SETD7 Promotes Cell Proliferation and Migration via Methylation-mediated TAF7 in Clear Cell Renal Cell Carcinoma

Abstract

SET domain containing 7(SETD7), a member of histone methyltransferases, is abnormally expressed in multiple tumor types. However, the biological function and underlying molecular mechanism of SETD7 in clear cell renal cell carcinoma (ccRCC) remain unclear. Here, we explored the biological effects of SETD7-TAF7-CCNA2 axis on proliferation and metastasis in ccRCC. We identified both SETD7 and TAF7 were up-regulated and significantly promoted the proliferation and migration of ccRCC cells. Concurrently, there was a significant positive correlation between the expression of SETD7 and TAF7, and the two were colocalized in the nucleus. Mechanistically, SETD7 methylates TAF7 at K5 and K300 sites, resulting in the deubiquitination and stabilization of TAF7. Furthermore, re-expression of TAF7 could partially restore SETD7 knockdown inhibited ccRCC cells proliferation and migration. In addition, TAF7 transcriptionally activated to drive the expression of cyclin A2 (CCNA2). And more importantly, the methylation of TAF7 at K5 and K300 sites exhibited higher transcriptional activity of CCNA2, which promotes formation and progression of ccRCC. Our findings reveal a unique mechanism that SETD7 mediated TAF7 methylation in regulating transcriptional activation of CCNA2 in ccRCC progression and provide a basis for developing effective therapeutic strategies by targeting members of SETD7-TAF7-CCNA2 axis.

Keywords: SETD7; TAF7; clear cell renal cell carcinoma; lysine methylation; oncogene.

Figure 1

SETD7 is upregulated in ccRCC and silencing of SETD7 inhibits the biological function of ccRCC cells. A, The expression level of SETD7 in 31 Pan-cancer which analyzed by TCGA databases. B, Representative immunohistochemical images of SETD7 in human renal clear cell carcinoma tissues and adjacent normal tissues. C, The mRNA and protein expressions of SETD7 were detected in ccRCC cell lines (786-O, CAKI-1) and normal tubular epithelial cell line (HK-2), analyzed by qRT-PCR and western blotting. D, The mRNA expression of SETD7 were detected to confirm the knockdown efficiency of two siRNAs in 786-O and CAKI-1 cells, analyzed by qRT-PCR. E, MTT assay was used to detect the effect of silencing SETD7 on the proliferation of 786-0 and CAKI-1 cells. F, Colony formation assays was used to detect the effect of silencing SETD7 on the clonogenic ability of 786-0 and CAKI-1 cells. G, Flow cytometry was performed to detect the effects of silencing SETD7 on cell cycle progression in 786-O and CAKI-1 cells, and the percentages of G1, S, G2 phase of cell cycle were calculated. H, Flow cytometry was performed to detect the effects of silencing SETD7 on cell apoptosis in 786-O and CAKI-1 cells, and the percentage of cell apoptosis was calculated. I, The effects of silencing SETD7 on 786-O and CAKI-1 cells migration were determined by wound-healing assay and transwell assay (J). K, Expression changes of cell cycle, apoptosis and migration-related molecules by silencing SETD7 in 786-O and CAKI-1 cells were detected by western blotting. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2

