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Fig. 5 | Molecular Cancer

Fig. 5

From: N6-methyladenosine-modified circIGF2BP3 inhibits CD8+ T-cell responses to facilitate tumor immune evasion by promoting the deubiquitination of PD-L1 in non-small cell lung cancer

Fig. 5

circIGF2BP3 elevates PKP3 expression by acting as an miRNA sponge for miR-328-3p and miR-3173-5p. A Schematic diagram illustrating the binding sites of circIGF2BP3 and its predicted miRNAs. miRNAs marked in red denote miRNAs containing binding sites for both circIGF2BP3 and PKP3, as predicted by starBase. B The relative luciferase activity of the circIGF2BP3 reporter in A549 cells transfected with the indicated miRNA mimics. Firefly luciferase activity was normalized to Renilla luciferase activity. C The relative expression of PKP3 in A549 cells transfected with the indicated miRNA mimics was detected by qRT-PCR. D FISH shows the colocalization of circIGF2BP3 with miR-328-3p and miR-3173-5p. circIGF2BP3 FISH (red), miR-328-3p FISH (green), miR-3173-5p FISH (green), nuclear DAPI staining (blue). Scale bars, 20 μm. E AGO2 IP assay to detect circIGF2BP3 in A549 cells transfected with miR-328-3p mimics (upper) or miR-3173-5p mimics (lower). F PCR detection of circIGF2BP3 enriched with 3′-end biotinylated miR-328-3p or 3′-end biotinylated miR-3173-5p. G The relative luciferase activity of the circIGF2BP3 reporter and its mutant in A549 cells transfected with miR-328-3p mimics (left) or miR-3173-5p mimics (right). Firefly luciferase activity was normalized to Renilla activity. H PCR detection of PKP3 enriched with 3′-end biotinylated miR-328-3p (upper) or 3′-end biotinylated miR-3173-5p (lower). I The relative luciferase activity of the PKP3–3’UTR reporter and its mutant in A549 cells transfected with miR-3173-5p mimics (left) or miR-328-3p mimics (right). Firefly luciferase activity was normalized to Renilla activity. J The relative luciferase activity of the PKP3–3’UTR reporter in circIGF2BP3-silenced (left) and circIGF2BP3-overexpressing (right) NSCLC cells. K The relative expression of PKP3 in A549 cells transfected with the indicated miRNA mimics alone or cotransfected with circIGF2BP3. L-N FACS analysis of PBMC-mediated elimination of NSCLC cells transfected with the indicated constructs after treatment with miR-328-3p mimics (L), miR-3173-5p mimics (M), or miR-328-3p and miR-3173-5p mimics (N). O. K-M OS analysis of NSCLC patients in the TCGA data set according to the expression levels of miR-328-3p (left) and miR-3173-5p (right). Data represent the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. P values were determined by unpaired Student’s t test (G, I and J), one-way ANOVA with Tukey’s post hoc test (B, C, K, L, M and N) and the log-rank test (O)

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