miR-485-3p通过靶向TLR1／NF-κB信号通路调节胃癌细胞放射敏感性

miR-485-3p regulates radiosensitivity of gastric cancer cells by targeting TLR1/NF-κB signaling pathway

目的：研究miR-485-3p是否通过靶向TLR1对胃癌细胞放射敏感性起作用。方法分别用qRT-PCR和蛋白印迹法检测miR-485-3p和TLR1的表达变化，DIANA、TargetScan和miRanda软件预测和双荧光素酶报告实验验证 miR-485-3p 对 TLR1的靶向作用。将 miR-485-3p mimic 或TLR1 siRNA转染到胃癌MGC803细胞中，放射处理细胞，凋亡实验、克隆形成实验和MTT检测细胞放射敏感性的变化。双荧光素酶报告实验检测miR-485-3p上调和TLR1沉默对NF-κB活性的影响。蛋白免疫印迹实验探究miR-485-3p上调和TLR1沉默对NF-κB靶基因的影响。结果放射处理后胃癌细胞miR-485-3p表达下调，TLR1表达上调。靶基因预测软件发现TLR1可能是miR-485-3p的靶基因，双荧光素酶报告实验进一步验证了TLR1是miR-485-3p的直接靶点。 miR-485-3p负调控TLR1的表达。 miR-485-3p的过表达提高了细胞凋亡率，降低了细胞克隆形成和细胞群体增殖能力，增强了细胞的放射敏感性，且TLR1的沉默也具有相同作用。 miR-485-3p上调和TLR1沉默均降低了NF-κB的活性，下调了NF-κB多个靶基因的表达。结论 miR-485-3p可能通过靶向TLR1调控NF-κB信号通路增强胃癌细胞的放射敏感性。

Objective To investigate whether miR-485-3p plays a role in regulation of radiosensitivity of gastric cancer cells by targeting TLR1. Methods Quantitative real-time PCR and Western blot were used to determine the expression of miR-485-3p and TLR1, respectively. The interaction between miR-485-3p and TLR1 was verified by target prediction software （ DIANA, TargetScan, and miRanda） and dual luciferase reporter assay. Gastric cancer MGC803 cells transfected with miR-485-3p mimic or TLR1 siRNA were exposed to irradiation. Apoptosis assay, colony formation assay, and MTT assay were used to evaluate the changes in radiosensitivity of gastric cancer cells. Dual luciferase reporter assay was used to determine the effects of miR-485-3p overexpression and TLR1 silencing on the activity of NF-κB. Western blot was used to study the effects of miR-485-3p overexpression and TLR1 silencing on NF-κB target genes. Results In gastric cancer cells exposed to radiation, the expression of miR-485-3p was downregulated and the expression of TLR1 was upregulated. TLR1 was predicted to be the target of miR-485-3p by target prediction software. Dual luciferase reporter assay further confirmed TLR1 as the direct target of miR-485-3p. miR-485-3p negatively regulated the expression of TLR1. The overexpression of miR-485-3p, as well as TLR1 silencing, increased the apoptosis rate of cells, reduced colony formation and cell proliferation, and enhanced the radiosensitivity of the cells. Both miR-485-3p overexpression and TLR1 silencing reduced the activity of NF-κB and downregulated the expression of multiple NF-κB target genes. Conclusions miR-485-3p enhances the radiosensitivity of gastric cancer cells probably by targeting TLR1 and regulating the NF-κB signaling pathway.