Silencing of SETD7 inhibits tumor growth and metastasis in vivo. A, The mRNA and protein expression of SETD7 in LV-shSETD7 cells was detected by qRT-PCR and western blotting. B, Gross morphology of tumors injected with either LV-Control or LV-shSETD7 cells after 31 days (n = 4). C, Morphology of excised tumors from nude mice. D, Growth curves of tumor volume were generated every 4 d for 31 d. E, Weight statistics of excised tumors. F, Volume statistics of excised tumors. G, The mRNA and protein expression levels of SETD7 were analyzed by qRT-PCR and western blotting in removed tumors. H, Image analysis was performed on nude mice to assess tumor metastasis on the 21th day after injection. I, Organs were resected and images of metastasis were shown. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Binding and co-localization between SETD7 and TAF7. A, The interaction between SETD7 and TAF7 was analyzed by STRING database. B, The correlation between the expression of SETD7 and TAF7 in ccRCC was analyzed by GEPIA database. C, Representative images of protein docking analysis. SETD7 is shown in green, and TAF7 is shown in blue. Protein-protein interaction positions marked by yellow lines. D, Representative images of immunofluorescence staining of DAPI, SETD7 and TAF7 in 786-O and CAKI-1 cells showed co-localization between SETD7 and TAF7 in the nucleus. E, Representative images of immunofluorescence staining of DAPI, Flag-SETD7 and His-TAF7 in 786-O and CAKI-1 cells. Results indicated co-localization between Flag and His in the nucleus. F, Co-immunoprecipitation (Co-IP) assays were used to detect interaction of endogenous SETD7 and TAF7 in 786-O and CAKI-1 cells. G, HEK-293T cells were co-transfected with Flag-SETD7 and His-TAF7 expression vectors. Co-IP assays were performed to detect protein interactions between exogenous SETD7 and TAF7.

Figure 4

SETD7 methylates TAF7 at lysine K5 and K300. A, Sequence alignment of TAF7 domain containing the SETD7 recognized [K/R]-[A/S/T]-K-X motif from diverse species. TAF7 residues at K5 and K300 were denoted in the protein sequences. The positions of the K5 and K300 were highlighted in red, and nearby identifiable lysine sites were highlighted in blue. B, Pan-lysine methylation of TAF7 in HEK-293T cells was demonstrated using Co-IP. C, Co-IP showed the pan-lysine methylation status of TAF7 after overexpressing SETD7 in HEK-293T cells. D, HEK-293T cells were co-transfected with Flag-SETD7 and different His-TAF7 WT or K5R, K300R, 2KR mutant vectors, respectively. Cell lysates were immunoprecipitated with anti-His antibody, followed by immunoblotting analysis by anti-lysine (K) methyl antibody to detect pan-lysine methylation status of TAF7.

Figure 5

TAF7 K5 and K300 methylation promote the stabilization of TAF7. A, Western blotting analyses indicated protein levels of SETD7 and TAF7 in NC and SETD7 knockdown 786-O and CAKI-1 cells treated with CHX for the indicated time. B, 786-O and CAKI-1 cells were transfected with NC or SETD7 siRNA-1 and then treated with MG132 or DMSO for 10 h. Western blotting analyses were performed to assess protein levels of SETD7 and TAF7. C, HEK-293T cells were co-transfected with His-TAF7+NC or His-TAF7+siSETD7-1 and then treated with MG132 for 10 h. Cell lysates were immunoprecipitated by anti-His antibody, followed by immunoblotting analysis with anti-V5 antibody to examine ubiquitination status of TAF7. D, HEK-293T cells that transfected with TAF7-WT or TAF7-K5R, K300R, 2KR mutants were subjected to CHX treatment for the specified time. Levels of SETD7 and TAF7 proteins were detected by western blotting analysis. E, HEK-293T cells were co-transfected with TAF7-WT+NC, TAF7-MUT+NC, TAF7-WT+siSETD7-1 or TAF7-MUT+siSETD7-1 and then treated with MG132 for 10 h. Co-IP assay with anti-His antibody followed by immunoblotting using anti-V5 antibody to detect ubiquitination levels of TAF7.

