干扰Kelch样家族蛋白21对黑色素瘤细胞增殖、迁移的影响

Effects of interfering Kelch-like family member 21 on proliferation and migration of melanoma cells

目的:研究干扰Kelch样家族蛋白21(KLHL21)后对黑色素瘤细胞增殖和迁移的影响。方法:采用qRT-PCR和Western blot法检测黑色素瘤细胞A375和正常细胞中KLHL21 mRNA和蛋白表达水平。将A375细胞分为3组:正常对照组、siNC组(转染阴性对照siRNA)和siKLHL21组(转染KLHL21特异性siRNA)。转染6 h后,采用CCK-8法、EdU染色法检测细胞增殖能力,采用Transwell小室检测细胞迁移能力。从TCGA数据库下载458例黑色素瘤患者的RNA-seq数据,利用TIME分析黑色素瘤患者中KLHL21表达水平与免疫细胞浸润水平的关系。选择黑色素瘤治疗中常用的8个免疫检查点抑制剂相关基因,应用Spearman相关性分析其与KLHL21表达水平的相关性。结果:黑色素瘤细胞中KLHL21 mRNA和蛋白表达水平均升高。与其他2组相比,siKLHL21组细胞中KLHL21 mRNA和蛋白表达水平降低,细胞增殖、迁移能力降低(P<0.05)。KLHL21表达与免疫浸润性细胞CD4^(+)T细胞浸润水平呈负相关(r_(S)=-0.110,P=0.019),与CD8^(^(+))T细胞、树突细胞浸润水平呈正相关(r_(S)=0.213、0.143,P<0.05)。基因表达相关性分析显示KLHL21与免疫检查点抑制剂相关基因PD-1、PD-L2、CTLA4、CD4、CD8A和CD80等的表达水平呈负相关(P<0.05)。结论:KLHL21在黑色素瘤细胞中高表达,下调KLHL21表达能够抑制黑色素瘤细胞的增殖和迁移,且KLHL21与免疫检查点相关基因相关。

Aim:To investigate the role of kelch-like family member 21(KLHL21) in the proliferation and migration of cutaneous melanoma cells.Methods:qRT-PCR and Western blot were used to detect mRNA and protein expression levels of KLHL21 in melanoma A375 cells and normal cells.A375 cells were divided into three groups, the cells transfected with KLHL21 siRNA were the interference group, the cells transfected with negative control siRNA were the negative control group, and the untransfected cells were the normal control group.After 6 hours, the proliferation and migration of A375 cells were detected using CCK-8 assay, EdU staining, and Transwell assay.RNA-seq data of 458 melanoma patients were downloaded from the TCGA database, and the relationship between KLHL21 and the level of immune cell infiltration in melanoma patients was analyzed using TIME.8 immune checkpoint inhibitor-related genes commonly used in melanoma treatment were selected and Spearman correlation analysis was applied to assess their correlation with KLHL21 expression levels.Results:Both mRNA and protein expression levels of KLHL21 were increased in A375 cells.Compared with the other two groups, the expression levels of KLHL21 mRNA and protein in the interference group were decreased, and the ability of cell proliferation and migration were decreased(P<0.05).Further analysis revealed that KLHL21 was negatively correlated with the infiltration level of CD4^(+)T cells(r_(S)=-0.110,P=0.019),and positively correlated with the infiltration level of CD8^(+)T cells and dendritic cells(r_(S)=0.213,0.143,P<0.05).Gene expression correlation analysis showed that KLHL21 was significantly negatively correlated with immune checkpoint inhibitor-related genes PD-1,PD-L2,CTLA4,CD4,CD8A and CD80(P<0.05).Conclusion:KLHL21 is highly expressed in the melanoma cells.Down-regulation of KLHL21 expression could inhibit the proliferation and migration of melanoma cells.