RNA免疫沉淀法检测RNA结合情况。采用集落形成试验检测细胞增殖。采用流式细胞术评估细胞凋亡。建立异种移植模型评估肿瘤生长。
结果显示，与正常结肠上皮细胞系相比，结直肠癌细胞中hsa_circ_0136666和程序性死亡受体配体1（PD-L1）表达增加，而微小RNA-497（miR-497）表达降低。研究表明，hsa_circ_0136666直接靶向miR-497，miR-497也通过结合其3'非翻译区（3'UTR）来调控PD-L1。进一步的机制研究表明，hsa_circ_0136666通过靶向miR-497并调节PD-L1表达来控制细胞增殖和凋亡。值得注意的是，hsa_circ_0136666通过miR-497/PD-L1轴及其在Treg细胞中的下游信号通路刺激Treg细胞。最后，发现hsa_circ_0136666在体内加速肿瘤生长。
我们的研究结果表明，hsa_circ_0136666通过抑制结直肠癌中miR-497水平来促进PD-L1表达，从而诱导Treg细胞激活并导致肿瘤免疫逃逸，为结直肠癌发病机制提供了新的机制性见解。
