Fig 2: Effect of direct and indirect inhibition of MYB on PCa cells growth and expression of cell cycle–associated genes. LNCaP (A) and C4-2 cells (B) were transfected with nontargeted scramble (NT-Scr) or MYB- specific siRNA (si-MYB) for 24 h, followed by low and high DHT treatment for 72 h. After incubation, cell growth was measured by WST-1 assay. LNCaP and C4-2 cells were transfected with either miR-150 inhibitor (miR150-Inh) (C and D) or miR-150 mimic (E and F) along with their respective negative controls for 24 h, and then cells were exposed to low and high DHT for 48 and 72 h. After that, cell growth was measured by WST-1 assay, and data were presented as a bar graph. LNCaP and C4-2 cells were transfected with nontargeted scramble (NT-Scr) or MYB- specific siRNA (si-MYB) for 24 h, followed by low and high DHT treatment for an additional 24 h. Total RNA was collected, and cell cycle–specific gene expression (CCNA1, CCNE1, and CCND1) was analyzed by qRT-PCR (G and H). Similarly, the expression of CCNA1, CCNE1, and CCND1 was also analyzed in miR-150 inhibitor–transfected LNCaP and C4-2 cells treated with a low or high dose of DHT. ACTB was used as an internal control (I and J). ∗p = < 0.05. DHT, dihydrotestosterone; PCa, prostate cancer.

Fig 3: MYB repression by high dose androgen occurs late due to the reduced stability of its mRNA.A and B, LNCaP and C4-2 cells were incubated with DHT (1 nM and 100 nM) for 6 h prior to the treatment with Actinomycin D. After 1 h, total RNA was isolated at different time intervals for up to 120 min, and MYB mRNA levels were analyzed by qRT PCR. 18s rRNA was used as an internal control. Percent mRNA decay was calculated from the earliest time point of analysis (0 min), and the data was presented as a line graph (mean ± S.D.). C–F, prostate cancer cells were treated with low (1 nM) or high (100 nM) doses of DHT for increasing time intervals (0–48 h). MYB expression was analyzed at both mRNA and protein levels by qRT-PCR and immunoblot assays, respectively. ACTB (qRT-PCR) and β-actin (immunoblot) were used as internal controls. Relative differences in MYB mRNA and protein levels are presented in the bar graph (mean ± S.D.), ∗p = < 0.05. G and H, LNCaP and C4-2 cells were incubated with DHT (1 nM and 100 nM) for 18 h prior to treatment with Actinomycin D. After 1 h, total RNA was isolated at different time points (0–120 min), and MYB mRNA expression was analyzed by qRT PCR. 18s rRNA was used as an internal control. Percent mRNA decay was calculated, and the data was presented as a line graph (mean ± S.D.). ∗p < 0.05; n.s. not significant. DHT, dihydrotestosterone.

Fig 4: MicroRNA29B (MIR29B) mediates HBG reactivation via inhibiting MYB in KU812 cells. (A) Western blot analysis of MYB and HbF protein expression relative to β-actin protein in KU812 cells transfected with increasing concentrations of MYB plasmid DNA (μg). Fold change of (B) MYB mRNA relative to β-actin, (C) MIR29B expression relative to RNU48, and (D) HBG-to-HBB mRNA ratio as a function of HBG/HBG + HBB mRNA expression in KU812 cells following transfection for 48 h with MIR29B mimic (50 or 100 nm) alone or in combination with MYB overexpression plasmid vector (2 μg). (E) Western blot of MYB, HbF, and HbA protein expression with β-actin internal control. (F) Bar graphs showing quantification of the ratio of HbF-to-HbA protein in KU812 cells transfected with MIR29B alone or in combination with MYB. *P < 0.05 **P < 0.005, ***P < 0.0005, and ****P < 0.0001 is statistically significant.

Fig 5: Effect of miR-150 inhibition and restoration on MYB expression in low- and high-dose DHT-treated prostate cancer cells.A and B, LNCaP and C4-2 cells were transfected with negative control-inhibitor (NC-Inh) or miR-150 inhibitor (miR150-Inh) for 24 h, followed by low and high doses treatment of DHT for another 24 h. MYB expression was analyzed by Western blotting. β-actin was used as a loading control. C and D, cells were transfected with negative control miRNA mimic (miR-NC) or miR-150 mimic (miR-150) for 24 h, followed by DHT (1.0 nM) treatment for 24 h and MYB expression analysis. E–G, prostate cancer cells were transfected with a luciferase reporter plasmid carrying MYB-3ʹUTR-WT (WT) sequence containing a putative-binding site for miR-150 or MYB-3ʹUTR-Mut (Mutant) where miR-150 binding site is mutated. Cells were also cotransfected with Renilla luciferase plasmid (pRL/TK) to control transfection efficiency. After 24 h, cells received treatment with low and high doses of DHT, and luciferase activity was examined after 24 h of incubation. The data is presented as normalized luciferase activity (mean ± SD), ∗p = < 0.05. H and I, LNCaP and C4-2 cells were cotransfected with pGL3-MYB-3′UTR WT or pGL3-MYB-3′UTR mutant (Mut) luciferase along with either control-inhibitor (NC-Inh) or miR-150 inhibitor (miR150-Inh) for 24 h. Thereafter, cells were treated with low and high DHT doses for 24 h. Luciferase activity was examined, and the data presented as the normalized luciferase activity (mean ± SD), ∗p = < 0.05, n.s. not significant. DHT, dihydrotestosterone.