Fig. 6: HuR regulates the expression of CDK6 and IGF1R mRNAs expression.

a HuR RNA-binding protein associates with CDK6 and IGF1R mRNAs. RIP assays were performed in SW-13 cells using HuR antibody to detect the association between HuR protein and CDK6 or IGF1R. IgG served as negative control. *P < 0.05 versus IgG group. b, c Knockdown of HuR expression reduces CDK6 and IGF1R mRNA and protein levels. HuR siRNA was transfected into SW-13 cells at the concentration of 50 nM and CDK6 and IGF1R mRNAs expression were detected by qRT-PCR (b), protein levels were detected by western blot (c). *P < 0.05 versus NC group. d, e Inhibition of HuR reduced the mRNA stability of CDK6 and IGF1R. HuR siRNA was transfected into adrenocortical carcinoma SW-13 cells, actinomycin D was used to treat cells at different time points 48 h post transfection. RNA expression was detected by qRT-PCR. *P < 0.05 versus NC group. f, g The effect of ASB16-AS1 and HuR on CDK6 and IGF1R mRNAs stability. SW-13 cells were transfected with ASB16-AS1 siRNA1 (AS1 siRNA1) or ASB16-AS1 siRNA2 (AS1 siRNA2), respectively, or simultaneously transfected with HuR siRNA, actinomycin D was then used to treat cells at different time points 48 h post-transfection. RNA was detected by qRT-PCR. *P < 0.05. h–j Inhibition of HuR attenuates ASB16-AS1 knockdown induced CDK6 and IGF1R up-regulation. ASB16-AS1 siRNA alone or with HuR siRNA were transfected into SW-13 cells, the expression of CDK6 and IGF1R mRNAs were analyzed by qRT-PCR (h and i), protein levels were detected by western blot (j). *P < 0.05. The experiments were performed in triplicate. The data are represented as mean ± SEM from three independent experiments.