Fig 2: LINC01094 binds to SPI1 to regulate CCL7 expression. (a) Correlation of LINC01094 with CCL7 predicted in the TCGA-LUAD database; (b) binding relationship between LINC01094 and SPI1 validated in TCGA-LUAD database; binding between LINC01094 and SPI1 examined by RIP (c) and biotin-LINC01094-based RNA pull down (d) assays; (e) subcellular localization of LINC01094 and SPI1 in H125 and A549 cells examined by FISH assay; mRNA (f) and protein (g) levels of CCL7 in A549 and H125 cells after LINC01094 knockdown examined by qPCR and WB analyses. Repetition = 3. Data are presented as the mean ± SD. **p < 0.01.

Fig 3: SPI1 promotes CCL7 transcription. (a) Transcription factors that can bind to CCL7 promoter with an over 0.5 correlation coefficient in TCGA-LUAD analyzed using the R TFBS package; (b) binding sites between SPI1 and CCL7 promoter; (c) conservative binding sequence of SPI1; (d) a sketch map for the fragmentation of CCL7 promoter sites using the Crispr-Cas9 system; mRNA (e) and protein (f) levels of CCL7 in A549 cells after knockout of A, B, and C fragments alone or simultaneously; (g) CCL7 promoter fragments enriched by anti-SPI1 examined by the ChIP-qPCR assay; (h) construction of the pGL4-Luc luciferase vector containing the CCL7 promoter sequence for the examination of SPI1-CCL7 binding by luciferase assay. Repetition = 3. Data are presented as the mean ± SD. **p < 0.01.

Fig 4: CCL7 is highly expressed in LUAD and linked to increased TAM infiltration. (a) CCL7 expression in LUAD and normal lung tissues in the Oncomine database; (b) CCL7 expression in TCGA-LUAD database of tumor tissues and in GTex database of normal lung tissues; (c) correlation between CCL7 expression and patient's prognosis; (d) correlation of CCL7 expression with the immune cell infiltration in LUAD patients; (e) correlations of CCL7 expression with the M2 macrophage markers ARG1 and CD206.

Fig 5: CCL7 knockdown suppresses EMT and mobility of LUAD cells. mRNA (a) and protein (b) levels of CCL7 in H1299, A549, H358 (low-invasive), and H125 (high-invasive) cells detected by qPCR and WB analyses; mRNA (c) and protein (d) levels of CCL7 in A549 and H125 cells after overexpression plasmid or shRNA transfection determined by qPCR and WB analyses; mRNA (e) and protein (f) levels of epithelial marker ZO-1 and mesenchymal markers Twist1, Snai1, and Slug in A549 and H125 cells examined by qPCR and WB analyses; (g) 24-h migration rate of A549 and H125 cells analyzed by the scratch test; (h) invasiveness of A549 and H125 cells analyzed by Transwell assay. Repetition = 3. **p < 0.01.