Fig. 9

RREB1 regulates the transcription of SNHG4 in PCa cells, and SNHG4 modulates RREB1 expression through a SNHG4/let-7a-5p/RREB1 positive feedback loop. a Venn diagram showing that there were five genes across two gene sets (predicted SNHG4 upstream TFs and potential let-7 targeted mRNAs). b The table shows the prediction score, p value, q-value and number of binding sites of the five predicted SNHG4 upstream TFs. c The canonical motif of RREB1. d Spearman’s correlation coefficient test showed the correlation between the expression of SNHG4 and RREB1 in PCa tumor samples (n = 499, R = 0.45, p < 0.001). Western blot (e) and qRT‒PCR analysis (f) showed that RREB1 expression was significantly knocked down by either pair of siRNAs against RREB1. g Construction of luciferase reporter plasmids containing the wild-type SNHG4 promoter or either of the three BS-mutated SNHG4 promoters is shown here. A dual-luciferase reporter assay showed that mutation of #3 BS significantly reduced the interactions between RREB1 and the SNHG4 promoter. h A dual-luciferase reporter assay was performed to assess the interactions between RREB1 and the SNHG4 promoter. si-RREB1 significantly inhibited the luciferase activity of luciferase plasmids containing the wild-type SNHG4 promoter but had no effect on the transcription of the #3 BS mutated SNHG4 promoter. i ChIP assay suggested that #3 BS was significantly enriched in the DNA products pulled down by anti-RREB1 antibody in 22Rv1 cells, whereas no significant enrichment was observed in #1 and #2 BS. j RREB1 was overexpressed in 22Rv1 cells, and ChIP assays showed that overexpression of RREB1 increased the enrichment of #3 BS in the pulldown product. k Predicted sequence of binding sites between SNHG4, let-7a and the 3’UTR of RREB1. l and m qRT‒PCR and western blot showed that let-7a mimics significantly depleted RREB1 expression; in contrast, let-7a inhibitor notably increased RREB1 expression. n and o qRT‒PCR and western blot showed that SNHG4 overexpression significantly increased RREB1 expression, whereas restoration of let-7a partially neutralized the RREB1 overexpression that was induced by SNHG4 overexpression alone. p Dual luciferase reporter assay shows that let-7a overexpression inhibited the transcription of luciferase reporter vectors harboring the 3’UTR of RREB1 in 22Rv1 and LNCaP cells. ns indicates not significant, * indicates p < 0.05, ** indicates p < 0.01