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. 2016 May 5;7:11406. doi: 10.1038/ncomms11406

Figure 3. miR-424(322) regulates T-cell cytokine secretions by blocking PD-L1 in a Skov3 (CP)/T-cell co-culture model.

Figure 3

(a,b) RT-PCR was performed to determine miR-424(322) and PD-L1 expression in 21 platinum-sensitive and 21 platinum-resistant ovarian tumours. (a) miR-424(322) levels were significantly decreased (t-test, **P≤0.01) and (b) PD-L1 levels were significantly increased (t-test, **P≤0.01) in the platinum-resistant tumours compared with the platinum-sensitive tumours. (c,d) RT-PCR was performed to determine miR-424(322) and PD-L1 expression in Skov3 and Skov3 (CP) cells, respectively. (c) miR-424(322) levels were significantly decreased (t-test, **P≤0.01) and (d) PD-L1 levels were significantly increased (t-test, **P≤0.01) in the Skov3 (CP) cells compared with the Skov3 cells. The results represent the mean±s.e.m. from three independent experiments. (e) Skov3 (CP) cells with stable overexpression of miR-424(322). miR-424(322) mRNA levels were determined via qRT-PCR assay. t-test, *P≤0.05. The results represent the mean±s.e.m. from three independent experiments. PD-L1 protein levels were determined by western blotting. One representative experiment of three experiments is shown. (f–l) Skov3 (CP) cells with stable overexpression of miR-424(322) with or without PD-L1 blocking antibody (anti-PD-L1). After 24 h, T cells were subsequently co-cultured with mitomycin C-treated Skov3 (CP) cells for 24 h. (f) Skov3 (CP) cells were sorted by FACS. The relative expression levels of PD-L1 in the Skov3 (CP) cells were determined via qRT-PCR assay. t-test, **P≤0.01. The results represent the mean±s.e.m. from three independent experiments. (g–l) Co-culture media were assayed for TNF-α, IFN-γ, IL-2, IL-10, IL-1β and TGF-β by cytokine ELISA assay. t-test, **P≤0.01. The results represent the mean±s.e.m. from three independent experiments.