circFAT1对胶质母细胞瘤细胞生物过程的机制研究  

The mechanisms of circFAT1 on the biological process of GBM cells

作　　者：张立群 乌日吉木斯 郑晓明[2] 汪琳 韩玉秀 张薇 阎涛 ZHANG Liqun;WURI Jimusi;ZHENG Xiaoming;WANG Lin;HAN Yuxiu;ZHANG Wei;YAN Tao(Tianjin Neurological Institute,Tianjin Medical University General Hospital,Tianjin 300052,China;Department of Obstertrics and Gynecology,Tianjin First Central Hospital)

机构地区：[1]天津市神经病学研究所,天津医科大学总医院,300052 [2]天津市第一中心医院妇产科

出　　处：《天津医药》2023年第8期797-802,共6页Tianjin Medical Journal

摘　　要：目的探讨circFAT1吸附miR-1182调控转化生长因子(TGF)-β/Smad信号通路对胶质母细胞瘤(GBM)增殖、侵袭、上皮-间充质转化(EMT)的调控作用及机制。方法收集50例GBM样本和5例非瘤脑组织(NBT)样本,随机抽取3例GBM和3例NBT样本进行组织RNA测序。体外培养GBM细胞系U87、U251、T98G、LN229及星形胶质细胞系NHA,通过qRT-PCR检测组织和细胞系中circFAT1表达。通过RNase R选择性降解线性转录本以明确circFAT1成环特性。将GBM细胞分为sh-对照组和sh-circFAT1组,采用慢病毒进行转染。转染后集落形成实验、Transwell实验、Western blot实验分别检测细胞增殖、侵袭及EMT能力。荧光原位杂交(FISH)检测circFAT1在GBM细胞内定位,通过双萤光素酶报告基因和RNA免疫共沉淀(RIP)实验验证circFAT1和miR-1182的相互作用。Western blot实验验证敲低circFAT1后或抑制miR-1182表达对TGF-β/Smad信号通路的调控作用。结果circFAT1在GBM组织和细胞中表达增加(P<0.05)。RNase R可减少线性FAT1表达(P<0.05),而不影响circFAT1。敲除circFAT1抑制了GBM细胞的增殖、侵袭和EMT(P<0.05)。双萤光素酶报告基因和RIP实验证实circFAT1靶向结合miR-1182。敲低circFAT1后TGFB2、p-SMAD2、p-SMAD3蛋白表达下降,抑制miR-1182表达后GFB2、p-SMAD2、p-SMAD3蛋白表达升高。结论敲除circFAT1可抑制GBM细胞增殖、侵袭和EMT,其作用机制可能与吸附miR-1182调控TGF-β/Smad信号通路有关。Objective To investigate the regulation and mechanism of circFAT1 on proliferation,invasion and epithelial-mesenchymal transition(EMT)of glioblastoma(GBM).Methods Fifty GBM samples and 5 non-tumor brain tissue(NBT)samples were collected,and 3 GBM and 3 NBT samples were randomly selected for tissue RNA sequencing.The expression levels of circFAT1 in U87,U251,T98G,LN229 GBM cell lines and NHA astrocyte lines were detected by qRT-PCR.The cyclic properties of circFAT1 were determined by selective degradation of linear transcripts by RNase R.GBM cells were divided into the sh-control group and the sh-circFAT1 group.Cell proliferation,invasion and EMT were detected by colony formation assay,transwell assay and Western blot assay after lentivirus transfection.Fluorescence in situ hybridization(FISH)was used to detect the localization of circFAT1 in GBM cells,and the interaction between circFAT1 and miR-1182 was verified by dual-luciferase reporter and RNA immunocoprecipitation(RIP)assay.Finally,Western blot assay was performed to verify the regulatory effect of circFAT1 knockdown or inhibition of miR-1182 expression on TGF-β/Smad signaling pathway.Results The expression of circFAT1 was increased in GBM tissue and cells(P<0.05).RNase R reduced linear FAT1 expression without affecting circFAT1(P<0.05).Knockout of circFAT1 inhibited the proliferation,invasion and EMT of GBM cells(P<0.05).Dual-luciferase reporter and RIP assay confirmed that circFAT1 targeting on miR-1182.The protein levels of TGFB2,p-SMAD2 and p-SMAD3 were decreased after circFAT1 knockdown,while protein expressions of TGFB2,p-SMAD2 and p-SMAD3 increased after inhibiting the expression of miR-1182(all P<0.05).Conclusion Knockout of circFAT1 can inhibit proliferation,invasion and EMT of GBM cells,and its mechanism may be related to the regulation of TGF-β/Smad signaling pathway by sponge miR-1182.

关 键 词：胶质母细胞瘤 细胞增殖 转化生长因子Β 微RNAS circFAT1 上皮间充质转化 

分 类 号：R739.41[医药卫生—肿瘤]