Fig 2: Circ-IARS served as a ceRNA for miR-1252-5p to regulate HDGF in NSCLC cells. (a) Circinteractome website and StarBase website together presented 12 miRNAs that were complementary to circ-IARS. (b and c) RT-qPCR detected the relative expression levels of 8 miRNAs in H1299 and A549 cells transfected with si-circ-IARS_1 or si-NC. (d) Schematic diagram showing the predicted binding sites between WT-circ-IARS and miR-1252-5p sequences. (e) Dual-luciferase reporter assay measured the relative luciferase activity of WT-circ-IARS reporter vector and MUT-circ-IARS reporter vector in HEK293T cells transfected with miR-1252-5p mimic or NC mimic. (f and g) RIP assay evaluated the relative enrichment of circ-IARS and miR-1252-5p by AGO2 or IgG in H1299 and A549 cells, normalized to the input of cells. (h and i) RT-qPCR measured the relative miR-1252-5p expression level in (h) BEAS-2B, H1299, A549, and H460 cells, and (i) H1299 and A549 cells transfected with si-NC, si-circ-IARS_1, si-circ-IARS_1 + miR-1252-5p inhibitor, or si-circ-IARS_1 + NC inhibitor. (j) Schematic diagram showing the predicted binding sites between WT-HDGF 3′UTR and miR-1252-5p sequences. (k) Dual-luciferase reporter assay measured the relative luciferase activity of WT-HDGF 3′UTR reporter vector and MUT-HDGF 3′UTR reporter vector in HEK293T cells transfected with miR-1252-5p mimic or NC mimic. (l and m) RIP assay evaluated the relative enrichment of HDGF and miR-1252-5p by AGO2 or IgG in H1299 and A549 cells, normalized to the input of cells. (n) RT-qPCR measured the relative miR-1252-5p expression level in H1299 and A549 cells transfected with miR-1252-5p mimic, miR-1252-5p inhibitor, NC mimic, or NC inhibitor. (o–r) Western blotting detected the relative HDGF protein expression in (o and p) H1299 and A549 cells transfected with HDGF vector, pcDNA vector, miR-1252-5p mimic, NC mimic, miR-1252-5p mimic + HDGF vector, or miR-1252-5p mimic + pcDNA vector, and (q) BEAS-2B, H1299, A549, and H460 cells, as well as (r) H1299 and A549 cells transfected with si-NC, si-circ-IARS_1, si-circ-IARS_1 + miR-1252-5p inhibitor, or si-circ-IARS_1 + NC inhibitor. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 3: Circ-IARS was upregulated in NSCLC patients. (a–c) RT-qPCR detected the relative expression of circ-IARS, miR-1252-5p, and HDGF mRNA in NSCLC tumor tissues (NSCLC; N = 44), normalized to that in normal para-carcinoma tissues (Normal; N = 44). (d–f) Spearman’s rank correlation analysis determined the linear correlation among circ-IARS, miR-1252-5p, and HDGF mRNA expression in NSCLC tumors. (g) TEM showed the exosomes isolated from the serum of NSCLC patients. (h) Western blotting measured the expression of TSG101, CD9, and CD63 in isolated exosomes from NSCLC serum and NSCLC cell medium. (i) RT-qPCR detected the relative exo-circ-IARS expression in the serum of NSCLC patients (NSCLC; N = 22), normalized to serum from healthy people (Healthy; N = 22). ***P < 0.001.

Fig 4: Silencing circ-IARS retarded the tumor growth of NSCLC in vivo. (a) Tumor volume was monitored every 5 days after inoculation of sh-circ-IARS or sh-NC-transfected H1299 cells into nude mice (N = 5). (b) Tumor weight was examined at the end day of the xenograft experiment. (c and d) RT-qPCR detected the relative circ-IARS and miR-1252-5p expression, and (e) western blotting measured the relative HDGF protein expression in tumor tissues from nude mice. (f) IHC examined the HDGF and Ki67 expression in paraffin-embedded xenograft tumor tissues. ***P < 0.001.