Identification of miRNA-125a-3p as a target of ANRIL. A. Changes in the expression of ANRIL in cytoplasm and nucleus of ANRIL siRNA NC- or siRNA 1/2-transfected HN6 and CAL27 cells. B. Relative expression (miR-125a-3p, miR-145-3p, miR-181d-5p, and miR-339-5p) in ANRIL siRNA- or OE-transfected HN6 and CAL27 cells. C. The correlation between ANRIL transcript level and miR-125a-3p mRNA level was measured in 49 HNSCC tissues. The ΔCt values (normalized to GADPH) were subjected to Pearson correlation analysis. D. The miR-125a-3p expression levels in normal oral epithelial cells (titled Normal), HN6 and CAL27 cells. E. Associations of miR-125a-3p and ANRIL with Ago2. HN6 and CAL27 cell lysates were collected for RIP using an anti-Ago2 antibody. miR-125a-3p and ANRIL were detected by qRT-PCR. F. Alignment of potential ANRIL base pairing with miR-125a-3p as identified by Starbase v2.0 (http://starbase.sysu.edu.cn/mirLncRNA.php). Mutant ANRIL at the putative binding site. Luciferase activity in CAL27 cells co-transfected with miR-NC or miR-125a-3p mimics and luciferase reporters containing nothing, ANRIL, NC mutant transcripts or ANRIL mutant transcripts. The results are presented as the relative ratio of firefly luciferase activity to Renilla luciferase activity. G. Change in miR-125a-3p expression levels in miR-NC-, miR-125a-3p mimic-, and miR-125a-3p inhibitor-transfected HN6 and CAL27 cells. Cell growth and viability were assayed in shANRIL NC-, shANRIL, shANRIL+miR-125a-3p mimic-NC-, shANRIL+miR-125a-3p mimic-, shANRIL+miR-125a-3p inhibitor-NC-, and shANRIL+miR-125a-3p inhibitor-transfected HN6 and CAL27 cells by CCK-8 and colony formation assays. H-J. *P < 0.05; **P < 0.01; n.s., not significant.