Fig. 4: RBM30 upregulates PD-L1 expression in HCC via the STAT1 signaling pathway.

A ChIP-qPCR assays confirmed that there is an interaction between RBM30 and the STAT1 DNA region. B Dual-luciferase reporter gene assays revealed that the activity of the STAT1 signaling pathway was significantly decreased in Lm3 cells after knocking out RBM30, and vice versa in Hep3B cells (n = 3, ***P < 0.0001). C qPCR assays demonstrated that the STAT1 mRNA content was dramatically increased in Hep3B cells after RBM30 overexpression, and vice versa in Lm3 cells (n = 3, ***P < 0.0001). D Western blotting assays indicated that RBM30 knockout resulted in a decrease in the expression of both STAT1 protein and its phosphorylated active form, p-STAT1, in HCC cells, as well as vice versa. However, this manipulation did not have any impact on the levels of other crucial proteins within the STAT1 signaling pathway or endogenous negative regulatory proteins. E The distribution of STAT1 and p-STAT1 on the chromatin genome in the constructed Hep3B-ov-RBM30 and control cell lines was examined using the Cut-Tag approach. F The visualization results of IGV indicated that the enrichment degree of STAT1 and p-STAT1 in the PD-L1 region was significantly enhanced after overexpression of RBM30. G Western blotting assays and qPCR assays were conducted in Lm3-sg-RBM30 cells, in which STAT1-wild type (WT), STAT1 Y701D (phosphomimetic mutant form), and STAT1 Y701F (dominant-negative mutant form) plasmids were transfected, showing that there was no significant rebound in the level of PD-L1 in HCC cells caused by RBM30 knockdown when transfected with inactivated STAT1 Y701F plasmid (n = 3, ***P < 0.0001). H FACS assays were conducted in Lm3-sg-RBM30 cells, in which STAT1-WT, STAT1 Y701D, and STAT1 Y701F plasmids were transfected, revealing that the RBM30 knockout-induced decrease in the expression of PD-L1 on Lm3 cell membranes was restored only when STAT1-WT/STAT1 Y701D plasmids were transferred (n = 3, ***P < 0.0001). I Western blotting and qPCR assays confirmed that blocking the STAT1 signaling pathway or interfering with STAT1 rescued the increased expression of PD-L1 caused by RBM30 overexpression in Hep3B cells (n = 3, ***P < 0.0001, ns indicates no statistical significance). J FACS assays revealed that RBM30 relies on the STAT1 signaling pathway to promote an increase in surface PD-L1 expression in HCC cells (n = 3, ***P < 0.0001, ns indicates no statistical significance).