丝氨酸精氨酸蛋白激酶1在体外促进HepG2细胞干性的获得  

Serine arginine protein kinase 1 promotes stemness acquisition of HepG2 cells in vitro

作　　者：肖绮雯 石永杰[2] 周强[2] 黄思聪[2] 康嘉乐[2] 嘉红云[2] 彭永正[1,3] XIAO Qiwen;SHI Yongjie;ZHOU Qiang;HUANG Sicong;KANG Jiale;JIA Hongyun;PENG Yongzheng(Department of Laboratory Medicine,Zhujiang Hospital,Southern Medical University,Guangzhou 510280,China;Department of Clinical Examination,The Second Affiliated Hospital of Guangzhou Medical University,Guangzhou 510260,China;Department of Transfusion Medicine,Zhujiang Hospital of Southern Medical University,Guangzhou 510280,China)

机构地区：[1]南方医科大学珠江医院检验医学部,广东广州510280 [2]广州医科大学附属第二医院检验科,广东广州510260 [3]南方医科大学珠江医院输血科,广东广州510280

出　　处：《西部医学》2022年第12期1729-1735,1742,共8页Medical Journal of West China

基　　金：广东省医学科学技术研究基金项目(A2021053,A2019189);广州市卫生健康科技项目(20211A011079,20191A010057)。

摘　　要：目的研究丝氨酸精氨酸蛋白激酶1(SRPK1)对肝癌细胞株HepG2肿瘤干性的作用。方法从GEPIA2数据库获得TCGA中关于肝细胞肝癌(LIHC)的数据,分析SRPK1表达在癌组织及正常样本的差异,以及与患者生存时间、临床分期的关系。采用TIMER数据库分析SRPK1表达与肝癌组织中浸润免疫细胞的相关性。构建SRPK1过表达和抑表达的HepG2稳转细胞株,并根据SRPK1表达差异分为SRPK1组及对照的Vector组,shRNA组及对照的Scramble组,Western blot检测4组细胞株SRPK1的蛋白水平。体外成球实验检测肿瘤细胞的成球能力,流式细胞术检测侧群(SP)细胞在细胞群中的比例。实时荧光定量PCR比较细胞肿瘤干性标记物Nanog、Oct4、CD133及Bmi1的mRNA水平。基因集合富集分析(GSEA)预测SRPK1与Wnt/β-catenin通路的相关程度。结果SRPK1表达在LIHC患者肿瘤组织中显著高于正常组织(P<0.05),在患者各个分期中表达有差异(P<0.05),高表达SRPK1患者生存率低于低表达SRPK1患者(P<0.05),肝癌组织中SRPK1与B细胞、CD8^(+)T细胞、CD4^(+)T细胞、巨噬细胞、中性粒细胞和树突细胞的浸润水平均相关(P<0.05)。Western blot结果显示SRPK1组SRPK1蛋白相对表达量高于Vector组(P<0.01),shRNA组则低于Scramble组(P<0.01)。SRPK1组肿瘤细胞成球率(SFE)、SP细胞比例、Nanog、Oct4、CD133及Bmi1的mRNA相对表达量均高于Vector组(P<0.05),shRNA组低于Scramble组(P<0.05)。GSEA结果显示LIHC中SRPK1高表达与Wnt/β-catenin通路活化正相关(P<0.05)。结论SRPK1促进HepG2细胞肿瘤干性的获得。Objective To investigate the effect of Serine-Arginine protein kinase 1(SRPK1)on tumor stemness in HepG2 cells.Methods GEPIA2(gene expression profiling interactive analysis 2)database was used to obtain the data about LIHC(liver hepatocellular carcinoma)in TCGA(The Cancer Genome Atlas)and analyze the difference in SRPK1 expression between liver cancer and normal samples,as well as the relationship with patient’s survival time and clinical stage.TIMER(Tumor Immune Estimation Resource)database was used to explore the correlation between SRPK1 expression and infiltrated immune cells in liver cancer tissues.HepG2 cells with overexpression and inhibition of SRPK1 protein were constructed and divided into the SRPK1 group,Vector group,shRNA group,and Scramble group.The protein level of SRPK1 was detected by Western blot.Sphere formation assay was used to detect the spheroidization ability of tumor cells,and flow cytometry was used to detect the proportion of side population(SP)cells in the cell population.The mRNA levels of tumor stemness markers,including Nanog,Oct4,CD133,and Bmi1,were compared by real-time PCR.GSEA was used to evaluate the relationship between SRPK1 and the Wnt/β-catenin pathway in LIHC.Results TCGA data showed that the expression of SRPK1 mRNA in LIHC tissues was significantly higher than that of normal controls(P<0.05),and there was a difference in the expression of SRPK1 mRNA in various stages of patients(P<0.05).The overall survival of patients with high expression of SRPK1 was significantly poorer than those with low SRPK1 expression(P<0.05).The expression of SRPK1 was significantly correlated with the infiltration levels of B cells,CD8^(+)T cells,CD4^(+)T cells,macrophages,neutrophils,and dendritic cells in liver cancer tissues(P<0.05).Western blot showed that the SRPK1 protein in the SRPK1 group was higher than that in the Vector group(P<0.01).SRPK1 group was higher than the Vector group in Tumor cell sphere formation efficiency(SFE),SP cell ratio,and mRNA of Nanog,Oct4,CD133,and Bmi1(P<0.05

关 键 词：丝氨酸精氨酸蛋白激酶1 HEPG2细胞 肿瘤干性 WNT/Β-CATENIN通路 

分 类 号：R735.7[医药卫生—肿瘤]