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.
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。