Fig. 2: EBV-miR-BART17-3p upregulates PD-L1 expression by targeting PBRM1. | Nature Communications

Fig. 2: EBV-miR-BART17-3p upregulates PD-L1 expression by targeting PBRM1.

From: EBV miRNAs BART11 and BART17-3p promote immune escape through the enhancer-mediated transcription of PD-L1

Fig. 2

a HONE1, AGS, HONE-EBV, AGS-EBV, C666-1, and SNU-719 cells were co-transfected with the PBRM1-WT or the PBRM1-MT vectors and the EBV-miR-BART17-3p mimics or inhibitors. The effect of EBV-miR-BART17-3p on the luciferase reporter activity of the PBRM1 3′-UTR is shown. n = 3 biologically independent samples. b RNA pull-down assays followed by qRT-PCR were performed to examine the binding effect of EBV-miR-BART17-3p on the 3′-UTR of PBRM1 in HONE1 and AGS cells transfected with the biotin-labeled or unbiotin-labeled EBV-miR-BART17-3p probes. n = 3 biologically independent samples. c After transfection of EBV-miR-BART17-3p mimics or negative control into EBV-negative HONE1 and AGS cells, anti-AGO2 antibody was used for the RIP experiment followed by qRT-PCR to detect the binding of EBV-miR-BART17-3p to the PBRM1 3′-UTR via AGO2. d Western blotting was used to detect whether EBV-miR-BART17-3p could regulate the PD-L1 protein via PBRM1 in the HONE1, AGS, HONE1-EBV, AGS-EBV, C666-1, and SNU-719 cells transfected with the PBRM1 overexpression vector, siPBRM1, EBV-miR-BART17-3p mimics or inhibitors, or co-transfected with EBV-miR-BART17-3p mimics and the PBRM1 overexpression vector, or EBV-miR-BART17-3p inhibitors and siPBRM1. GAPDH was used as an internal control. e Immunofluorescence assays were used to identify PD-L1 expression in HONE1 cells transfected with the PBRM1 overexpression vector, siPBRM1, EBV-miR-BART17-3p mimics, or co-transfected EBV-miR-BART17-3p mimics and the PBRM1 overexpression vector. PBRM1: green; PD-L1: red; merge: signal superimposed image of DAPI, PBRM1, and PD-L1; magnification: ×400; scale bars = 20 µm. f Flow cytometry analysis of PD-L1 expression in HONE1 and HONE1-EBV cells transfected with the PBRM1 overexpression vector, siPBRM1, EBV-miR-BART17-3p mimics or inhibitors, or co-transfected with EBV-miR-BART17-3p mimics and the PBRM1 overexpression vector or EBV-miR-BART17-3p inhibitors and siPBRM1. Each group was analyzed using 3 independent replicates, and the mean fluorescence intensity (MFI) of PD-L1 was calculated. The original results are shown in Supplementary Fig. 5b. Data are presented as mean ± s.d., and p-values are calculated by unpaired two-sided t-test in a, b, f. Source data are provided as a Source Data file.

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