AR inhibition on celastrol-induced autophagy is related with miR-101 transactivation.

<p>To see whether miR-101 could affect AR suppression on celastrol-induced autophagy, AR positive LNCaP cells were transfected with miR-101 mimic or negative control (NC) for 24 h (<b>A</b>), followed by additional 24 h treatment with celastrol (2.0 μM, CEL). MiR-101 levels were determined by RT-PCR. #, <i>P</i><0.05 <i>versus</i> NC transfection without celastrol treament. **, <i>P</i><0.01 between miR-101 and NC transfections with celastrol treatment. The protein levels of LC3 and p62 as well as AR were determined by Western blotting using GAPDH as a loading control. <b>B</b>, AR negative DU145 cells were transfected with pEGFP-C1-AR (EGFP-AR) or empty vector (EGFP-V) in the presence of miR-101 inhibitor (miR-101 Inh) or negative control (NC). Cells were incubated in celastrol (2.0 μM, CEL) for an additional 24 h. MiR-101 levels were determined by RT-PCR. #, <i>P</i><0.05 <i>versus</i> EGFP-V plus NC transfections. *, <i>P</i><0.05 between EGFP-V and EGFP-AR in the presence of miR-101 inhibitor. The protein levels of AR, LC3and p62 were detected by Western blotting. <b>C</b>, DU145 cells were transfected with pGL3-B-miR-101-W (with wild type AR binding site) or pGL3-B-miR-101-M (with mutant AR binding site), along with AR expression vector (EGFP-AR) or empty vector (EGFP-V) for 24 h, then treated with DMSO or celastrol (CEL, 2.0 μM) for additional 24 h. MiR-101 levels were determined by RT-PCR. #, <i>P</i><0.05 <i>versus</i> EGFP-V plus pGL3-B-miR-101-M transfections. *, <i>P</i><0.05 between pGL3-B-miR-101-M and pGL3-B-miR-101-W transfections. The protein levels of AR, LC3 and p62 were detected by Western blotting using GAPDH as a loading control.