Figure 3.

WWOX-AS1 acts as a ceRNA to sponge miR-20b-5p and upregulate WWOX expression in HCC. (a) According to subcellular fractionation assay, WWOX-AS1 was mainly distributed in cytoplasm of HepG2 and Huh7 cells. (b) DIANA and starBase predicted seven potential miRNAs that could bind with both WWOX-AS1 and WWOX. (c) The expression levels of potential miRNAs were detected in transfected cells via qRT-PCR after 48 h of transfection. (d) MiR-20b-5p expression in HCC tissues and adjacent non-tumor tissues was detected by qRT-PCR. (e) MiR-20b-5p expression in HCC cell lines and one control cell line was examined through western blot. (f) RIP revealed the significant enrichment of WWOX-AS1, miR-20b-5p and WWOX in anti-Ago2 group after incubation for overnight. (g) The predicted binding sequences between miR-20b-5p and WWOX-AS1 (or WWOX). (h) qRT-PCR was used to detect the overexpression efficiency of miR-20b-5p after 48 h of transfection. (i) Luciferase reporter assay was performed to examine the interaction between miR-20b-5p and WWOX-AS1 (or WWOX) in HepG2 and Huh7 cells after 48 h of transfection. (j) Expression relevance between miR-20b-5p and WWOX-AS1 (or WWOX) in HCC tissues was analyzed via Pearson’s correlation analysis. **P < .01