核转运蛋白α2基因沉默对黑色素瘤细胞增殖和侵袭能力的影响

The effects of the nuclear transporter karyopherin alpha2 gene silencing on the cell proliferation and invasion of melanoma cells

目的 基于肿瘤基因组图谱(TCGA)数据库探讨核转运蛋白α2(KPNA2)在黑色素瘤中的表达及KPNA2基因沉默后对黑色素瘤细胞增殖、侵袭的影响。方法 通过TCGA数据库检索黑色素瘤组织中KPNA2 mRNA表达水平及其与患者预后的关系,分析黑色素瘤中KPNA2的共表达基因并对基因进行基因本体论(GO)、京都基因与基因组百科全书(KEGG)富集分析。检测黑色素瘤细胞系中KPNA2表达水平,选择KPNA2高表达细胞系A375,瞬时转染小干扰RNA敲低KPNA2的表达。采用CCK-8法、Transwell实验和蛋白印迹法(Western blot)分析KPNA2基因沉默对黑色素瘤细胞系A375增殖、侵袭能力和磷酸化P53(p-P53)蛋白表达水平的影响。结果 黑色素瘤组织中KPNA2 mRNA相对表达水平为6.439(5.800, 6.958),高于正常组织的4.035(1.726, 3.538),差异有统计学意义(P<0.001)。KPNA2高表达患者中位生存时间为63.9个月,短于KPNA2低表达患者的104.6个月,差异有统计学意义(P=0.011)。GO、KEGG富集分析显示,黑色素瘤中KPNA2共表达基因涉及的信号通路主要包括P53信号通路、细胞循环等,涉及的细胞组分包括细胞间桥等,生物学过程包括细胞循环周期等,分子功能包括DNA复制起点结合等。KPNA2在黑色素瘤细胞系A375、A875和SK-MEL-28中均有表达,且在A375细胞系中表达量更高,故选用A375细胞进行后续研究。CCK-8法检测结果显示,培养72、96 h后,KPNA2基因沉默后的A375细胞增殖能力低于空白对照细胞和空载细胞,差异有统计学意义(P<0.05);Transwell实验结果显示,KPNA2基因沉默后的A375细胞侵袭能力低于空白对照细胞和空载细胞,差异有统计学意义(P<0.05)。Western bolt结果显示,KPNA2基因沉默后的A375细胞中p-P53蛋白表达水平高于空白对照细胞和空载细胞,差异有统计学意义(P<0.05)。结论 KPNA2 mRNA在黑色素瘤中显著高表达,且与患者的不良预后有关。KPNA2基因沉默可促进抑癌基因P53的磷酸化,从而调控P53信号通路参与黑色素瘤细胞的增殖和侵袭。

Objective To investigate the nuclear transporter karyopherin alpha2(KPNA2)mRNA expression in melanoma based on the Cancer Genome Atlas(TCGA)database and to further explore the efficacy on proliferation and invasion of melanoma cells after KPNA2 gene silencing.Methods The TCGA database was used to search the literatures relating KPNA2 mRNA expression level in melano⁃ma tissues and its relationship with prognosis of patients.The co⁃expressed genes of KPNA2 in mela⁃noma were analyzed by Gene Ontology(GO),Kyoto Encyclopediaof Genes and Genomes(KEGG)enrichment analysis.The expression level of KPNA2 mRNA in the melanoma cell line was detected,and the KPNA2 higher expression cell line A375 was selected.The small interfering RNA was transi⁃ently transfected to knock down the expression of KPNA2.The CCK⁃8,Transwell assay and Western blot methods were used to analyze the efficacy on the proliferation, invasion and phosphorylated P53(p⁃P53) protein expression level in melanoma cell line A375 after KPNA2 gene silencing. Results Therelative expression level of KPNA2 mRNA in melanoma tissues were 6. 439 (5. 800, 6. 958), whichwas significantly higher than 4. 035 (1. 726, 3. 538) in the normal tissues(P <0. 001). The medi⁃an survival time of KPNA2 high expression patients was 63. 9 months, which was lower than 104. 6months in the low expression patients(P = 0. 011). The KEGG signaling pathway involved inKPNA2 co⁃expressed genes in melanoma were P53 signaling pathway and cell cycle. The cellularcomponents involved included intercellular bridges, etc. , the biological process included the cellcycle, etc. , and the molecular function included the origin of DNA replication combine, etc.KPNA2 were expressed in the melanoma cell lines A375, A875 and SK⁃MEL⁃28, and with a higherexpression in the A375 cell line. Therefore, A375 cells were selected for follow⁃up study. AfterKPNA2 gene silencing in melanoma A375 cell line, CCK⁃8 test results showed that the cell prolifera⁃tion ability was significantly decreased at 72 h and 96 h (P <0. 05). Transwell results showed thatthe cell invasion ability was significantly decreased compared with blank control cells and emptycells (P <0. 05). The Western blot results showed that after KPNA2 gene silencing in melanomaA375 cell line, the expression level of p⁃P53 protein was significantly increased compared withblank control cells and empty cells (P <0. 05). Conclusion KPNA2 mRNA is significantly over⁃expressed in melanoma and is associated with poor prognosis of melanoma patients. KPNA2 gene si⁃lencing can promote the phosphorylation of tumor suppressor gene P53, thereby regulating the P53signaling pathway to participate in the proliferation and invasion of melanoma cells.