KMT2B up-regulates SNHG12 by regulating the H3K4me3 modification of the SNHG12 promoter
As shown in Figure 3A, there was a large amount of H3K4me3 enrichment in the SNHG12 promoter. It is reported that KMT2B mediates the transcriptional activation of H3K4me3 modification [26]. Moreover, through ENCORI database analysis, KMT2B was highly expressed in RCC (Figure 3B) and positively correlated with SNHG12 expression (Figure 3C). Therefore, KMT2B may regulate SNHG12 by mediating the H3K4me3 modification of its promoter region.
KMT2B mRNA (t = 32.217, P < 0.001) and protein expression (t = 26.836, p<0.001) increased in RCC tissues compared with adjacent tissues (Figure 3D). Compared with human normal kidney cell line HK-2, KMT2B (A498: q=8.968, p<0.001; 786-O: q=18.341, p<0.001; Caki-1: q=9.740, p<0.001; 769-P: q=4.657, p=0.049) and protein expression (A498: q=11.443, p<0.001; 786-O: q=17.322, p<0.001; Caki-1: q=8.446, p<0.001; 769-P: q=5.656, p=0.017) in RCC cells was increased (Figure 3E). Next, we knocked down KMT2B in 786-O cells and found reduced SNHG12 expression (t=11.509, p<0.001) (Figure 3F). ChIP showed that knocking down KMT2B reduced modification of SNHG12 promoter H3K4me3 (t=11.413, p<0.001) (Figure 3G).
SNHG12 recruits E2F1 to promote CEP55 expression
In RCC, SNHG12 was positively correlated with CEP55 expression (r=0.234, p<0.001) (Figure 4A), which is highly expressed in RCC and can promote cancer cell proliferation, invasion, and migration [24]. Analysis with LncMAP database showed that E2F1 was one of the transcription factors involved in regulation of SNHG12 on CEP55 in RCC (Table 3). Additionally, ENCORI database analysis showed that E2F1 was highly expressed in RCC (Figure 4B) and positively correlated with CEP55 (r=0.542, p<0.001) (Figure 4C). Therefore, SNHG12 may regulate CEP55 through E2F1.
Table 3
The transcription factors of SNHG12 regulating CEP55 in RCC predicted by LncMAP.
LncRNA Symbol | TF Symbol | Gene Symbol |
SNHG12 | E2F1 | CEP55 |
SNHG12 | PBX3 | CEP55 |
To further verify this hypothesis, we showed that SNHG12 was mainly located in the nucleus (Figure 4D). RIP results showed enriched SNHG12 when using anti-E2F1 antibody (t=11.889, p<0.001) (Figure 4E). Meanwhile, CEP55 promoter region was bound by anti-E2F1 antibody (t=9.566, p<0.001) (Figure 4F). Furthermore, overexpression of SNHG12 promoted binding of E2F1 with CEP55 promoter region, whereas SNHG12 inhibited this binding (oe-NC vs. oe-SNHG12: q=8.724, p=0.001; sh-NC vs. sh-SNHG12: q=11.861, p<0.001) (Figure 4G). Then, we predicted the binding site between E2F1 and CEP55 promoter region through Jaspar (Table 4). Dual luciferase reporter assay showed that the truncation or mutation of site 1 did not affect the luciferase activity of the oe-E2F1 group, while sites 2 and 3 were mutated, the luciferase activity increased (Figure 4H and 4I). The above results revealed that site 1 was the main site for E2F1 to act on the CEP55 promoter region.
Table 4
JASPAR analysis of the binding site of the E2F1 in the promoter region of CEP55.
Matrix ID | Name | Score | Relative score | Sequence ID | Start | End | Strand | Predicted sequence |
MA0024.1 | E2F1 | 7.17655 | 0.813289007993 | CEP55 | 292 | 299 | + | tttgcccc |
MA0024.1 | E2F1 | 7.17655 | 0.813289007993 | CEP55 | 1091 | 1098 | - | ttagccgc |
MA0024.1 | E2F1 | 6.81352 | 0.800797328565 | CEP55 | 836 | 843 | - | tttcccga |
Finally, in the 786-O cells, CEP55 expression was increased by SNHG12 overexpressing (q=20.319, p<0.001), but reduced by further silencing E2F1 (q=6.508, p=0.008). When overexpressing SNHG12 and silencing E2F1 at the same time, CEP55 expression was reduced (q=19.571, p<0.001) (Figure 4J).
