Fig. 6: AR mutations activated fat metabolism via the SREBP1.
a Shown are 4 ARE motifs based on the JASPAR ChIP-seq database (http://jaspar.genereg.net/) in SREBP1 promoter. b AR bond to SREBP1 promoter. MHCC-97H cells stably expressing HA-AR mutation or HA-AR WT were assayed for AR binding to SREBP1 promoter by ChIP. A random sequence was used as a negative control (NC). Data (Mean ± SD, n = 3) was measured by qPCR and analyzed by one-way ANOVA. * p < 0.05. c Mutations activated the transcription of AR to SREBP1 promoter. HA-AR WT, HA-ARQ62L and HA-ARE81Q were stably expressed in MHCC-97H cells carrying SREBP1 promoter-Luc reporter and measured for luciferase activity with or without 24 hours 10^ (-8) M R1881 treatment. Data (Mean ± SD, n = 3) was analyzed by one-way ANOVA. ** p < 0.01, *** p < 0.001. d AR N-term mutations up-regulated the expression of SREBP1 in hepatoma cells. The expression of SREBP1 was detected in MHCC-97H cells by immunoblot and RT-qPCR. H3 and GAPDH were used respectively as loading control. The protein level of SREBP1 was examined relative to H3. HA was run on a separate gel (gel #1). AR were from a separate gel (gel #2). H3, SREBP1 were from another gel (gel #3). The molecular weights of H3 bands were estimated from the manufacturer guidelines. The mRNA data (Mean ± SD, n = 3) was normalized by GAPDH and was analyzed by one-way ANOVA. *p < 0.05, ***p < 0.001. e AR N-term mutations up-regulated the expression of SREBP1 in HDT mice. The IHC stain of SREBP1 of different HDT mice and control liver tissues were shown. N: normal liver, T: tumor liver (Scale bar = 200/50 μm). f AR N-term mutations activated the fat metabolism pathway. The expression level of fat metabolism associated genes (FASN/ACLY/S1P/S2P) were detected by immunoblot in MHCC-97H cells and GAPDH was as loading control. The molecular weights of GAPDH bands were estimated from the manufacturer guidelines. g, h SREBP1 knockdown inhibits the growth and survival of AR-Q62L and AR-E81Q expressing hepatoma cells. MHCC-97L cells stably expressing AR-Q62L and AR-E81Q were treated with SREBP1-specific siRNA (siSREBP1 1/2) or a control siRNA (siNC). The molecular weights of protein bands were estimated from the manufacturer guidelines. Cell growth was measured by CCK8 assay. Data (mean ± SD, n = 3) were analyzed by Repeated measures ANOVA. * p < 0.05, *** p < 0.001.
