Fig 2: SLC4A4 expression is downregulated in ccRCC and predicts a poor prognosis. SLC4A4 mRNA levels in 72 normal tissues and 533 ccRCC tissues were downloaded from the dataset of TCGA-KIRC. (A) SLC4A4 mRNA levels were lower in cancer tissues than in para-cancer tissues. (B) SLC4A4 levels in three additional ccRCC datasets. (C–H) SLC4A4 levels were compared in ccRCC patients according to the following clinicopathological parameters: (C) distant metastasis, (G) T stage, (E) TNM stage, (F) grade, (G) OS and (H) DFS. Data are shown as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001; ## p < 0.01; ### p < 0.001.

Fig 3: SLC4A4 overexpression attenuates the effects of miR-223-3p mimics on cellular processes. (A) MiR-223-3p and (B) SLC4A4 levels were measured by qRT-PCR in 786-O and Caki-1 cells. Overexpression of SLC4A4 impaired the effects of miR-223-3p mimics on (C) KRAS expression, (D) cell proliferation, (E) migration and (F) invasion in Caki-1 cells. Data are shown as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig 4: SLC4A4 is a direct target of miR-223-3p. (A) Western blotting and (B) qRT-PCR analysis of SLC4A4 expression in 786-O and Caki-1 cells transfected with miR-223-3p mimics versus the corresponding NC. (C) Western blotting and (D) qRT-PCR analysis of SLC4A4 expression in 786-O and Caki-1 cells transfected with miR-223-3p inhibitors versus the corresponding NC. (E) The predicted binding sites for miR-223-3p in the SLC4A4 3′-UTR. The red nucleotides are the seed-pairing target sites of miR-223-3p. (F) Luciferase reporter assays demonstrate that the reporter activity of 786-O and Caki-1 cells decreased by approximately 50% upon co-transfection of the wild-type SLC4A4 3′-UTR reporter construct and miR-223-3p mimics. Data are shown as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig 5: SLC4A4 overexpression inhibits cell proliferation, migration and invasion in vitro. SLC4A4 was downregulated in ccRCC tissues, as shown by (A) qRT-PCR and (B) immunohistochemistry analysis. (C) SLC4A4 was downregulated in ccRCC tissues, as shown by immunohistochemistry analysis from the Human Protein Atlas. (D) Gene set enrichment analysis comparing low and high SLC4A4 expression groups in the database of TCGA. Enrichment curves are shown for activated gene sets related to the epithelial-mesenchymal transition and KRAS signaling. (E) and (F) SLC4A4 mRNA and protein levels were successfully upregulated in 786-O and Caki-1 cells, and SLC4A4 overexpression inhibited KRAS expression. (G–I) SLC4A4 overexpression inhibited (G) proliferation, (H) migration and (I) invasion in Caki-1 cells. Data are shown as the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001.