RNF43通过β-catenin抑制黑色素瘤细胞PD-L1表达并促进CD8^(+)T细胞介导的抗肿瘤免疫反应

RNF43 inhibits PD-L1 expression viaβ-catenin in melanoma cells and promotes CD8^(+)T cell-mediated anti-tumor immune reaction

目的探讨环指蛋白43(RNF43)对黑色素瘤中CD8^(+)T细胞介导的抗肿瘤免疫反应的调节作用。方法利用慢病毒感染技术对小鼠黑色素瘤细胞株B16-OVA进行RNF43基因过表达与敲低;通过小鼠皮下成瘤实验检测Lv-Ctrl-OE组、Lv-RNF43-OE组、Lv-Ctrl-KD组和Lv-RNF43-KD组小鼠黑色素瘤细胞株B16-OVA体内增殖情况,通过流式细胞术检测黑色素瘤肿瘤免疫微环境中CD8^(+)T细胞穿孔素和γ干扰素(IFN-γ)的表达水平;采用实时荧光定量PCR实验检测细胞中β-catenin和程序性死亡受体配体1(PD-L1)mRNA的表达水平;通过双荧光素酶报告基因实验检测RNF43对PD-L1的转录调控作用。结果本研究成功构建RNF43稳定过表达与敲低的小鼠黑色素瘤细胞株Lv-RNF43-OE和Lv-RNF43-KD。小鼠皮下成瘤实验结果显示,Lv-RNF43-OE组瘤体质量为(0.08±0.06)g,明显小于Lv-Ctrl-OE组的(1.04±0.52)g,差异有统计学意义(t=3.71,P=0.032);Lv-RNF43-KD组瘤体质量为(1.94±0.29)g,与Lv-Ctrl-KD组的(1.15±0.74)g相比差异无统计学意义(t=-1.70,P=0.164)。流式细胞术结果显示,Lv-RNF43-OE组CD8^(+)T细胞穿孔素荧光强度为9034±2628,明显高于Lv-Ctrl-OE组的3847±1637,差异有统计学意义(t=-3.35,P=0.015);Lv-RNF43-KD组CD8^(+)T细胞穿孔素荧光强度为966±247,明显低于Lv-Ctrl-KD组的2226±646,差异有统计学意义(t=3.16,P=0.034);Lv-RNF43-OE组CD8^(+)T细胞IFN-γ荧光强度为2422±429,明显高于Lv-Ctrl-OE组的1688±324,差异有统计学意义(t=-2.73,P=0.034);Lv-RNF43-KD组CD8^(+)T细胞IFN-γ荧光强度为614(454,863),与Lv-Ctrl-KD组的1159(1152,2068)相比差异有统计学意义(Z=-1.96,P=0.050)。实时荧光定量PCR实验结果显示,Lv-RNF43-OE组β-catenin mRNA相对表达量为0.67±0.16,明显低于Lv-Ctrl-OE组的1.00±0.11,差异有统计学意义(t=2.98,P=0.041);Lv-RNF43-OE组PD-L1 mRNA相对表达量为0.32±0.09,明显低于Lv-Ctrl-OE组的1.00±0.09,差异有统计学意义(t=9.13,P=0.001)。双荧光素酶报告基因实验结果显示,pCMV6-NC组、RNF43组、RNF43^(+)β-catenin组和β-catenin组PD-L1启动子荧光素酶活性分别为1.00±0.00、0.84±0.00、1.49±0.00和1.57±0.03(F=2218.33,P<0.001),进一步两两比较发现,与pCMV6-NC组相比,RNF43组PD-L1启动子荧光素酶活性明显降低(P<0.001),RNF43^(+)β-catenin组和β-catenin组PD-L1启动子荧光素酶活性明显升高(P<0.001;P=0.003);与RNF43组相比,RNF43^(+)β-catenin组PD-L1启动子荧光素酶活性明显升高(P<0.001)。结论RNF43可能通过抑制β-catenin的表达降低黑色素瘤中PD-L1 mRNA的表达并促进CD8^(+)T细胞介导的抗肿瘤免疫反应。

Objective To investigate the regulatory effects of ring finger protein 43(RNF43)on CD8^(+)T cell-mediated anti-tumor immune reaction in melanoma.Methods RNF43 gene was over-expressed and knockdown in mouse melanoma cells line B16-OVA by lentivirus infection;In vivo proliferation 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 interferonγ(IFN-γ)in tumor immune microenvironment of melanoma were detected by flow cytometry;The expression levels ofβ-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.Results Stable 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 9034±2628,which was significantly higher than that in the Lv-Ctrl-OE group(3847±1637),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(2226±646),with a statistically significant difference(t=3.16,P=0.034);The fluorescence intensity of IFN-γof CD8^(+)T cell in the Lv-RNF43-OE group was 2422±429,which was significantly higher than that of 1688±324 in the Lv-Ctrl-OE group,with a statistically significant difference(t=-2.73,P=0.034);The fluorescence intensity of IFN-γof 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 1159(1152,2068)in the Lv-Ctrl-KD group.The results of quantitative real-time PCR showed that the relative expression level ofβ-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 andβ-catenin groups were 1.00±0.00,0.84±0.00,1.49±0.00 and 1.57±0.03(F=2218.33,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 andβ-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).Conclusion RNF43 may reduce the expression of PD-L1 mRNA in melanoma by inhibiting the expression ofβ-catenin and promote CD8^(+)T cell-mediated anti-tumor immune reaction.