泛素结合酶2S参与调控黑素瘤行为机制的生物信息学分析

Mechanisms regulating melanoma by the ubiquitin-conjugating enzyme E2S:a bioinformatics analysis

目的揭示黑素瘤中可能与泛素结合酶2S(UBE2S)存在相互作用的基因,探讨UBE2S参与调控黑素瘤细胞生物学行为的分子机制。方法将A375黑素瘤细胞分为基因敲降组和阴性对照组,分别用含LV-UBE2S-RNAi(14011-1)的慢病毒和CON053慢病毒转染细胞构建UBE2S敲降细胞株和阴性对照细胞株。提取两组细胞株RNA,并将其纯化及片段化,与芯片探针杂交洗染获取芯片数据。采用Ingenuity路径分析软件对获得的差异表达基因的芯片数据进行进一步解析,鉴定可能与UBE2S存在相互作用的基因,应用免疫印迹法对UBE2S调控的下游分子进行验证。采用双尾t检验筛选差异基因。结果在基因敲降组与阴性对照组之间共筛选出差异倍数绝对值>2且P<0.05的差异基因512个,其中上调基因247个,下调基因265个。差异基因信息的Ingenuity路径分析结果证实,干扰素信号和肝X受体/视网膜X受体活化通路显著激活,真核起始因子2和核因子κB信号通路则被抑制。预测上游调控因子中IFNA2为强烈激活,核因子κB(复合物)被抑制。综合以上生物信息学分析结果,推测黑素瘤细胞UBE2S基因可通过调节IFITM1、STAT1、ISG15和TNFRSF11B基因的表达发挥功能。免疫印迹显示,A375细胞UBE2S基因敲降前后,IFITM1蛋白均未表达,敲降后ISG15蛋白表达上调19.94倍,STAT1蛋白表达上调1.47倍,TNFRSF11B蛋白表达下调79.1%。结论UBE2S可能通过与STAT1、ISG15和TNFRSF11B的相互作用,共同调控黑素瘤肿瘤细胞生物学行为。

Objective To identify genes interacting with the ubiquitin-conjugating enzyme E2S(UBE2S)in melanoma,and to explore the molecular mechanisms regulating the biological behavior of melanoma cells by UBE2S.Methods Cultured A375 melanoma cells were divided into 2 groups to be transfected with LV-UBE2S-RNAi(14011-1)-containing lentivirus(gene knockdown group)and a negative control lentivirus CON053(negative control group),respectively,and the UBE2S gene-knockdown cell line and negative control cell line were established.RNA was extracted from the two cell lines,purified,fragmented,and hybridized with GeneChip probes,followed by washing and dyeing,and then microarray data were obtained.Ingenuity pathway analysis(IPA)software was used to further analyze the gene expression profiling data to identify potential genes interacting with UBE2S.Western blot analysis was performed to verify the downstream molecules regulated by UBE2S.A two-tailed t test was used to screen for differentially expressed genes.Results A total of 512 differentially expressed genes with a|fold change|>2 and a P value<0.05 were screened out between the gene knockdown group and negative control group,including 247 upregulated genes and 265 downregulated genes.IPA of differentially expressed genes revealed that interferon signaling and liver X receptor(LXR)/retinoid X receptor(RXR)activation pathways were significantly activated,while eukaryotic initiation factor-2(EIF2)signaling and nuclear factor-κB(NF-κB)signaling pathways were inhibited.Among the upstream regulators,IFNA2 was predicted to be strongly activated,and NF-κB(complex)to be strongly inhibited.Based on the above bioinformatics analysis results,it was suggested that the UBE2S gene may exert some effects in melanoma cells by regulating the expression of IFITM1,STAT1,ISG15 and TNFRSF11B genes.Western blot analysis showed that IFITM1 protein was not expressed in A375 cells before and after the UBE2S gene knockdown.After the UBE2S gene knockdown,ISG15 protein expression was upregulated by 19.94 times,STAT1 protein expression was upregulated by 1.47 times,and TNFRSF11B protein expression was downregulated by 79.1%.Conclusion UBE2S may interact with STAT1,ISG15 and TNFRSF11B to regulate the biological behavior of melanoma cells.