异甘草素通过hsa_circ_0027107/miR-651-5p/FOXN2轴抑制肺腺癌的恶性进展

Isoglycyrrhizin Inhibits Malignant Progression of Lung Adenocarcinoma via hsa_circ_0027107/miR-651-5p/FOXN2 Axis

目的探讨circ_0027107在肺腺癌发生、发展中的作用及分子机制,同时探究异甘草素(Isoliquiritigenin,ISL)通过hsa_circ_0027107对肺腺癌的影响。方法肺腺癌基因芯片筛选出显著高表达的hsa_circ_0027107,通过实时荧光定量(Quantitative Real-time PCR,qRT-PCR)检测肺腺癌患者及健康人血浆中,癌细胞和正常细胞中hsa_circ_0027107的表达情况,荧光原位杂检测hsa_circ_0027107在肺腺癌及癌旁组织中的表达情况,体外培养肺腺癌细胞A549和H1299,采用CCK-8和克隆形成检测细胞增殖能力,Transwell实验检测细胞迁移和侵袭能力,划痕实验检测细胞迁移能力,评估hsa_circ_0027107在肺腺癌中的功能;通过miRanda、TargetScan数据库预测circ_0027107下游的miR-651-5p,与miRDB、miRTarBase、TargetScan数据库预测miR-651-5p下游蛋白FOXN2,双荧光素酶基因实验报告确定circ_0027107与miR-651-5p、miR-651-5p与FOXN2之间的相互作用关系,并通过相关功能实验验证。结果肺腺癌基因芯片结果显示环状RNA hsa_circ_0027107差异表达明显,荧光原位杂交结果显示肺腺癌患者组织中hsa_circ_0027107的平均荧光强度为低于癌旁组织,miR-651-5p的平均荧光强度为高于癌旁组织,hsa_circ_0027107的低表达与肺腺癌患者的淋巴结转移和临床分期有关。核质分离结果显示hsa_circ_0027107、miR-651-5p都主要在细胞质中表达。过表达hsa_circ_0027107抑制肺腺癌细胞的增殖、迁移和侵袭能力,而过表达miR-651-5p可逆转circ_0027107对肺腺癌细胞增殖、迁移、侵袭的影响,同时,发现在NCL-H1299、A549中FOXN2表达降低,敲低FOXN2可逆转miR-651-5p对肺腺癌细胞增殖、迁移、侵袭的影响。研究发现ISL能够明显促进Hsa_circ_0027107的表达,并明显抑制体外肺癌细胞的增殖、迁移和侵袭能力,同时,功能实验也证实敲低Hsa_circ_0027107的表达能够拯救ISL对肺癌细胞的增殖、迁移和侵袭能力的抑制。结论肺腺癌组织、细胞、血浆中hsa_circ_0027107低表达,hsa_circ_0027107通过调控miR-651-5p作用于FOXN2抑制肿瘤的恶性生物学表型,ISL通过hsa_circ_0027107抑制肺腺癌的恶性进展。

Objective To explore the functionality and molecular mechanism of circ_0027107 in lung adenocarcinogenesis and the effect of isoliquiritigenin on lung adenocarcinoma via hsa_circ_0027107.Methods Lung adenocarcinoma gene chip was screened for significantly high expression of hsa_circ_0027107,and the expression of hsa_circ_0027107 in plasma of lung adenocarcinoma patients and healthy people,cancer cells and normal cells was detected by Quantitative Real-time PCR(qRT-PCR),and fluorescence in situ heterogeneity was detected for the expression of hsa_circ_0027107 in lung adenocarcinoma and paracancer tissues.Lung adenocarcinoma cells A549 and H1299 were cultured,and the function of hsa_circ_0027107 in lung adenocarcinoma was assessed by using CCK-8 and clone formation to detect cell proliferation ability.Transwell assay was used to detect cell migration and invasive ability,and scratch assay to detect cell migration ability.The function of hsa_circ_0027107 in lung adenocarcinoma was predicted.Through the miRanda and TargetScan database,miR-651-5p in downstream of circ_0027107 was predicted,and miRDB,miRTarBase and TargetScan database to predict miR-651-5p downstream protein FOXN2,dual luciferase gene assay report to determine the interaction between circ_0027107 and miR-651-5p,miR-651-5p and FOXN2 interactions and verified by relevant functional experiments.Results The results of lung adenocarcinoma gene microarray showed that the cyclic RNA hsa_circ_0027107 was significantly differentially expressed,and the results of fluorescence in situ hybridisation showed that the average fluorescence intensity of hsa_circ_0027107 in the tissues of lung adenocarcinoma patients was lower than that of the cancerous tissues,and that of miR-651-5p was higher than that of the cancerous tissues,and that low expression of hsa_circ_0027107 was associated with the low expression of miR-651-5p and FOXN2.0027107 was associated with lymph node metastasis and clinical staging in lung adenocarcinoma patients.Nucleoplasmic separation showed that both hsa_circ_0027107 and miR-651-5p were mainly expressed in the cytoplasm.Overexpression of hsa_circ_0027107 inhibited the proliferation,migration and invasion ability of lung adenocarcinoma cells,whereas overexpression of miR-651-5p reversed the effects of circ_0027107 on proliferation,migration and invasion of lung adenocarcinoma cells.Meanwhile,it is found that FOXN2 expression was reduced in NCL-H1299 and A549 and knockdown of FOXN2 reversed the effect of miR-651-5p on proliferation,migration and invasion of lung adenocarcinoma cells.In the study,it is found that isoliquiritigenin could significantly promote the expression of hsa_circ_0027107 and significantly inhibit the proliferation,migration and invasion ability of lung adenocarcinoma cells in vitro,meanwhile,the functional experiments also confirmed that knocking down the expression of hsa_circ_0027107 could rescue isoliquiritigenin inhibiting proliferation,migration and invasion of lung adenocarcinoma cells.Conclusion Lung adenocarcinoma tissues,cells and plasma have low expression of hsa_circ_0027107,which inhibits the malignant biological phenotype of tumour by regulating miR-651-5p acting on FOXN2,it may become a potential diagnostic and therapeutic marker for lung adenocarcinoma.Isoglycyrrhizin inhibits malignant progression of lung adenocarcinoma via hsa_circ_0027107.