Fig. 7: ASB16-AS1 post-translationally represses HuR expression through BTRC-mediated ubiquitination.

a Inhibition of ASB16-AS1 expression attenuates HuR protein degradation. SW-13 cells were transfected with ASB16-AS1 siRNA1 or siRNA2 or its negative control (NC) and then treated with 50 μg/ml cycloheximide (CHX) for the indicated period of time. HuR protein levels were detected by western blot. b ASB16-AS1 promotes HuR degradation via proteasomal degradation. SW-13 cells were transfected with a plasmid encoding ASB16-AS1 or pcDNA3.1 control and cells were treated with 20 μM MG132 for 24 h. HuR protein levels were detected by western blot. c ASB16-AS1 promotes HuR protein ubiquitination. SW-13 cells were co-transfected with HA-Ub, Flag-HuR, ASB16-AS1 siRNA1 (Si1), or ASB16-AS1 siRNA2 (Si2) or its negative control (NC), the cells were then treated with 20 μM MG132 for 24 h. The cells were then lysed for immunoprecipitation. Immunoprecipitation was performed using anti-Flag antibody. IgG served as negative control. Western blot was used to detect ubiquitinated HuR protein using anti-HA antibody. d Inhibition of ASB16-AS1 expression attenuates the interaction between HuR and the ubiquitin E3 ligase BTRC. SW-13 cells were transfected with ASB16-AS1 siRNA1 (Si1) or ASB16-AS1 siRNA2 (Si2), immunoprecipitation was performed using either anti-HuR or anti-BTRC antibody, western blot was performed to detect the association between HuR and BTRC protein. e Knockdown of BTRC abolished down-regulation of HuR, CDK6, IGF1R upon enhanced expression of ASB16-AS1. Adrenocortical carcinoma SW-13 cells were transfected with plasmid encoding ASB16-AS1 or in combination with siRNA targeting BTRC, 48 h post-transfection the expression of HuR, CDK6, and IGF1R were detected by western blot. f Schematic of ASB16-AS1 regulating adrenocortical carcinoma cell proliferation. Three independent experiments were performed.