miR-138 negatively regulates the expression of CD44 through direct targeting its 3′ UTR. (A) Overexpression of CD44 in human GBM patient samples (11.06 ± 0.1995, n = 9) compared to control samples (7.168 ± 0.7913, n = 4) as measured by SyBr-Green real-time quantitative PCR (qRT-PCR) (***p < 0.001). (B) TCGA database showed the overexpression of CD44 in GBM (2329 ± 76.09, n = 200) compared to control samples (299.9 ± 50.01, n = 9) (***p < 0.001). (C) Paired expression correlation between miR-138 and CD44 in human GBM patient samples (Slope = − 0.08235, R2 = 0.8863, p < 0.001, n = 13 (9 GBM and 4 controls)). The expression levels of miR-138 and CD44 were measured by TaqMan miRNA expression assay or SyBr-Green real-time quantitative PCR (qRT-PCR), respectively. (D) Enrichment plot by Gene Set Enrichment Analysis (GSEA) showed the enriched genes including CD44 involving in hyaluronan metabolic pathway. (E) Western blotting on GBM cells with transient overexpression of miR-138. The protein expression levels of CD44 were significantly lower than miR-Ctrl treated GBM cells. (F) Detection of CD44 positive cells by flow cytometry on GBM cells after miR-138 overexpression in comparison to control groups with no miR or miR-Ctrl overexpression. Transiently transfected GBM cells with miR-138 mimics were stained with FITC-labeled anti-CD44 antibodies and analyzed. FICT-negative cell population was gated by analyzing GBM cells transfected with siRNAs against CD44 (Supplementary material Fig. S5). (G) Schematic diagram of 3′ UTR sequences of CD44 containing two predicted miR-138 binding sites and the reporter constructs showing the mutated sequences in the seed regions (CD44-mut1 and CD44-mut2). (H) Luciferase reporter assays to test directing binding of miR-138 to the CD44 3′ UTR regions in GBM cells. GBM cells were transfected with luciferase reporter gene expressing DNAs containing CD44 3′ UTR, CD44-mut1 or CD44-mut2 sequences respectively. Next day, the cells were further transfected with miR-138 or miR-Ctrl for 48 h. Passively lysed cell lysates were assayed for luciferase activity by Dual Luciferase Assay kit. The relative luciferase activity values were normalized to Renilla luciferase activity as internal control. All error bars indicates standard deviations (n = 3), and the p-values were determined by two-tailed student t-test. ***p < 0.001.