Abstract:

ObjectiveTo investigate the regulatory effects of ring finger protein 43 （RNF43） on CD8⁺T cell-mediated anti-tumor immune reaction in melanoma.MethodsRNF43 gene was over-expressed and knockdown in mouse melanoma cells line B16-OVA by lentivirus infection；In vivoproliferation of mouse melanoma cells line B16-OVA in the Lv-Ctrl-OE，Lv-RNF43-OE，Lv-Ctrl-KD and Lv-RNF43-KD groups was detected by subcutaneous tumorigenesis assay in mice，and the expression levels of CD8⁺T cells perforin and interferon γ （IFN-γ） in tumor immune microenvironment of melanoma were detected by flow cytometry； The expression levels of β-catenin and programmed death-ligand 1 （PD-L1） mRNA in cells were detected by quantitative real-time PCR assay； The effect of RNF43 on the transcriptional regulation of PD-L1 was detected by dual-luciferase reporter gene assay.ResultsStable RNF43 over-expressing and RNF43 knockdown mouse melanoma cells lines Lv-RNF43-OE and Lv-RNF43-KD were successfully constructed. The results of subcutaneous tumorigenesis experiment in mice showed that the tumor mass of the Lv-RNF43-OE group was （0.08±0.06） g，which was significantly smaller than that of the Lv-Ctrl-OE group [（1.04±0.52） g]，with a statistically significant difference （t=3.71，P=0.032）； The tumor mass of Lv-RNF43-KD group was （1.94±0.29） g，with no statistically significant difference （t=-1.70，P=0.164） compared with that of the Lv-Ctrl-KD group （1.15±0.74） g. The flow cytometry results showed that the fluorescence intensity of CD8⁺T cell perforin in the Lv-RNF43-OE group was 9 034 ± 2 628，which was significantly higher than that in the Lv-Ctrl-OE group （3 847 ±1 637），with a statistically significant difference （t=-3.35，P=0.015）； The fluorescence intensity of CD8⁺T cell perforin in the Lv-RNF43-KD group was 966±247，which was significantly lower than that in the Lv-Ctrl-KD group （2 226±646），with a statistically significant difference （t=3.16，P=0.034）； The fluorescence intensity of IFN-γ of CD8⁺T cell in the Lv-RNF43-OE group was 2 422±429，which was significantly higher than that of 1 688±324 in the Lv-Ctrl-OE group，with a statistically significant difference （t=-2.73，P=0.034）； The fluorescence intensity of IFN-γ of CD8⁺T cell in the Lv-RNF43-KD group was 614 （454，863），with a statistically significant difference （Z=-1.96，P=0.050） compared with 1 159 （1 152，2 068） in the Lv-Ctrl-KD group. The results of quantitative real-time PCR showed that the relative expression level of β-catenin mRNA in the Lv-RNF43-OE group was 0.67±0.16，which was significantly lower than that of 1.00±0.11 in the Lv-Ctrl-OE group，with a statistically significant difference （t=2.98，P=0.041）； The relative expression level of PD-L1 mRNA in the Lv-RNF43-OE group was 0.32±0.09，which was significantly lower than that of 1.00±0.09 in the Lv-Ctrl-OE group，with a statistically significant difference （t=9.13，P=0.001）. The results of the dual-luciferase reporter gene assay showed that the PD-L1 promoter luciferase activity in the pCMV6-NC，RNF43，RNF43+β-catenin and β-catenin groups were 1.00±0.00，0.84±0.00，1.49±0.00 and 1.57±0.03 （F=2 218.33，P<0.001）. Further pairwise comparison showed that compared with the pCMV6-NC group，PD-L1 promoter luciferase activity was significantly lower in the RNF43 group （P<0.001） and significantly higher in the RNF43+β-catenin and β-catenin groups （P<0.001；P=0.003）； compared with the RNF43 group，PD-L1 promoter luciferase activity was significantly higher in the RNF43+β-catenin group （P<0.001）.ConclusionRNF43 may reduce the expression of PD-L1 mRNA in melanoma by inhibiting the expression of β-catenin and promote CD8⁺T cell-mediated anti-tumor immune reaction.

Key words:Melanoma,Beta catenin,Ring finger protein 43