Fig. 4: AC104041.1 competes with miR-6817-3p to sustain Wnt2B expression in HNSCC.

a Luciferase activity in SCC4 cells co-transfected with luciferase reporter containing Wnt2B and miR-6817-3p mimics or in combination with AC104041.1 overexpression. Data are presented as the mean values ± SEM, the experiment was performed in triplicates and repeated three times, ***P < 0.001, compared with control cells (one-way ANOVA). b Luciferase activity in SCC4 cells co-transfected with luciferase reporter containing Wnt2B and miR-6817-3p inhibitors or in combination with AC104041.1 knockdown. Data are presented as the mean values ± SEM, the experiment was performed in triplicates and repeated three times, ***P < 0.001, compared with control cells (one-way ANOVA). c Downregulation of Wnt2B mRNA stability caused by silencing of AC104041.1 was reversed by miR-6817-3p inhibitors. Data are mean values ± SEM, the experiment was performed in triplicates and repeated three times, **P < 0.01 (two-way ANOVA). d Upregulation of Wnt2B mRNA stability caused by overexpression of AC104041.1 was reversed by miR-6817-3p mimics. Data are mean values ± SEM, the experiment was performed in triplicates and repeated three times, **P < 0.01 (two-way ANOVA). e Immunoblotting for Wnt2B levels in empty-vector and ectopic AC104041.1-expressing SCC4 cells in the presence of miR-6817-3p mimics or knockdown of AC104041.1 cells in the presence of miR-6817-3p inhibitors. f AGO2 competing assay using AGO2 antibody-mediated RNA-IP in the indicated groups. RNA levels of precipitated AC104041.1, miR-6817-3p, and Wnt2B are tested using qPCR assay. Data are presented as the mean values ± SEM, *P < 0.05, ***P < 0.001 (one-way ANOVA). g Relative expression of Wnt2B in HNSCC tissues compared with their matched non-tumorous adjacent tissues in TCGA cohorts (n = 500). Each symbol represents one patient. ***P < 0.001 (Student’s t-test). h Relative expression levels of Wnt2B in paired normal and HNSCC tissues (n = 94). Each dot represents the average of qPCR results performed in triplicates. Horizontal lines represent mean values. ***P < 0.001 (paired t-test). i Kaplan–Meier analysis of the correlation between Wnt2B expression levels and overall survival. Higher expression of Wnt2B is significantly correlated with poor OS (**P < 0.01, log-rank test). j Association of AC104041.1 and Wnt2B with overall survival in HNSCC. k Positive correlation between AC104041.1 and Wnt2B levels in HNSCC tissues (n = 94, Spearman correlation r = 0.7684, P < 0.001). Each dot represents the average of qPCR results performed in triplicates. l Negative correlation between miR-6817-3p and Wnt2B levels in HNSCC tissues (n = 94, Spearman correlation r = −0.7157, P < 0.001). Each dot represents the average of qPCR results performed in triplicates.