SRPK1激活Wnt/β-catenin通路促进肝癌细胞上皮间充质转化  

Serine arginine protein kinase 1 promotes epithelial mesenchymal transformation in hepatoma cells through Wnt/β-catenin pathway activation

作　　者：石永杰[1] 陈旖鹛 黄思聪[1] 嘉红云[1] 周强[1] 魏洁[1] 肖绮雯[1] 康嘉乐[1] SHI Yongjie;CHEN Yimei;HUANG Sicong;JIA Hongyun;ZHOU Qiang;WEI Jie;XIAO Qiwen;KANG Jiale(Department of Clinical Examination,The Second Affiliated Hospital of Guangzhou Medical University,Guangzhou 510260,China;Department of Health And Physical Examination,The Second Affiliated Hospital of Guangzhou Medical University,Guangzhou 510260,China)

机构地区：[1]广州医科大学附属第二医院检验科,广东广州510260 [2]广州医科大学附属第二医院健康体检部,广东广州510260

出　　处：《西部医学》2023年第7期951-958,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)。

摘　　要：目的 探讨丝氨酸精氨酸蛋白激酶1(SRPK1)对肝癌细胞株HepG2上皮间充质转化(EMT)的作用。方法 通过Ualcan及TIMER 2.0数据库分析SRPK1 mRNA在肝细胞癌(LIHC)与正常样本、配对癌旁样本之间的表达差异,与生存时间、临床分期、病理分期、TP53变异、人种、性别、年龄、体重等临床资料的关系。用HPA数据库的免疫组化数据验证SRPK1蛋白在LIHC组织及正常对照间的表达差异。构建SRPK1过表达及抑表达的HepG2细胞,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。Western Blot、免疫荧光检测EMT分子标记物E-cadherin、Vimentin蛋白表达差异。核浆蛋白分离比较细胞β-catenin入核程度的变化。GEPIA2数据库分析在LIHC组织中SRPK1与Wnt/β-catenin通路下游基因Twist、MYC、MMP9的相关性,Real-time PCR验证SRPK1对Twist、MYC、MMP 9表达的影响。结果 SRPK1表达在LIHC组织中表达显著高于正常对照及配对癌旁样本(P<0.05),随疾病分期及病理分级增加而增加(P<0.05),高表达SRPK1组总生存期低于低表达组(P=0.035),LIHC患者TP53突变组SRPK1表达高于非突变组(P<0.05),在不同种族、性别、年龄、体重间无差别(P>0.05)。SRPK1过表达HepG2细胞E-cadherin表达下降,Vimentin表达增加(P<0.05);抑表达细胞E-cadherin增加,Vimentin下降(P<0.05)。SRPK1在LIHC组织中分别与Twist、MYC、MMP9表达呈正相关性(P<0.05);SRPK1过表达细胞β-catenin蛋白在细胞核中表达增加(P<0.05),Twist、MYC、MMP9表达增加(P<0.05);反之,抑表达细胞β-catenin在细胞核中表达下降(P<0.05),Twist、MYC、MMP9表达减少(P<0.05)。结论 SRPK1可能通过Wnt/β-catenin通路促进HepG2细胞EMT活化。Objective To investigate the effect of serine-Arginine protein kinase 1(SRPK1)on Epithelial mesenchymal transformation(EMT)in HepG2 cell line.Methods The differential expression of SRPK1 mRNA among liver hepatocellular carcinoma(LIHC)and normal samples and paired adjacent tissues were compared,and the relationship between the expression of SRPK1 and the clinicopathological characteristics of liver cancer including overall survival,clinical stage,pathological stage,TP53 mutant,race,sex,age,weight were analyzed by Ualcan and TIMER database.Immunohistochemical data from HPA database were used to verify the difference of SRPK1 protein expression between LIHC tissues and normal controls.HepG2 cells with SRPK1 overexpression and inhibition 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.The differential protein expressions of EMT molecular markers E-cadherin and Vimentin were detected by Western Blot and immunofluorescence.Nucleoplasmic protein isolation was used to compare the degree ofβ-catenin protein transport from Cytoplasm to Nucleus.GEPIA2 database was used to analyze the correlation between SRPK1 and Wnt/β-catenin pathway downstream genes including Twist,MYC and MMP9 in LIHC.Real-time PCR was used to verify the effect of SRPK1 on Twist,MYC and MMP9 expressions.Results The expression of SRPK1 in LIHC tissues was significantly higher than that in normal control and paired paracancer samples(P<0.05),and increased with worse stage and grade(P<0.05).The overall survival of high SRPK1 expression was lower than that of low SRPK1 expression(P=0.035),and the expression of SRPK1 in TP53 mutant group was higher than that in the TP53 non-mutant group(P<0.05).There was no difference among different races,genders,ages and weights(P>0.05).SRPK1 overexpressed HepG2 cells showed decreased E-cadherin and increased Vimentin(P<0.05).Consistently,E-cadherin increased and Vimentin decreased in SRPK1 inhibitied cells(P<0.05).SRPK1

关 键 词：丝氨酸精氨酸蛋白激酶1 肝细胞癌 HEPG2细胞 上皮间充质转化 WNT/Β-CATENIN通路 

分 类 号：R735.7[医药卫生—肿瘤]