PMC

TRIB3 expression in RCC cell lines and verification of transfection efficiency. A/B: TRIB3 was expressed at low levels in HK-2 cells and at high levels in multiple RCC cell lines. C/D: Transfection efficiency of 786-O and Caki-1 cells transfected with a TRIB3 overexpression lentivirus. E/F: Transfection efficiency of 769-P and A498 cells transfected with a TRIB3 knockdown lentivirus. “ ** ” represents P < 0.01.

In vivo study. A/B: The xenograft tumor volume was significantly higher in the TRIB3 overexpression group than in the negative control group (P < 0.001). C/D: The mRNA and protein expression levels of TRIB3 were higher in the TRIB3 overexpression group than in the control group.

Regulation of HIF-1α on TRIB3 expression. A/B: TRIB3 and HIF-1α expression are gradually increased in response to hypoxia and appear to be positively correlated. No obvious correlation was observed between HIF-2α and TRIB3 expression. C/D: Overexpression of HIF-1α in Caik-1 and HK-2 cells upregulates TRIB3 expression. E/F: HIF-1α directly binds to multiple regions of the TRIB3 promoter. Luciferase reporter assays revealed that excising HIF-1α binding sites in the TRIB3 promoter significantly attenuates the expression of TRIB3 (P < 0.01). “ * ” represents P < 0.05, “ ** ” represents P < 0.01, “ *** ” represents P < 0.001.

The molecular mechanisms by which TRIB3 promotes RCC cell proliferation and invasion. A: RNA-sequencing in Caki-1 cells to compare differential gene expression at the transcriptional level between the TRIB3 overexpression and control groups. B/C: Bioinformatics analysis revealed that the MAPK pathway was one of the most affected signaling pathways. D/E: The results of RNA-sequencing analysis were further verified by western blot.

TRIB3 expression in RCC tissues and its correlation with clinicopathological features. A/B/E: Immunochemical staining indicated that TRIB3 expression was higher in RCC tissues than in normal paracancerous tissues. TRIB3 expression was localized in nucleus. C/D: HIF‑1α and HIF‑2α are highly expressed in RCC tissues. F-H: TRIB3 is highly expressed in fresh RCC tissues at both the mRNA and protein level. J: Progression-free survival was better in the TRIB3 low/no expression group than in the high expression group, although this difference was not statistically significant (P = 0.493).

RCC cell proliferation, migration and invasion assays. A: CCK-8 assays show that TRIB3 overexpression significantly promotes the proliferation of 786-O (P = 0.0188) and Caki-1 cells (P = 0.009). B: A decreased proliferation rate was detected in TRIB3 knockdown cells compared with that in nontargeting control cells (P < 0.05). C/E: Wound healing assays and Matrigel-free transwell assays indicate that overexpression of TRIB3 promotes the migration of 786-O and Caki-1 cells (P < 0.05). D/F: Downregulation of TRIB3 also inhibits the migration of 769-P and A498 cells (P < 0.01). G: In Matrigel-coated transwell assays, overexpression of TRIB3 markedly increases the invasive ability of 786-O and Caki-1 cells (P < 0.01). H: TRIB3 knockdown decreases the invasive potential of 769-P and A498 cells (P < 0.05). “ * ” represents P < 0.05, “ ** ” represents P < 0.01, “ *** ” represents P < 0.001.