摘要
目的探讨丝氨酸-精氨酸蛋白激酶2(SRPK2)与前列腺癌细胞周期的相关性。方法通过皮尔森相关性分析探索癌症基因组图谱(TCGA)数据库中前列腺癌和SRPK2的相关基因集, 并进一步将SRPK2相关基因集导入蛋白质相互作用关系检索工具(STRING)蛋白互作网络在线分析网站, 通过Cytoscape软件进行可视化, 提取与SRPK2相关联蛋白子网络;将上述分析所得基因通过京都基因和基因组百科全书(KEGG)数据集分析出与SRPK2潜在相关的通路, 运用基因探针富集分析(GSEA)富集分析探索SRPK2所激活和抑制的相关通路;构建SRPK2敲低的前列腺癌细胞株DU145, 分为空白载体组(DU145-KO-NC)和敲低组(DU145-KO-SRPK2), 通过细胞周期检测试剂盒、细胞增殖-毒性检测试剂盒、划痕实验、侵袭实验检测细胞的周期变化、增殖、迁移和侵袭能力, 组间比较采用独立样本t检验。结果 TCGA数据库分析结果显示有2 158个基因在表达量上与SRPK2呈正相关, 779个基因与SRPK2呈负相关;通过STRING蛋白互作网络在线分析, SRPK2与SF3B1、SRSF10、PRPF40A、PRPF4B、DDX46、BCLAF1、NUDT21、CLASRP等基因除了在表达量上存在统计学相关性, 在蛋白层面上同样存在着相互作用的关系。对2 937个与SRPK2相关的基因进行功能和通路分析, 结果显示SRPK2与细胞周期相关通路相关, 如有丝分裂纺锤(Mitotic Spindle), G2~M期检查点(G2~M Checkpoint), E2F靶点(E2F Targets), Myc-V1靶点(Myc-V1 Targets)通路。KEGG数据集表明与SRPK2潜在相关的通路有细胞周期(Cell Cycle)、核糖体(Ribosome)、线粒体自噬(Mitophagy)、凋亡(Apoptosis)等。通过进一步GSEA富集分析, 结果显示SRPK2同样在前列腺癌中激活细胞周期相关通路, 如有丝分裂纺锤体(Mitotic Spindle), G2~M期检查点(G2~M Checkpoint), E2F靶点(E2F Targets)(NES>0)。为了进一步验证SRPK2对细胞周期的影响, 成功构建SRPK2敲低的DU145细胞株, 结果提示敲低SRPK2基因后, 敲低组的S期高于空白载体组(DU145-KO-NC比DU145-KO-SRPK2为27.053±0.860比33.297±0.883, t=-7.166, P<0.01), 而敲低组的增殖(DU145-KO-NC比DU145-KO-SRPK2为0.085±0.002比0.344±0.042, t=10.655;P<0.01)、迁移(30 h:DU145-KO-NC比DU145-KO-SRPK2为0.929±0.066比0.690±0.057, t=6.101, P<0.01)、侵袭能力(DU145-KO-NC比DU145-KO-SRPK2为81.667±4.163比42.333±7.572, t=5.750, P<0.05)低于空白载体组, 差异均具有统计学意义。结论 SRPK2可能通过促进细胞周期从而影响前列腺癌进展。
Objective To study the correlation between serine-arginine protein kinase 2(SRPK2)and prostate cancer cell cycle.Methods The relevant gene set of SRPK2 and prostate cancer in the TCGA database was explored by Pearson correlation analysis,and the SRPK2-related gene set was further imported into STRING website and Tshown by Cytoscape.The genes from the above analysis were imported into KEGG data set to analyze the potentially related pathways of SRPK2.Furthermore,the GSEA enrichment analysis was used to explore the relevant pathways of activation and inhibition in the high expression state of SRPK2.To check the findings above,we successfully constructed the SRPK2 knock-out prostate cancer cell strain on DU145 cancer cell line,which was divided into DU145-KO-NC group and DU145-KO-SRPK2 group.The periodic changes,proliferation,migration and invasive abilities of cancer cells were detected through cell cycle assay,CCK8 cell proliferation and toxicity detection kit,wound healing assay and Transwell experiment.The results were analyzed by independent-samples T test.Results The TCGA database analysis showed that 2158 genes were positively correlated with SRPK2 and 779 genes were negatively correlated with SRPK2.Online analysis was performed through the STRING website,revealing the genes of SRPK2 and SF3B1,SRSF10,PRPF40A,PRPF4P,DDX46,BCLAF1,NUDT21 and CLASRP were related with SRPK2 both in gene and protein levels.Functional and pathway analysis of 2,937 SRPK2-related genes showed that SRPK2 was associated with cell cycle-related pathways such as mitetic spindle,G2-M checkpoint,E2F targets,and Myc targets pathways.Through the KEGG data set we concluded that the pathways potentially related to SRPK2 were Ribosome,mitophagy,apoptosis,etc.Similarly,GSEA enrichment analysis showed that SRPK2 also activated cell cycle-related pathways in prostate cancer,such as matic spindle,G2-M checkpoint,E2F targets(NES>0).In order to further verify the effect of SRPK2 on cell cycle,the DU145 cell strain of SRPK2 knock-down was successfully constructed,and the results showed that after knocking down the SRPK2 gene,the S phase of DU145-KO-SRPK2 group was longer than that of DU145-KO-NC group(DU145-KO-NC vs.DU145-KO-SRPK2:27.053±0.860 vs.33.297±0.883,t=-7.166,P<0.01),while the proliferation of DU145-KO-SRPK2 group(DU145-KO-NC vs.DU145-KO-SRPK2:0.085±0.002 vs.0.344±0.042,t=10.655;P<0.01),migration(30 h:DU145-KO-NC vs.DU145-KO-SRPK2:0.929±0.066 vs.0.690±0.057,t=6.101,P<0.01),invasion ability(DU145-KO-NC vs.DU145-KO-SRPK2:81.7±4.16 vs.42.3±7.57,t=5.750,P<0.05)was significantly reduced as compared with that of DU145-KO-NC group.Conclusion SRPK2 may affect the progression of prostate cancer by promoting cell cycle.
作者
卓扬佳
张益勋
林俊东
冯源发
邹志豪
谢文杰
李玥娇
邹芬
钟惟德
Zhuo Yangjia;Zhang Yixun;Lin Jundong;Feng Yuanfa;Zou Zhihao;Xie Wenjie;Li Yuejiao;Zou Fen;Zhong Weide(Department of Urology,Guangzhou First People’s Hospital,the Second Affiliated Hospital of South China University of Technology,Guangzhou 510180,China)
出处
《中华实验外科杂志》
CAS
北大核心
2022年第2期303-307,共5页
Chinese Journal of Experimental Surgery
基金
广东省区域联合基金-青年基金(2020A1515110640)
广州市第一人民医院红棉计划资助项目(Q2019020)
广州市第一人民医院博士科研启动经费(KYQD0035)。
关键词
前列腺癌
细胞周期
生物信息分析
肿瘤进展
Prostate cancer
Cell cycle
Bioinformatics analysis
Tumor progression
