Fig 2: miR-218 affects chemoresistance by downregulating PPP2R5A. (A) UM1 and Cal27 cells transfected with miR-218, PPP2R5A or control were treated with cisplatin (2.5 µg/ml). The cell viability was assayed using the CCK-8 assay. (B) UM1cis cells were transfected with anti-miR-218, siRNA against PPP2R5A or negative control for 24 h, and then treated with a gradient concentration of cisplatin. The CCK-8 assay was performed 48 h after treatment. (C) Western blotting was used to detect PPP2AB56a protein (left panel). The expression level of PPP2AB56a was quantitated (right panel). Data represent the mean ± SD of 3 independent experiments; *P<0.05, **P<0.01.

Fig 3: PPP2R5A protein expression and its correlation with miR-218 in oral cancer tissues. (A) The protein expression of PPP2R5A was examined in primary tumors from patients with oral cancer who received chemotherapy prior to surgery and had a sensitive or non-sensitive outcome by immunohistochemistry. Scale bar, 100 µm. (B) The expression of PPP2R5A protein in chemosensitive tissues was higher than that in tissues with failure of chemotherapy. (C) A reverse correlation between PPP2R5A protein and miR-218 expression was observed.

Fig 4: PPP2R5A affects cisplatin treatment sensitivity in oral cancer cells. (A) qRT-PCR analysis of PPP2R5A expression was conducted in oral cancer cell lines. (B) A reverse correlation between PPP2R5A mRNA and miR-218 expression in oral cancer cell lines was observed. (C) UM1 and Cal27 cells were transfected with siRNA against PPP2R5A or its control (siNC), and total protein was extracted for immunoblotting (upper panel). The expression level of PPP2AB56a was quantitated (lower panel). (D) UM1cis and Cal27cis cells were infected with LV-PPP2R5A or its control (LV-control), and immunoblotting was performed and quantitated. The tumor cells were transfected with siRNA against (E) PPP2R5A or (F) LV-PPP2R5A and treated with cisplatin (2.5 µg/ml), and the apoptosis rate was quantitated by flow cytometry. Data represent the mean ± SD of 3 independent experiments; *P<0.05, **P<0.01.

Fig 5: miR-218 activates Wnt signaling in oral cancer cells by inhibiting PPP2R5A. (A) UM1 and Cal27 cells were transfected with miR-218 along with PPP2R5A or its control, and total protein was extracted from the cells for western blot analysis (upper panel). The protein level was quantitated (lower panel). (B) miR-218, PPP2R5A, or the control were transfected into tumor cells in the presence of cisplatin (2.5 µg/ml). Cell apoptosis was assessed by flow cytometry after 48 h. (C and D) siRNA against ß-catenin suppressed Wnt activation and inhibited the miR-218-induced protection against cell apoptosis. Three independent experiments were performed. Data represent the mean ± SD of 3 independent experiments; *P<0.05, **P<0.01.