Zebularine通过SFRP2/Dkk3去甲基化调控Wnt/β-catenin信号通路诱导食管癌细胞凋亡  

Zebularine induces apoptosis of esophageal cancer cells via demethylation SFRP2/Dkk3 to regulate Wnt/β-catenin signaling pathway

作　　者：张凤霞 张建东 单保恩[3] 储金秀 ZHANG Feng-xia;ZHANG Jian-dong;SHAN Bao-en;CHU Jin-xiu(Hebei Key Laboratory for Chronic Diseases of Basic Medical College,North China University of Science and Technology,Tangshan 063200,China;Department of Laboratory Medicine,the Third Hospital of Hebei Medical University,Shijiazhuang 050000,China;Research Center,the Fourth Hospital of Hebei Medical University,Shijiazhuang 050000,China;North China University of Science and Technology,Tangshan 063200,China)

机构地区：[1]华北理工大学基础医学院,河北省慢性疾病基础医学重点实验室,河北唐山063200 [2]河北医科大学第三医院检验科,河北石家庄050000 [3]河北医科大学第四医院科研中心,河北石家庄050000 [4]华北理工大学,河北唐山063200

出　　处：《药学学报》2021年第5期1384-1390,共7页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(81673642);河北省医学科学研究重点课题计划项目(20170640)。

摘　　要：研究去甲基化药物zebularine对食管癌细胞凋亡的影响及其作用机制。采用不同浓度(25、50、100、200和400μmol·L~(-1)) zebularine处理食管癌ECA109和KYSE170细胞, CCK-8法检测细胞活力。选用浓度为100μmol·L~(-1) zebularine处理细胞,流式细胞术检测凋亡率, Western blot检测凋亡蛋白(Bcl-2蛋白、Bax蛋白、cleaved-caspase-3蛋白和cleaved-PARP蛋白)和Wnt (wingless-related integration site)信号通路分子(β-catenin蛋白、cyclin D1蛋白和c-Myc蛋白)的表达量,实时荧光定量PCR检测Wnt信号通路上游负调控基因表达水平,甲基化特异性PCR(methylation specific PCR, MSP)法检测人分泌型卷曲相关蛋白2 (secreted frizzled related protein 2, SFRP2)和Dkk3(dickkopf 3)基因的甲基化状态。敲低SFRP2和Dkk3观察对zebularine诱导凋亡的影响。结果显示, zebularine能抑制ECA109和KYSE170细胞的活力,呈剂量和时间依赖性;zebularine可诱导细胞凋亡,下调Bcl-2蛋白,上调Bax、cleaved-caspase-3和cleaved-PARP蛋白的表达,并抑制β-catenin、cyclin D1和c-Myc蛋白表达,与阴性对照组比较,差异均具有统计学意义(P <0.05);zebularine作用后, Dkk3和SFRP2的mRNA表达水平明显升高, SFRP2和Dkk3启动子甲基化水平降低;敲低SFRP2和Dkk3可降低zebularine诱导的细胞凋亡。综上所述, zebularine降低SFRP2和Dkk3基因启动子甲基化水平,促进SFRP2和Dkk3基因表达,进而通过抑制Wnt/β-catenin信号通路诱导食管癌细胞凋亡。To explore the effect and mechanisms of demethylation drug zebularine on esophageal cancer cells apoptosis,ECA109 cells and KYSE170 cells were treated with zebularine at different concentrations(25,50,100,200,and 400μmol·L-1).The cell viability was measured by CCK-8.Flow cytometry was used to detect the cell apoptosis rate,Western blot was performed to determine the expression of apoptosis protein(Bcl-2,Bax,cleavedcaspase-3,and cleaved-PARP)and Wnt signal pathway molecules(β-catenin,cyclin D1,and c-Myc),real-time quantitative PCR was used to detect the expression level of negative regulatory genes of Wnt signaling pathway,methylation specific PCR(MSP)was used to detect the methylation status of secreted frizzled related protein 2(SFRP2)and dickkopf 3(Dkk3)genes.After knockdown of SFRP2 and Dkk3,the effect of zebularine on apoptosis was detected.The studies showed that zebularine could inhibit the activity of ECA109 and KYSE170 cells in a dose-dependent and time-dependent manner;zebularine could induce cell apoptosis,down-regulate the expression of Bcl-2 protein,up-regulate the expression of Bax,cleaved-caspase-3,and cleaved-PARP protein,and inhibit the expression ofβ-catenin,cyclin D1,and c-Myc protein(P<0.05);the mRNA expression levels of Dkk3 and SFRP2 were significantly up-regulated by zebularine,while the methylation levels of SFRP2 and Dkk3 promoters were decreased;knockdown of SFRP2 and Dkk3 could reduce the apoptosis induced by zebularine.In summary,zebularine could reduce the methylation level of SFRP2 and Dkk3 gene promoter,promote the expression of SFRP2 and Dkk3 gene,and then induce the apoptosis of esophageal cancer cells by inhibiting Wnt/β-catenin signaling pathway.

关 键 词：食管癌 ZEBULARINE 细胞凋亡 人分泌型卷曲相关蛋白2 dickkopf 3 WNT/Β-CATENIN信号通路 

分 类 号：R966[医药卫生—药理学]