Figure 4.

sTWEAK regulates oncomiR-221-3p expression and down-regulates NLK and TCF12 targets. (a) oncomiR-221-3p expression in PC-3 and LNCaP cell extracts and in extracellular vesicles (EVs) isolated from cell media. Different lettering over boxes indicates statistical differences. Significant differences are established at p < 0.05. Data are expressed as mean ± SEM (n = 4 experiments). (b) Selected targets for oncomiR-221-3p by 3 different target prediction algorithms. ^# conserved elements in multiply-aligned sequences. (c) qRT-PCR mRNA expression of selected oncomiR-221-3p targets in PC-3 cells before and after treatment with sTWEAK. Significant differences: ** p < 0.01; *** p < 0.001. Data are expressed as mean ± SEM (n = 6 experiments). Transcription factor 12 (TCF12); synaptosome associated protein 23 (SNAP23); dipeptidyl peptidase 8 (DPP8); aryl hydrocarbon receptor nuclear translocator (ARNT); Nemo-like kinase (NLK); ZFP36 ring finger protein-like 2 (ZFP36L2); folliculin interacting protein 2 (FNIP2); estrogen receptor 1 (ESR1); homeodomain interacting protein kinase 1 (HIPK1); and scavenger receptor class B-ember 2 (SCARB2). (d) Expression of TCF12, NLK and Fn14 protein in PC-3 cells transfected with the oncomiR-221-3p inhibitor and further treated with sTWEAK. Representative Western blots are presented (top). The membranes were tested with the corresponding antibody. iNC: inhibitor negative control, i221-3p: inhibitor miR-221-3p; Nemo-like kinase (NLK); transcription factor 12 (TCF12); fibroblast growth factor 14 (Fn14). Relative protein expression levels are shown (bottom), which were normalized to the corresponding control β-actin. Different lettering over boxes indicates statistical differences. Significant differences are established at p < 0.05. Data are expressed as mean ± SEM (n = 3 experiments). Full-length blots and gels are presented in Figure S4.