Figure 6

Silencing of TAF7 inhibits the proliferation, migration and promoted apoptosis of ccRCC cells. A, TCGA databases analyzed the expression of TAF7 in 31 Pan-cancer. B, TAF7 protein expression in ccRCC tissues vs normal tissues was confirmed by using immunohistochemistry assays. C, The mRNA and protein expressions of TAF7 in 786-O, CAKI-1 and HK-2 cells were detected by qRT-PCR and western blotting. D, The mRNA expression level of TAF7 in 786-O and CAKI-1 cells after si-TAF7 transfection, analyzed by qRT-PCR. E, F, The effect of silencing TAF7 on ccRCC cell proliferation was detected by MTT assay and colony formation assay. G, H, Flow cytometry statistical analysis was performed to detect the effects of silencing TAF7 on cell cycle progression and cell apoptosis in ccRCC cells. I, J, The effect of silencing TAF7 on ccRCC cell migration was detected by wound-healing assay and transwell assay. K, Expression changes of cell cycle, apoptosis and migration-related molecules in ccRCC cells after silencing TAF7, analyzed by western blotting. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7

Overexpression of TAF7 partially reverses the inhibition of ccRCC cell biological function caused by silencing SETD7. A, B, MTT assays and colony formation assays were performed to determine the effect of TAF7-WT, TAF7-K5R, TAF7-K300R, or TAF7-2KR vectors treatment on cell viability in 786-O and CAKI-1 cells. C, D, Wound-healing assays and transwell analysis represented the migration capacity of ccRCC cells transfected with TAF7-WT, TAF7-K5R, TAF7-K300R or TAF7-2KR vectors, respectively. E, F, Cell activity levels of 786-O and CAKI-1 cells were examined by MTT and colony formation assays after co-transfection with NC+Ctrl, NC+ovTAF7, siSETD7+Ctrl, siSETD7+ovTAF7. G, H, The migration capacity was detected by wound-healing and transwell assays after co-transfected with NC+Ctrl, NC+ovTAF7, siSETD7+Ctrl, siSETD7+ovTAF7 in ccRCC cells. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 8

TAF7 methylation enhances CCNA2 transcriptional and oncogenic activity. A, Volcano plots for differentially expressed genes (DEGs) of ccRCC tumor versus normal kidney tissue samples. Red dots, significantly upregulated genes. Green dots, significantly downregulated genes. Grey dots, nondifferentially expressed genes. B, KEGG enrichment analysis for DEGs. C, GSEA snapshots of KEGG pathway enrichment analysis: Cell cycle. D, The expression of CCNA2 in ccRCC tissues and adjacent noncancerous kidney tissues in TCGA database. E, Scatterplot showed the correlation analysis between TAF7 and CCNA2 expression by Pearson's r. F, UCSC database analysis showed that TAF7 could bind to the promoter region of CCNA2 to regulate its transcription. G, Luciferase reporter assay were performed in HEK-293 cells to explore the effect on co-transfection with NC+pGL3-CCNA2-luc-WT, siSETD7+pGL3-CCNA2-luc-WT, NC+ pGL3-CCNA2-luc-MUT and siSETD7+pGL3-CCNA2-luc-MUT. Renilla luciferase served as the internal control. H, HEK-293 cells were co-transfected with NC+pGL3-CCNA2-luc-WT, siTAF7+pGL3-CCNA2-luc-WT, NC+pGL3-CCNA2-luc-MUT and siTAF7+pGL3-CCNA2-luc-MUT. Luciferase activity was determined at 48 h post-transfection. I, Luciferase assays were performed in HEK-293 cells after co-transfection with TAF7-WT or TAF7-MUT and pGL3-CCNA2-luc vector. Introduction of TAF7 mutants, including TAF7-K5R, TAF7-K300R and TAF7-2KR attenuated the transcriptional activation of the CCNA2 luciferase reporter compared with TAF7-WT. J, Luciferase activity in HEK-293 cells was measured 48 h after transfection with increasing amounts of SETD7. Introduction of SETD7 increased the transcriptional activation with CCNA2 in a dose-dependent manner. K, Proposed model for the regulation of ccRCC progression by the SETD7-TAF7-CCNA2 axis. SETD7 methylates TAF7 at K5 and K300, enhancing its binding to TAF7 and protecting TAF7 from ubiquitination degradation. The stabilization of TAF7 stimulates transcriptional expression activation of the target gene CCNA2, which consequently promotes the progression of ccRCC tumors.