TRIM27 interacts with Iκbα to promote the growth of human renal cancer cells through regulating the NF-κB pathway

Abstract

Background: The tripartite motif (TRIM) family proteins exhibit oncogenic roles in various cancers. The roles of TRIM27, a member of the TRIM super family, in renal cell carcinoma (RCC) remained unexplored. In the current study, we aimed to investigate the clinical impact and roles of TRIM27 in the development of RCC.

Methods: The mRNA levels of TRIM27 and Kaplan-Meier survival of RCC were analyzed from The Cancer Genome Atlas database. Real-time PCR and Western blotting were used to measure the mRNA and protein levels of TRIM27 both in vivo and in vitro. siRNA and TRIM27 were exogenously overexpressed in RCC cell lines to manipulate TRIM27 expression.

Results: We discovered that TRIM27 was elevated in RCC patients, and the expression of TRIM27 was closely correlated with poor prognosis. The loss of function and gain of function results illustrated that TRIM27 promotes cell proliferation and inhibits apoptosis in RCC cell lines. Furthermore, TRIM27 expression was positively associated with NF-κB expression in patients with RCC. Blocking the activity of NF-κB attenuated the TRIM27-mediated enhancement of proliferation and inhibition of apoptosis. TRIM27 directly interacted with Iκbα, an inhibitor of NF-κB, to promote its ubiquitination, and the inhibitory effects of TRIM27 on Iκbα led to NF-κB activation.

Conclusions: Our results suggest that TRIM27 exhibits an oncogenic role in RCC by regulating NF-κB signaling. TRIM27 serves as a specific prognostic indicator for RCC, and strategies targeting the suppression of TRIM27 function may shed light on future therapeutic approaches.

Keywords: Apoptosis; Iκbα; NF-κB; Proliferation; Renal cell carcinoma; TRIM27.

Fig. 1

TRIM27 is upregulated and correlated with poor prognosis in human renal cancer. A. The mRNA level of TRIM27 was upregulated in renal cancer. Data were collected from the TCGA database. ***p < 0.001 vs pare-non-cancer tissues B. Prognosis of RCC (KIRC) patients with high or low expression of TRIM27 derived from the TCGA database. C. The relative mRNA level of TRIM27 was upregulated in human renal cancer tissues. n = 20, ***p < 0.001 vs pare-non-cancer tissues. D–F. KEGG, REACTOME, and DUTTA analysis indicated that cell apoptosis was positively correlated with elevated TRIM27 expression in RCC from the database GSE53757. NES: Normalized enrichment score

Fig. 2

TRIM27 silencing suppressed the proliferation and promoted apoptosis of human renal cells. A & B. TRIM27 siRNAs deeply suppressed the relative mRNA and protein content of TRIM27 in human Caki-2 and 786–0 cells, respectively. ***p < 0.001 vs siNC. The mRNA levels of TRIM27 was used 2^−ΔΔCt method and normalized to that of GAPDH. C & D. siTRIM27–1 and siTRIM27–2 significantly suppressed the proliferation of Caki-2 and 786–0 cells. *p < 0.05 vs siNC, ***p < 0.001 vs siNC. E. Apoptosis of Caki-2 and 786–0 cells was significantly increased following transfecting with siTRIM27–1 and siTRIM27–2 at 48 h, respectively. ***p < 0.001 vs siNC. F & G. Western blot was used to examine the protein contents of TRIM27, cleaved caspase-3, NF-κB (cytoplasm), NF-κB (nuclear) and IkBa in Caki-2 cells (F) and 786–0 cells (G) transfected with siNC, siTRIM27–1, or siTRIM27–2 at 48 h respectively. *p < 0.05 vs siNC, **p < 0.01 vs siNC, ***p < 0.001 vs siNC

Fig. 3

oeTRIM27 rescues the function of TRIM27 in siTRIM27-transfected cells. A and B. The proliferations of Caki-2 and 786–0 cells transfected with siTRIM27, oeTRIM27, or siTRIM27 + oeTRIM27 at 0, 12, 24, and 48 h respectively. *p < 0.05 vs Control, ***p < 0.001 vs Control. C and D. Flow cytometric evaluation of the apoptosis of Caki-2 and 786–0 cells transfected with siTRIM27, oeTRIM27, or siTRIM27 + oeTRIM27 at 48 h respectively, ***p < 0.001 vs Control. D and E. Western blot was used to examine the relative proteins of TRIM27, cleaved caspase-3, NF-κB (cytoplasm), and NF-κB (nuclear) in Caki-2 and 786–0 cells transfected with siTRIM27, oeTRIM27, or siTRIM27 + oeTRIM27 at 48 h respectively. ***p < 0.001 vs Control

Fig. 4

TRIM27 silencing reduced the tumorigenicity of human Caki-2 cells in vivo. A & B. Knockdown of TRIM27 in Caki-2 cells deeply suppressed the tumor volume and weight in vivo. **p < 0.01 vs siNC, ***p < 0.001 vs siTRIM27. C. TUNEL staining assays were used to determine tumor cell apoptosis. D. TUNEL quantification of siNC and siTRIM27 tumors, ***p < 0.001 vs siNC

Fig. 5

TRIM27 overexpression promoted the tumorigenicity of human Caki-1 cells in vivo. A & B. Overexpression of TRIM27 in Caki-2 cells significantly increased the tumor volume and weight in vivo. *p < 0.05 vs oeNC, ***p < 0.001 vs oeNC. C. TUNEL staining assays were used to determine tumor cell apoptosis. D. TUNEL quantification of oeNC and oeTRIM27 tumors, ***p < 0.001 vs oeNC

Fig. 6

The NF-κB inhibitor PDTC suppressed the function of oeTRIM27 in Caki-1 cells. A & B. oeTRIM27 significantly upregulated the relative mRNA and protein levels of TRIM27 in Caki-1 cells. ***p < 0.001 vs oeNC. The mRNA levels of TRIM27 was used 2^−ΔΔCt method and normalized to that of GAPDH. C. The proliferation of oeTRIM27 transfected cells was deeply downregulated in the presence of the inhibitor PDTC. *p < 0.05 vs oeNC + DMSO, ***p < 0.001 vs oeNC + DMSO;!p < 0.05 vs oeTRIM27 + DMSO,!!p < 0.01 vs oeTRIM27 + DMSO,!!!p < 0.001 vs oeTRIM27 + DMSO. D. The inhibitor PDTC significantly reduced the apoptosis of oeTRIM27 transfected cells. *p < 0.05 vs oeNC + DMSO, ***p < 0.001 vs oeNC + DMSO;!!!p < 0.001 vs oeTRIM27 + DMSO. E. The nuclear translocation of NF-κB and IkBa in oeNC or oeTRIM27 transfected cells were deeply suppressed by the inhibitor PDTC. ***p < 0.001 vs oeNC + DMSO;!!!p < 0.001 vs oeTRIM27 + DMSO. F. Correlation analysis between TRIM27 and NF-κB in human renal cancer tissues (n = 20)

Fig. 7

TRIM27 interacted with Iκbα and was positively associated with its ubiquitination in human Caki-2 cells. A. Western blot was used to examine the protein content of TRIM27 and Iκbα in oeNC or oeTRIM27 with or without the MG132 treatment. ***p < 0.001 vs oeNC + DMSO;!!!p < 0.001 vs oeTRIM27 + DMSO. B. TRIM27 interacted with Iκbα in human Caki-2 cells. C. TRIM27 silencing suppressed the ubiquitination of Iκbα in human Caki-2 cells