Circle_0056992 promotes the proliferation,invasion and metastasis of lung adenocarcinoma by targeting microRNA-136-5p/forkhead box protein N2 signal axis
Objective To investigate the role of circular RNA(circRNA)_0056992 in the devel-opment of lung adenocarcinoma by regulating the microRNA(miR)-136-5p/forkhead box protein 2 signa-ling axis.Methods Plasma from LUAD patients(21 cases)treated in Tangshan People's Hospital from 2021 to 2022 and plasma from healthy individuals(21 cases)were collected,and the expression of circ_0056992 was detected in LUAD plasma and cells by real-time quantitative polymerase chain reaction assay.Ribonuclease R and actinomycin D assays were performed to verify the cyclic structure of circ_0056992.Fluorescence in situ hybridization assay and nucleoplasmic separation assay were performed to verify the cellular localization of circ_0056992,miR-136-5p and FOXN2.LUAD cells were divided into sh-NC group and shR-circ_0056992 group,pSilencer-NC+ASO-NC group,pSilencer-NC+ASO-136-5p group,shR_circ_0056992+ASO-NC group and shR_circ_0056992+ASO-136-5p group.Cell proliferation,inva-sion and migration were detected using cell counting reagent,clone formation,5-ethynyl-2'-deoxyuridine nucleoside,Transwel and scratch assays.miRanda website predicted the binding sites of circ_0056992 with miR-136-5p and miR-136-5p with FOXN2.Dual luciferase assays were performed to validate the targeting relationship between circ_0056992 and miR-136-5p and between miR-136-5p and FOXN2.One-way ANOVA was used for multiple group comparisons,and independent samples t-test was used for comparisons between two groups.Results Circ_0056992 was lowly expressed in LUAD plasma(1.048±0.108 vs.0.363±0.266,t=10.950,P＜0.05)and cells(0.193±0.019 vs.1.001±0.063,F=21.220,P＜0.05).circ_0056992 was cyclic,and circ_0056992 and miR-136-5p co-localized in the cytoplasm,and FOXN2 was more abundant in the cytoplasm.Cell counting kit-8(CCK-8),clonogenic and EdU results showed that the cell proliferative capacity of the shR-circ_0056992 group was higher than that of the NC group(1.708±0.102 vs.1.000±0.014;1.600±0.177 vs.1.000±0.046;1.476±0.050 vs.1.000±0.051,t=11.910,5.679,11.590,all P＜0.05).Scratch results showed that the cell migration ability of shR-circ_0056992 group was higher than that of NC group after 24 h and 48 h(0.485±0.009 vs.0.636±0.046;0.254±0.024 vs.0.442±0.016,t=6.933,5.553,all P＜0.05).Transwell results showed that the cell invasion ability of shR-circ_0056992 group was higher than that of NC group(907.700±67.020 vs.565.700±67.010,t=11.160,P＜0.05).miRanda website prediction showed that there was a binding site between circ_0056992 and miR-136-5p.CCK-8,clone formation and EdU results showed that the cell proliferation ability of shR_circ_0056992+ASO-NC group was higher than that of pSilencer-NC+ASO-NC group and shR_circ_0056992+ASO-136-5p group(1.267±0.017 vs.0.761±0.004,0.719±0.006;1.466±0.212 vs.1.000±0.039,0.800±0.057;1.497±0.042 vs.1.000±0.073,0.833±0.053,F=2 605.000,21.050,108.100,all P＜0.05),and that in the pSilencer-NC+ASO-136-5p group was lower than in the pSilencer-NC+ASO-NC group and the shR_circ_0056992+ASO-136-5p group(0.458±0.004 vs.0.761±0.004,0.719±0.006;0.170±0.023 vs.1.000±0.039,0.800±0.057;0.418±0.064 vs.1.000±0.073,0.833±0.053,F=4 123.000,319.900,65.820,all P＜0.05).Transwell results showed that the cell invasion ability of shR_circ_0056992+ASO-NC group was higher than pSilencer-NC+ASO-NC group and shR_circ_0056992+ASO-136-5p group(1.574±0.090 vs.1.000±0.030,0.841±0.046,F=119.800,all P＜0.05),and that in the pSilencer-NC+ASO-136-5p group was lower than pSilencer-NC+ASO-NC group and shR_circ_0056992+ASO-136-5p group(0.598±0.134 vs.1.000±0.030,0.841±0.046,F=17.630,P＜0.05).The scratch results showed that the migration residual distance of cells in the shR_circ_0056992+ASO-NC group was shorter than that in the pSilencer-NC+ASO-NC group and the shR_circ_0056992+ASO-136-5p group after 24 h and 48 h(0.540±0.017 vs.0.707±0.012,0.735±0.026;0.360±0.036 vs.0.509±0.053,0.564±0.042,F=92.710,17.150,all P＜0.05).The migration residual distance of cells in pSilencer-NC+ASO-136-5p group was longer than pSilencer-NC+ASO-NC group and shR_circ_0056992+ASO-136-5p group(0.886±0.040 vs.0.707±0.012,0.735±0.026;0.714±0.030 vs.0.509±0.053,0.564±0.042,F=35.280,18.640,P＜0.05).miRanda website prediction showed that miR-136-5p had a binding site with FOXN2.Conclusion Circ_0056992 can promote lung adenocar-cinoma proliferation,invasion,and metastasis by regulating the miR-136-5p/FOXN2 signaling axis.
Circular RNAMicroRNAForkhead box protein 2Lung adenocarcinoma
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NSTL
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