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Abstract：
BACKGROUND: Colorectal cancer (CRC) is ranked as the third most commonly diagnosed cancer and the third cause of cancer related deaths. CRC is greatly attributed to genetic and epigenetic mutations and immune dysregulation. Tumor aberrant expression of Toll-like Receptors (TLRs) can contribute to tumorigenesis. Recent studies suggested that microRNAs act as direct ligands of TLRs altering their expression and signaling pathways.
OBJECTIVE: To prove our concept that specific miRNA mimics may act as antagonists of their specific toll like receptors inhibiting their expression that could limit the release of pro-inflammatory and pro-tumorigenic cytokines leading to apoptosis of tumor cells.
METHODS: From public microarray databases, we retrieved TLRs and miRNAs related to CRC followed by in silico docking of the selected miRNA ligands into the TLRs. Clinical validation after co-immunoprecipitation of TLRs and their interacting miRNA ligands was done. Expression of TLRs 1, 7,8 was determined by ELISA while miRNAs was measured by RT-qPCR. In addition, microRNA mimics of the down regulated miRNAs were transfected into human CRC cell lines.
RESULTS: Our data demonstrate that TLRs 1, 7, 8 are up regulated in CRC compared to controls. Further, three miRNAs (-122, -29b and -15b) are relatively downregulated, while 4 miRNAs (-202, miRNA-98, -21 and -let7i) are upregulated in CRC patients compared to those with benign tumor and healthy controls. Transfection of down regulated miRNA mimics into CRC cell lines resulted in a significant reduction of the number and viability of cells as well as down regulating the expression of TLRs 1, 7 and 8 with ultimate reduction of downstream effector IL6 protein, suggesting that these miRNAs are negative regulators of carcinogenesis.
CONCLUSIONS: MicroRNAs could act as antagonistic ligands of TLRs limiting the inflammatory tumor microenvironment.
OBJECTIVE: To prove our concept that specific miRNA mimics may act as antagonists of their specific toll like receptors inhibiting their expression that could limit the release of pro-inflammatory and pro-tumorigenic cytokines leading to apoptosis of tumor cells.
METHODS: From public microarray databases, we retrieved TLRs and miRNAs related to CRC followed by in silico docking of the selected miRNA ligands into the TLRs. Clinical validation after co-immunoprecipitation of TLRs and their interacting miRNA ligands was done. Expression of TLRs 1, 7,8 was determined by ELISA while miRNAs was measured by RT-qPCR. In addition, microRNA mimics of the down regulated miRNAs were transfected into human CRC cell lines.
RESULTS: Our data demonstrate that TLRs 1, 7, 8 are up regulated in CRC compared to controls. Further, three miRNAs (-122, -29b and -15b) are relatively downregulated, while 4 miRNAs (-202, miRNA-98, -21 and -let7i) are upregulated in CRC patients compared to those with benign tumor and healthy controls. Transfection of down regulated miRNA mimics into CRC cell lines resulted in a significant reduction of the number and viability of cells as well as down regulating the expression of TLRs 1, 7 and 8 with ultimate reduction of downstream effector IL6 protein, suggesting that these miRNAs are negative regulators of carcinogenesis.
CONCLUSIONS: MicroRNAs could act as antagonistic ligands of TLRs limiting the inflammatory tumor microenvironment.
摘要：
背景：结直肠癌（CRC）被列为第三大最常诊断的癌症，也是癌症相关死亡的第三大原因。CRC主要归因于遗传和表观遗传突变以及免疫失调。Toll样受体（TLR）的肿瘤异常表达可能导致肿瘤发生。最近的研究表明，microRNAs作为TLRs的直接配体，改变其表达和信号通路。
目的：为了证明我们的观点，即特定的miRNA模拟物可以作为其特定的toll样受体的拮抗剂，抑制其表达，从而限制促炎和促瘤细胞因子的释放，导致肿瘤细胞凋亡。
方法：来自公共微阵列数据库，我们检索了与CRC相关的TLRs和miRNAs,然后将所选的miRNA配体进行了计算机模拟对接。在TLR及其相互作用的miRNA配体的共免疫沉淀后进行临床验证。通过ELISA测定TLR1、7、8的表达,而通过RT-qPCR测定miRNA。此外,将下调的miRNA的微RNA模拟物转染到人CRC细胞系中。
结果：我们的数据表明，与对照组相比，TLR1、7、8在CRC中上调。Further,三个miRNA（-122，-29b和-15b）相对下调，而4种miRNA（-202，miRNA-98，-21和-let7i）在CRC患者中与良性肿瘤和健康对照相比上调。将下调的miRNA模拟物转染到CRC细胞系中导致细胞数量和活力的显着减少，并下调TLRs1、7和8的表达，最终减少下游效应物IL6蛋白。提示这些miRNA是致癌作用的负调节因子。
结论：MicroRNAs可以作为TLRs的拮抗配体限制炎性肿瘤微环境。
目的：为了证明我们的观点，即特定的miRNA模拟物可以作为其特定的toll样受体的拮抗剂，抑制其表达，从而限制促炎和促瘤细胞因子的释放，导致肿瘤细胞凋亡。
方法：来自公共微阵列数据库，我们检索了与CRC相关的TLRs和miRNAs,然后将所选的miRNA配体进行了计算机模拟对接。在TLR及其相互作用的miRNA配体的共免疫沉淀后进行临床验证。通过ELISA测定TLR1、7、8的表达,而通过RT-qPCR测定miRNA。此外,将下调的miRNA的微RNA模拟物转染到人CRC细胞系中。
结果：我们的数据表明，与对照组相比，TLR1、7、8在CRC中上调。Further,三个miRNA（-122，-29b和-15b）相对下调，而4种miRNA（-202，miRNA-98，-21和-let7i）在CRC患者中与良性肿瘤和健康对照相比上调。将下调的miRNA模拟物转染到CRC细胞系中导致细胞数量和活力的显着减少，并下调TLRs1、7和8的表达，最终减少下游效应物IL6蛋白。提示这些miRNA是致癌作用的负调节因子。
结论：MicroRNAs可以作为TLRs的拮抗配体限制炎性肿瘤微环境。
