摘要
目的探究敲低序列相似性83蛋白质家族成员D(FAM83D)对调节宫颈癌细胞干细胞特性的作用及其机制。方法收集手术切除的宫颈癌组织及对应癌旁组织,培养宫颈癌细胞株Hela、Siha、C33A及正常宫颈上皮细胞株H8,检测FAM83D及干细胞标志基因Sox2、Oct4的mRNA及蛋白表达水平。对Hela细胞进行分组,转染阴性对照(NC)siRNA、FAM83D siRNA,感染NC shRNA慢病毒、FAM83D shRNA慢病毒,联合使用对照溶剂二甲基亚砜(DMSO)或β-catenin通路激动剂SKL2001,检测FAM83D及干细胞标志基因Sox2、Oct4的mRNA及蛋白表达水平,并采用瘤球形成实验检测肿瘤干细胞特性。结果宫颈癌组织中FAM83D、Oct4、Sox2 mRNA相对表达水平均明显高于癌旁组织(P<0.001),且宫颈癌组织中FAM83D mRNA与Oct4、Sox2 mRNA相对表达水平均呈负相关(r=-0.351、-0.332,P<0.05)。Hela、Siha、C33A细胞中FAM83D、Oct4、Sox2 mRNA和蛋白相对表达水平均明显高于H8细胞(P<0.05),并且Hela细胞中FAM83D、Oct4、Sox2 mRNA和蛋白相对表达水平均明显高于Siha、C33A细胞(P<0.05)。转染siRNA或感染shRNA慢病毒敲低FAM83D后,Hela细胞的细胞球形成率及Oct4、Sox2、β-catenin mRNA和蛋白相对表达水平均明显降低(P<0.05);联合使用β-catenin通路激动剂SKL2001,Hela细胞的细胞球形成率及Oct4、Sox2 mRNA和蛋白相对表达水平均明显升高(P<0.05)。结论敲低FAM83D对宫颈癌细胞干细胞特性具有抑制作用,该作用与抑制Wnt/β-catenin通路有关。
Objective To investigate the effect and mechanism of knockdown of FAM83D on regulating stem cell characteristics of cervical cancer cells.Methods Surgically resected cervical cancer tissues and corresponding paracancerous tissues were collected,the cervical cancer cell lines Hela,Siha,C33A and normal cervical epithelial cell line H8 were cultured,the mRNA and protein expression levels of FAM83D and stem cell marker genes Sox2 and Oct4 were detected.The Hela cells were divided into the groups,transfected with negative control(NC)siRNA and FAM83D siRNA,infected with NC shRNA lentivirus and FAM83D shRNA lentivirus,and combined with the treatment of control solvent dimethyl sulfoxide(DMSO)or β-catenin pathway agonist SKL2001.The mRNA and protein expression levels of FAM83D and stem cell marker genes Sox2 and Oct4 were detected,and the characteristics of tumor stem cells were detected by the tumor sphere formation experiment.Results The relative mRNA expression levels of FAM83D,Oct4 and Sox2 in cervical cancer tissues were significantly higher than those in paracancerous tissues(P<0.001),moreover the FAM83D mRNA relative expression level in cervical cancer tissues was negatively correlated with Oct4 and Sox2 mRNA relative expression levels(r=-0.351,-0.332,P<0.05).The relative mRNA and protein expression levels of FAM83D,Oct4 and Sox2 in Hela,Siha and C33A cells were significantly higher than those in H8 cells(P<0.05),moreover the relative mRNA and protein expression levels of FAM83D,Oct4 and Sox2 in Hela cells were higher than those in Siha and C33A cells(P<0.05).After transfecting siRNA or infecting shRNA lentivirus to knock down FAM83D,the cell sphere formation rate and the mRNA and protein relative expression levels of Oct4,Sox2 andβ-catenin were significantly decreased(P<0.05).The combined use ofβ-catenin pathway agonist SKL2001,the cell sphere formation rate of Hela cells and the mRNA and protein relative expression levels of Oct4 and Sox2 were significantly increased(P<0.05).Conclusion Knockdown of FAM83D has
作者
张岚
王静依
刘述江
叶礼翠
吴欣瑜
ZHANG Lan;WANG Jingyi;LIU Shujiang;YE Licui;WU Xinyu(Department of Gynaecology and Obstetrics,Second Affiliated Hospital of Chengdu Medical College/Nuclear Industry 416 Hospital,Chengdu,Sichuan 610057,China)
出处
《重庆医学》
CAS
2023年第14期2107-2114,共8页
Chongqing medicine
基金
成都市卫生建康委员会科研课题(2021226)。
