DOK1 facilitates the advancement of ccRCC

Abstract

Background: Renal cell carcinoma (RCC) is one of the most common human cancers. Clear cell renal cell carcinoma (ccRCC) is a major subtype of RCC. However, the molecular mechanisms underlying ccRCC oncogenesis require further investigation. Docking protein 1 (DOK1) is a putative tumor suppressor gene; however, its role in ccRCC remains unclear. Methods: Bioinformatic analysis was used to illustrate the poor prognosis associated with DOK1 expression and its role in tumor development in ccRCC in patients. qPCR (quantitative polymerase chain reaction) and western blotting assays were used to validate DOK1 expression in ccRCC cells. In vitro experiments were performed to further elucidate the biological role of DOK1 in ccRCC. Results: DOK1 was overexpressed in ccRCC tissues and cells at both mRNA and protein levels. High DOK1 expression closely correlated with poor survival in patients with ccRCC. DOK1 expression significantly accelerated ccRCC proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Through PI3K (phosphatidylin-ositol-3-kinase)/AKT (protein kinase B)/GSK3β (glycogen synthase kinase 3 beta) signaling, DOK1 may control the progression of ccRCC. Conclusion: DOK1 has the potential to serve as a valuable biomarker and target for treatment in ccRCC through its regulation of PI3K/AKT/GSK3β signaling to promote ccRCC progression.

Keywords: DOK1; KIRC; PI3K-AKT; ccRCC; epithelial-mesenchymal transition.

Figure 1

DOK1 overexpression predicts poor ccRCC prognosis. (A and B) DOK1 expression detected using the TCGA database. (C and D) DOK1 expression in cancerous and paracancerous tissues using unpaired (C) and paired tests (D) found using the GSE53757 dataset. (E) Upregulated DOK1 predicts poor survival as shown by results from the TCGA database. (F and G) Visualization of DOK1 using IHC in ccRCC (G) and healthy tissues (F), the results for kidney tissues obtained using the HPA online database. (H) Univariate and multivariate Cox analyses for DOK1 in ccRCC. (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 2

Determination of DOK1 expression in ccRCC cell lines and DOK1 knockdown efficiency. (A and B) qRT-PCR (A) and western blotting (B) were used to determine mRNA and protein levels of DOK1 in ccRCC cell lines. (C and D) qRT-PCR results showing knockdown efficiency of DOK1 in OSRC2 (C) and 786-O (D) cells. (E and F) western blotting showing siRNA II knockdown efficiency in OSRC2. (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 3

DOK1 accelerates ccRCC cell proliferation and metastasis. (A and B) CCK-8 assays were used to determine 786-O (A) and OSRC2 (B) cells proliferation. (C and D) EdU assays were used to further verify 786-O (C) and OSRC2 (D) cell proliferation (×200). (E and F) Wound healing assays were used to show 786-O (E) and OSRC2 (F) cell migration. (G and H) Transwell assays showing 786-O (G) and OSRC2 (H) cell migration and invasion (×100). Bar = 100 μm. (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 4

DOK1 promotes EMT by regulating PI3K-AKT-GSK3β signaling in ccRCC. (A and B) KEGG analysis (A) and hallmark of GSEA (B) of upregulated DEGs. (C and D) Western blotting results showing PI3K, p-PI3K, AKT, p-AKT, GSK3β, and p-GSK3β protein levels. (E and F) Western blotting results showing EMT markers. (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5

DOK1 may target the PI3K/AKT/GSK3β pathway to regulate ccRCC EMT, and methylation of DOK1 revealed poor prognosis. (A and B) DOK1 showed high methylation in ccRCC tissues. (C) DOK1 may be a promising candidate molecule regulating EMT in ccRCC via regulation of the PI3K/AKT/GSK3β signaling pathway.