小鼠前脂肪细胞分化过程中STAU1介导Foxp1 mRNA降解机制初探

A study on mechanism of STAU1-mediated Foxp1 mRNA degradation during differentiation of mouse preadipocytes

目的 探究小鼠前脂肪细胞分化过程中STAU1对Foxp1 mRNA转录后的调控作用。方法 培养3T3-L1前脂肪细胞,采用鸡尾酒法诱导其分化为成熟脂肪细胞,分别收取第0天和第4天样本进行RNA测序筛选出差异倍数(FC)>2的转录因子;提取第0、1、2、3、4天细胞总RNA进行实时荧光定量聚合酶链式反应(RT-qPCR)实验验证。RBPDB数据库预测Foxp1的RNA结合蛋白。转染空载体质粒和STAU1过表达质粒后将细胞分为对照组和STAU1过表达组,提取第0、12、24、36、48小时细胞总RNA。利用RT-qPCR及Western-Blot实验检测两组3T3-L1细胞STAU1过表达效率、分化过程中Foxp1的mRNA和蛋白质表达水平。10只雄性2~3周龄C57BL/6小鼠的腹股沟白色脂肪组织(inguinal white adipose tissue, iWAT)分离培养的血管基质部分(Stromal vascular fraction, SVF)细胞,鸡尾酒法诱导分化,感染慢病毒,利用免疫荧光实验检测SVF细胞中Foxp1的蛋白质表达水平,用catRAPID软件进行预测。RNA免疫共沉淀(RNA binding protein immunoprecipitation, RIP)实验检测STAU1与Foxp1 mRNA 3′非翻译区(3′un-translated region, 3′UTR)STAU1结合位点(Staufen binding site, SBS)的结合情况。转染质粒24 h后加入放线菌素D,提取第0、1、2、4、6、8小时细胞总RNA,利用RT-qPCR实验检测两组3T3-L1细胞STAU1对Foxp1 mRNA稳定性的影响。结果 经RNA-seq筛选出5个表达上调和5个表达下调的转录因子;RT-qPCR验证结果显示,在成脂过程中Foxp1 mRNA的表达逐渐下降,Pparγ mRNA的表达逐渐上调;RBPDB数据库结果显示,STAU1是Foxp1的RNA结合蛋白;与对照组比较,STAU1过表达组STAU1的mRNA和蛋白质表达水平显著升高(P<0.05),Foxp1的mRNA和蛋白质表达水平均显著降低(P<0.05);免疫荧光结果显示,与对照组比较,STAU1过表达组中Foxp1相对荧光强度降低(P<0.05);RNA免疫沉淀实验后的PCR验证结果显示,STAU1与Foxp1 mRNA 3′UTR的6 714~7 000 bp存在结合;与对照组比较,STAU1过表达组Foxp1的mRNA稳定性显著下降(P<0.05)。结论 STAU1通过结合在Foxp1 mRNA 3′UTR上,启动STAU1介导的mRNA降解途径(Staufen-mediated decay, SMD)降解其mRNA,下调Foxp1的mRNA表达水平。

Objective To investigate the regulatory effect of STAU1 on transcription of Foxp1 mRNA during differentiation of mouse preadipocytes.Methods 3T3-L1 preadipocytes were cultured,and the differentiation into mature adipocytes were induced by cocktail method,and RNA sequencing was performed on day 0 and day 4 samples to screen out transcription factors with FC differential multiples>2.The total RNA of cells on days 0,1,2,3 and 4 were extracted for real-time quantitative polymerase chain reaction(RT-qPCR)experimental verification.Bioinformatics(RBPDB database)predicted RNA-binding protein of Foxp1.After transfection of empty vector plasmid and STAU1 overexpression plasmid,cells were divided into control group and STAU1 over expression group,and total RNA of cells at 0,12,24,36,and 48 hours were extracted.RT-qPCR and Western-Blot experiments were used to investigate the STAU1 over-expression efficiency and the mRNA and protein expression levels of Foxp1 during differentiation in two groups of 3T3-L1 cells.10 male 2~3 week-old C57BL/6 mice were isolated and cultured stromal vascular fraction(SVF)cells of inguinal white adipose tissue(iWAT),cocktail method induced differentiation,after infection with lentivirus,and the protein expression level of Foxp1 in SVF cells were detected by immunofluorescence experiments with using catRAPID software.RNA binding protein immunoprecipitation(RIP)experiments detected binding of STAU1 to Staufen binding site(SBS)of Foxp1 mRNA 3'untranslated region(3'UTR).Actinomycin D was added 24 hours after plasmid transfection,and the total RNA of cells at 0,1,2,4,6 and 8 hours were extracted,and the effect of STAU1 on the stability of Foxp1 mRNA in two groups of 3T3-L1 cells were detected by RT-qPCR experiment.Results RNA-seq screened out five transcription factors with up-regulated expression and five down-regulated expressions,and the RT-qPCR verification results showed that the expression of Foxp1 mRNA gradually was decreased during the adipogenic process,and the expression of PparγmRNA was gradually up-regulated.RBPDB database predictions showed that STAU1 was an RNA-binding protein of Foxp1.Compared with the control group,the expression levels of mRNA and protein of Foxp1 gene in the STAU1 overexpression group were significantly reduced(P<0.05).The immunofluorescence results showed that the relative fluorescence intensity of Foxp1 in the STAU1 overexpression group was reduced(P<0.05)compared with the control group.PCR results after RNA immunoprecipitation showed that STAU1 was bound to 6714-7000 bp of Foxp1 mRNA 3'UTR.Compared with the control group,the mRNA stability of Foxp1 in the STAU1 overexpression group was significantly reduced(P<0.05).Conclusion STAU1 initiates the staufen1-mediated mRNA degradation pathway(Staufen-mediated decay,SMD)to degrade its mRNA by binding to Foxp1 mRNA 3'UTR,thereby down-regulating the mRNA expression level of Foxp1.