Fig 2: lncRNA RP11-732M18.3 promotes the expression and secretion of VEGFA. (A, B) The expression of VEGFA was detected by western blot (WB) in indicated cells. lncRNA-RP11-732M18.3 increased the expression of VEGFA. All experiments were performed in triplicate (n = 5, *p < 0.05). (C) WB analysis of VEGFA in the concentrated supernatant of indicated cells. An equal volume (45 µL of concentrated supernatants from 5 mL cell culture supernatant) of concentrated supernatant were loaded. All experiments were performed in triplicate (n = 5, *p < 0.05). (D) Supernatant levels of VEGFA were estimated using enzyme-linked immunosorbent assay (ELISA). Upper panel: standard curve; Lower panel: statistical results. All experiments were performed in triplicate (n = 5, *p < 0.05). (E) Activation of the VEGFA downstream signaling pathway was detected using WB in ECs after 48 h of co-culture with lncRNA-RP11-732M18.3 knockdown cells. Knockdown of lncRNA-RP11-732M18.3 decreased phosphorylated VEGFR2 (Tyr1175), Src (Tyr416), and Akt (Ser473). All experiments were performed in triplicate (n = 5, *p < 0.05). (F) Activation of the VEGFA downstream signaling pathway was detected by WB in ECs after 48 h of co-culturing with lncRNA-RP11-732M18.3 overexpressing cells and lncRNA-RP11-732M18.3 knockdown cells. Overexpression of lncRNA-RP11-732M18.3 increased phosphorylated VEGFR2 (Tyr1175), Src (Tyr416), and Akt (Ser473). All experiments were performed in triplicate (n = 5, *p < 0.05).

Fig 3: lncRNA-RP11-732M18.3 increases VEGFA transcription through the 14-3-3β/and EP300 pathways. (A) EP300 mediates lncRNA-RP11-732M18.3 regulation of VEGFA transcription as detected by RT-qPCR in indicated cells. All experiments were performed in triplicate (n = 3, *p < 0.05). (B) EP300 mediates the regulation of VEGFA by lncRNA-RP11-732M18.3 as detected by WB in indicated cells. All experiments were performed in triplicate (n = 3, *p < 0.05). (C) The expression of EP300 in cytosolic and nuclear fractions was detected by WB in indicated cells. Lamin-B and β-actin were used as controls for cytosolic and nuclear fractions, respectively. lncRNA-RP11-732M18.3 promotes the nuclear translocate of EP300. All experiments were performed in triplicate (n = 3, *p < 0.05). (D) Immunofluorescence of EP300 in indicated treatment cells. (E) Quantification of immunofluorescence results. lncRNA-RP11-732M18.3 promotes the expression and nucleus translocation of EP300. All experiments were performed in triplicate (n = 3, *p < 0.05). (F) 14-3-3β/α mediates the regulation of EP300 by lncRNA-RP11-732M18.3 as detected by WB in indicated cells. All experiments were performed in triplicate (n = 3, *p < 0.05). #p < 0.05.

Fig 4: VEGFA as a key regulator of angiogenesis is an important prognostic factor for glioma patients. (A) The expression distribution of the VEGFA gene in glioma tissues using the Chinese Glioma Genome Atlas (CGGA) data (325 glioma samples). The horizontal axis represents different groups of samples, the vertical axis represents the gene expression distribution, different colors represent different groups, and the upper left corner represents the significance p-value test method. (B) Kaplan-Meier survival analysis on VEGFA expression in the CGGA data (222 glioma samples). (C) Kaplan-Meier survival analysis of the VEGFA gene signature. (D) Time-dependent receiver operating characteristic analysis of the VEGFA gene signature for predicting 2-year, 3-year, and 5-year survival. (E, F) Hazard ratio and p‐value of constituents involved in univariate and multivariate Cox regression and parameters of the VEGFA gene using TCGA data (663 glioma tumors). (G) Nomogram to predict the 1-year overall survival of glioma patients.

Fig 5: The correlation between DMDRMR and its ceRNA axis in ccRCC.A, B Representative images (A) and scores (B) of miR-378a-5p expression from ccRCC and adjacent tissues by ISH assays (scale bars, 50 µm). C, D Representative images (C) and scores (D) of DAB2IP expression from ccRCC and adjacent tissues by IHC assays (scale bars, 50 µm). E–G Spearman correlation analysis between miR-378a-5p and DMDRMR expression levels (E), DAB2IP and DMDRMR expression levels (F), DAB2IP and miR-378a-5p expression levels (G). H ROC curve analysis for the indicated parameters in ccRCC compared to adjacent tissues using the expression levels of DMDRMR, miR-378a-5p, DAB2IP, and combined their expression levels. I–L The qRT-PCR analysis of miR-378a-5p (I), EZH2 (J), SMURF1 (K), and VEGFA (L) expression levels in 48 paired ccRCC and adjacent tissues. Delta cycle threshold (ΔCt). M The correlation analysis of DMDRMR, miR-378a-5p, EZH2, SMURF1 and VEGFA expression levels. N Kaplan–Meier survival analysis was compared among the different combination of DMDRMR and DAB2IP expression. O Proposed model for the DMDRMR/miR-378a-5p/DAB2IP axis promoting the angiogenesis of ccRCC. The results are presented as mean ± SD. *p < 0.05 and ***p < 0.001.