m^(6)A修饰的circ_100367通过调控miR-885-5p轴介导甲状腺乳头状癌免疫逃逸

m^(6)A-modified circ_100367 mediates immune escape from papillary thyroid carcinoma by regulating the miR-885-5p axis

目的:探讨m^(6)A修饰的circ_100367对甲状腺乳头状癌(papillary thyroid carcinoma,PTC)免疫逃逸的影响,并阐述其可能的机制。方法:采用qRT-PCR法检测PTC患者癌组织及其癌细胞株中circ_100367和miR-885-5p的表达水平。将circ_100367干扰质粒(si-circ_100367)及其阴性对照干扰质粒(si-NC)、circ_100367过表达质粒(oe-circ_100367)及其阴性对照质粒(oe-NC)和miR-885-5p mimics及其阴性对照NC mimics分别转染至TPC1细胞中,分为Blank组、si-circ_100367组、si-NC组、oe-circ_100367组、oe-NC组、miR-885-5p mimics组和NC mimics组。采用qRT-PCR法检测各组TPC1细胞中circ_100367和miR-885-5p的表达水平;采用双荧光素酶报告系统验证circ_100367可靶向调控miR-885-5p;MeRIP-qPCR法检测circ_100367的m^(6)A甲基化水平;Transwell法检测细胞侵袭能力;细胞划痕实验检测细胞迁移能力;Western blot检测细胞FASL、IDO1、PD-L2和PD-L1表达水平。结果:circ_100367在PTC患者癌组织及其癌细胞株K1、TPC1和IHH4中表达水平明显升高(P<0.05),而miR-885-5p表达水平明显降低(P<0.05)。双荧光素酶报告系统结果说明:circ_100367靶向负调控miR-885-5p。MeRIP试验结果显示:circ_100367含有丰富的m^(6)A甲基化,且与NC mimics比较,miR-885-5p mimics细胞中circ_100367的m^(6)A甲基化水平明显降低(P<0.05)。此外,与Blank或si-NC比较,si-circ_100367细胞侵袭数量、细胞划痕愈合率和FASL、PD-L1、PD-L2、IDO1蛋白表达水平明显降低(P<0.05);与Blank或oe-NC比较,oe-circ_100367细胞侵袭数量、细胞划痕愈合率和FASL、PD-L1、PD-L2、IDO1蛋白表达水平明显升高(P<0.05);与Blank或NC mimics比较,miR-885-5p mimics细胞侵袭数量、细胞划痕愈合率和FASL、PD-L1、PD-L2、IDO1蛋白表达水平明显降低(P<0.05)。结论:m^(6)A甲基化修饰的circ_100367能诱导PTC细胞侵袭,影响PD-L1的表达进而促进免疫逃逸,其可能与m^(6)A甲基化修饰的circ_100367和miR-885-5p竞争性结合有关。

Objective:To investigate the effect of m^(6)A-modified circ_100367 on immune escape of papillary thyroid carcinoma(PTC)and its possible mechanism.Methods:Expression levels of circ_100367 and miR-885-5p in PTC patients'cancer tissues and their cancer cell lines were detected by qRT-PCR assay.si-circ_100367 interference plasmid(si-circ_100367)and its negative control interference plasmid(si-NC),circ_100367 overexpression plasmid(oe-circ_100367)and its negative control plasmid(oe-NC),and miR-885-5p mimics and negative control NC mimics were transfected into TPC1 cells and divided into Blank group,si-circ_100367 group,si-NC group,oe-circ_100367 group,oe-NC group,miR-885-5p mimics group and NC mimics group.The expression levels of circ_100367 and miR-885-5p in TPC1 cells of each group was detected by qRT-PCR assay.The dual-luciferase reporter system was used to verify the targeted regulation of miR-885-5p by circ_100367.The m^(6)A methylation level of circ_100367 was detected by MeRIP-qPCR.The invasive ability of cells was detected by Transwell method.The cell migration ability was detected by wounding healing assay.The expression levels of FASL,IDO1,PD-L2 and PD-L1 were detected by Western blot.Results:The expression level of circ_100367 was significantly increased in the cancer tissures of PTC patients and its cancer cell lines K1,TPC1 and IHH4(P<0.05),while the expression level of miR-885-5p was significantly decreased(P<0.05).The results of the dual-luciferase reporter system showed that circ_100367 targeted and negatively regulated miR-885-5p.The results of MeRIP assay showed that circ_100367 was rich in m^(6)A methylation,and compared with NC mimics,the m^(6)A methylation level of circ_100367 in miR-885-5p mimics cells was significantly lower(P<0.05).Compared with Blank or si-NC,si-circ_100367 cell invasion number,cell wound healing rate and FASL,PD-L1,PD-L2,IDO1 protein expression levels were significantly decreased(P<0.05).Compared with Blank or oe-NC,oe-circ_100367 cell invasion number,cell wound healing rate and protein expression levels of FASL,PD-L1,PD-L2,IDO1 were significantly increased(P<0.05).Compared with Blank or NC mimics,the invasion number,cell wound healing rate of miR-885-5p mimics and expression levels of FASL,PD-L1,PD-L2,IDO1 proteins were significantly decreased(P<0.05).Conclusion:The m^(6)A methylated circ_100367 can induce PTC cell invasion,affect the expression of PD-L1 and promote immune escape,which may be related to the competitive binding of m^(6)A methylated circ_100367 and miR-885-5p.