SNHG12 recruits E2F1 to promote RCC cell proliferation, migration, and invasion and HUVEC angiogenesis
Compared with oe-SNHG12 + sh-NC, E2F1 (q=11.902, p<0.001) and CEP55 (q=9.059, p<0.001) expression were decreased by oe-SNHG12 + sh-E2F1. Compared with sh-E2F1 + oe-NC, E2F1 expression remained not changed by sh-E2F1 + oe-CEP55 treatment (q=0.592, p=0.974), but CEP55 expression was elevated (q=7.761, p=0.003) (Figure 5A).
In addition, compared with the oe-SNHG12 + sh-NC group, cell proliferation ability (24h: q=0.904, p=0.918; 48h: q=9.545, p<0.001; 72h: q=18.003, p<0.001), cell migration and invasion ability (q=11.362, p<0.001; q=9.652, p<0.001), and capillary-like tube formation ability (q=8.552, p<0.001) of the oe-SNHG12 + sh-E2F1 group were decreased, while compared with the sh-E2F1 + oe-NC group, cell proliferation ability (24h: q=1.193, p=0.833; 48h: q=6.435, p=0.001; 72h: q=9.255, p<0.001), cell migration and invasion ability (q=5.429, p=0.021; q=7.011, p=0.005), and capillary-like tube formation ability (q=7.126, p=0.003) of the sh-E2F1 + oe-CEP55 group were increased (Figure 5B-E). The protein expression of MMP-2 (q=11.187, p<0.001), MMP-9 (q=11.251, p<0.001), and VEGF (q=15.538, p<0.001) in the oe-SNHG12 + sh-E2F1 group decreased compared with the oe-SNHG12 + sh-NC group, while the trend was opposite in the sh-E2F1 + oe-CEP55 group compared with the sh-E2F1 + oe-NC group (MMP-2: q=6.789, p=0.006; MMP-9: q=8.884, p=0.001; VEGF: q=8.970, p=0.001) (Figure 5F).
Knockdown of SNHG12 inhibits RCC growth and angiogenesis in vivo
As shown in Figure 6A-6C, compared with the NC, SNHG12 silencing slowed down tumor growth (3w: q=13.128, 4w: q=22.341, 5w: q=31.637, all p<0.001) and reduced tumor weight (q=16.945, p<0.001), while further E2F1 overexpression boosted tumor growth (3w: q=19.778, 4w: q=28.276, 5w: q=38.723, all p<0.001) and increased tumor weight (q=15.807, p<0.001). Compared with silencing E2F1 alone, simultaneous silencing E2F1 and overexpressing CEP55 enhanced tumor growth (3w: q=19.956, 4w: q=24.591, 5w: q=38.692, all p<0.001) and weight (q=20.071, p<0.001) .
Compared with NC, silencing SNHG12 reduced E2F1 (q=29.522, p<0.001) and CEP55 (q=19.445, p<0.001) expression, while further E2F1 overexpression reversed this trend (E2F1: q=26.221, p<0.001; CEP55: q=20.545, p<0.001). Compared with silencing E2F1 alone, simultaneous silencing E2F1 and overexpressing CEP55 up-regulated CEP55 (q=27.005, p<0.001) (Figure 6D).
Immunohistochemical detection showed that compared with NC, silencing SNHG12 decreased VEGF protein expression (q=15.484, p<0.001), which was reversed by further E2F1 overexpression (q=17.701, p<0.001). Compared with silencing E2F1 alone, simultaneous silencing E2F1 and overexpressing CEP55 increased VEGF expression (q=22.056, p<0.001) (Figure 6E). Meanwhile, compared with NC, silencing SNHG12 reduced MMP-2 (q=24.073, p<0.001), MMP-9 (q=23.464, p<0.001), VEGF (q=22.801, p<0.001) protein expression, which was neutralized by further E2F1 up-regulation(MMP-2: q=13.062, p<0.001; MMP-9: q=14.071, p<0.001; VEGF: q=15.967, p<0.001). Compared with silencing E2F1 alone, their expression was promoted after simultaneous silencing E2F1 and overexpressing CEP55 (MMP-2: q=15.733, p<0.001; MMP-9: q=16.792, p<0.001; VEGF: q=14.292, p<0.001) (Figure 6F).