Fig. 2

CircSERPINA3 regulates SERPINA3 through competitively binding to miR-653-5p. A, B Subcellular fractionation assay and FISH assay were conducted to identify the location of circSERPINA3 in PC-3 cells. C, D Subcellular fractionation assay and FISH assay were implemented to examine the distribution of circSERPINA3 in DU145 cells. E Potential miRNAs that could bind to circSERPINA3 were selected with the help of starBase. F Potential five miRNAs were selected and respectively overexpressed in pmirGLO-circSERPINA3 transfected HEK-293T cells and then dual luciferase reporter assays were conducted to detect the luciferase activity of pmirGLO-circSERPINA3 plasmids. G Dual luciferase reporter assay was carried out to verify the binding capacity between miR-653-5p and circSERPINA3 based on the luciferase activity of pmirGLO-circSERPINA3-WT/Mut vectors. H RNA pull down assay was implemented to evaluate the enrichment of miR-653-5p in Bio-circSERPINA3-WT/Mut. I Five downstream mRNAs for miR-653-5p was predicted through starBase. J, K RT-qPCR was utilized to examine the expression of the potential mRNAs in normal WPMY-1 cells and PCa cells. L The luciferase activity of pmirGLO-SERPINA3 3’UTR-WT/Mut was detected after mimic-miR-653-5p transfection by means of dual luciferase reporter assay to verify the binding capacity between miR-653-5p and SERPINA3. M RNA pull down assay was done to test the enrichment of miR-653-5p in Bio-SERPINA3 3ʹUTR-WT/Mut. N After PC-3 cells were transfected with sh-NC, sh-circSERPINA3-1, sh-circSERPINA3-1 + NC inhibitor or sh-circSERPINA3-1 + miR-653-5p inhibitor, a series of recue assays were conducted to detect the expression of SERPINA3. (NC negative control, WT wide-type, Mut Mutant, Bio biotinylated, sh short hairpin). ^*P < 0.05, ^**P < 0.01