Fig. 6

miR-455-5p regulates cisplatin resistance through direct regulation of PD-L1 expression. A TargetScan prediction of miR-455-5p binding (seed sequence in red) to human PD-L1 3’UTR. B Spearman correlation analysis of cell surface PD-L1 expression (reciprocal of mean fluorescence intensity values) and miR-455-5p levels (Cqn) in the panel of NSCLC cell lines. C Bar plot of PD-L1 expression (microarray log₂ intensity) in pN2 and pN0 patients (MED samples). Error bars represent SEM. P value was calculated by Limma moderated t test. D and E Distribution of PD-L1 expression (TPS [tumor proportion score]) and miR-455-5p levels (Cqn) in NSCLC primary tumors obtained from CSS cohort (D) and CIMA-CUN Cohort (E). F Correlation analysis of miR-455-5p levels with PD-L1 mRNA in tumors from TGCA-LUAD and TGCA-LUSC cohorts Left: Bubble plots report the correlation coefficients. Size of the bubbles indicates statistical significance. Right: Bar plot reporting the value of miR-455-5p normalized count for each tertile threshold in TGCA-LUAD and TGCA-LUSC cohorts. The number of patients was reported inside the bar. G Representative flow cytometry histogram plots (left) and quantification (right) of PD-L1 median fluorescence intensity (MFI) in the indicated cell lines treated with a miR-455-5p mimic (OE) or a negative control mimic (CTRL). Results are shown as fold change of MFI relative to CTRL cells. Data are mean ± SEM (N = 4 or 5). P values were calculated by one sample t test. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. H Representative flow cytometry histogram plots (left) and quantification (right) of cell surface PD-L1 MFI in CDDP-R cells transfected with a miR-455-5p mimic or a negative control in the presence of a scramble TSB or a PD-L1-specific miR-455-5p TSB. Data are reported as fold change in MFI relative to CDDP-R cells transfected with a CTRL miRNA mimic and with a scramble TSB. Data are mean ± SEM (N = 6). P values were calculated by one sample t test. *P < 0.05, **P < 0.001; ns, not significant. I Bar plot representing cell viability (Fold change relative to CTRL mimic in the presence of a scramble TSB) of CDDP-R cells transfected as in (G) and treated for 72 h with cisplatin at the indicated doses. Data are mean ± SEM (N = 5). P values were calculated by one sample t test. *P < 0.05, **P < 0.01; ns, not significant. J Bar plot representing cell surface PD-L1 expression in the indicated cell lines stimulated for 48 h with ± 40 ng/ml of IFN-γ. The result is shown as fold change in the MFI relative to Parental CTRL cells. Data are mean ± SEM (N = 3). P values were calculated by one sample t test. *P < 0.05, **P < 0.01, ***P < 0.001; ns, not significant. K Immunoblot analysis of pEGFR, EGFR and PD-L1 in BEAS-2B transfected with a miR-455-5p mimic or a negative control and treated for 36 h with ± 40 ng/ml of EGF. GAPDH was used as loading control. L Immunoblot analysis of pEGFR, EGFR and PD-L1 expression in NCI-H1975 transfected with a miR-455-5p mimic or a negative control. GAPDH was used as loading control