Fig. 1

Identification of circIGF2BP3 as a candidate circRNA that inhibits CD8⁺ TIL infiltration and antitumor immunity in NSCLC. A Heatmap showing the top 75 most upregulated circRNAs and the 75 most downregulated circRNAs in NSCLC tissues (T) and paired nontumor tissues (N) analyzed in GSE126533. hsa_circ_0079587 (circIGF2BP3) is marked with a red box. B Flowchart of the steps utilized in the present study to identify and validate circRNAs in NSCLC. C Topological overlap matrix (TOM) and clustering diagram of circRNAs presenting similarity based on topological overlap combined with assigned module color. D The relationship between circRNA modules and clinical traits. Each cell represents the correlation coefficient (upper) and p-value (lower) between the corresponding module eigengene and clinical trait. The eigengene of the turquoise module marked in the blue box is negatively correlated with CD8⁺ TIL infiltration. E Heatmap showing 87 mRNAs that were both upregulated in NSCLC and negatively correlated with CD8⁺ TIL infiltration predicted by at least 4 algorithms. F The constructed ceRNA network of circRNA-miRNA-mRNA interactions. Nodes in red, dark blue and light blue represent circRNAs, miRNAs and mRNAs, respectively. G The relative expression of the four indicated circRNAs in different NSCLC cell lines detected by qRT-PCR. GAPDH was used as an internal control. H RNase R treatment and qRT-PCR analyses were performed to validate the corresponding circRNAs. I Sanger sequencing was conducted to identify the back-splicing site of circIGF2BP3. J. Diagram illustrating that circIGF2BP3 is generated from back-splicing between exons 4 and 13 of linear IGF2BP3. K Time-course qRT-PCR analyses of the relative abundance of circIGF2BP3 and linear IGF2BP3 in A549 cells treated with actinomycin D (10 μg/ml). L qPCR analysis of circIGF2BP3 and its linear form in cDNA and gDNA amplified by convergent and divergent primers, respectively. M qRT-PCR analysis of circIGF2BP3 abundance in the cytoplasmic and nuclear fractions of A549 cells. GAPDH and U1 were used as positive controls in the cytoplasm and nucleus, respectively. N FISH indicating that circIGF2BP3 was mainly located in the cytoplasm. Scale bars, 20 μm. Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. P values were determined by unpaired Student’s t test (H and K)