Figure 4.

hsa-miR-329-3p inhibits PD-L1 expression by downregulating KDM1A levels in HCC cells. (a) The sequences of the KDM1A 3′ UTR wild type (WT), KDM1A 3′ UTR mutant (Mut), and miR-329-3p, and the schematic diagram of target prediction between KDM1A and miR-329-3p. (b) Binding of miR-329-3p to the 3′ UTR of KDM1A in HEK293T cells by luciferase reporter assay. (c, d) mRNA/miRNA expression of KDM1A, PD-L1, and miR-329-3p in SMMC7721^miRNA cells treated with the control (c) or in SMMC7721^miRNA cells treated with the inhibitor and control (d). (e) Top: proteins immunoprecipitated with anti-K267me were analyzed using anti-MEF2D. Bottom: protein expression in whole cell lysates from wild-type SMMC7221 cells, SMMC7721^miRNA cells, and SMMC7721^miRNA cells with the inhibitor. (f) The results of RGA reflecting the strength of T cell-mediated antitumor immunity in SMMC7221 cells stably overexpressing mi-NC, SMMC7721^miRNA cells, and SMMC7721^miRNA cells with the inhibitor or control. (g–i) The results of the cytotoxic rate, IFN-γ level, and IL-2 level in SMMC7221 cells stably overexpressing mi-NC, SMMC7721^miRNA cells, and SMMC7721^miRNA cells with the inhibitor or control. The data are presented as the means ± SD. n = 3 experiments in (a–i). ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.01.