DANCR在人胚胎干细胞向内胚层分化中的调控功能

DANCR Regulates hESC Differentiation Towards Definitive Endoderm

【目的】探讨DANCR在人胚胎干细胞(hESC)向内胚层(DE)分化中的作用。【方法】建立体外诱导hESC向DE分化的体系,检测DANCR的表达水平与DE分化的相关性;利用慢病毒体系敲低hESC的DANCR表达水平,将敲低DANCR的hESC进行DE分化;用qPCR和Western blot方法检测DE分子标志物SOX17和FOXA2,以及原条分子标志物Brachyury(T),EOMES,MIXL1和GSC的表达水平;用Dual luciferase reporter assay和qPCR证明在DE分化过程中DANCR与WNT通路的相互作用。【结果】体外诱导hESC向DE分化的体系能够有效模拟体内DE分化,DANCR的表达水平随DE分化进程逐渐下降。建立敲低DANCR的hESC细胞株,DANCR表达水平相比对照组显著降低(P<0.001)。干扰DANCR表达使分化早期的原条分子标志物Brachyury(T),EOMES,MIXL1和GSC,以及分化后期的DE分子标志物SOX17和FOXA2的mRNA表达水平相比对照组显著降低(全部P<0.05)。并且,在DE分化过程中,敲低DANCR组的WNT通路的转录活性相比对照组显著降低(P<0.05),表现为WNT通路下游基因FZD5,FZD8,SFRP1,FRZB和ANKRD6 mRNA表达水平明显减少(P<0.05)。然而,敲低DANCR对TGFβ通路的SMAD2/3和p-SMAD2蛋白表达水平没有显著影响(P>0.05)。人为激活细胞内β-CATENIN蛋白活性,能够有效回补由于敲低DANCR导致的DE分化缺陷。【结论】DANCR在DE分化过程中逐渐下调并促进DE分化发生,DAN⁃CR可能通过与WNT通路相互作用参与调控hESC向DE分化。

【Objective】To explore the function of DANCR during the differentiation of human embryonic stem cells(hESC)toward definitive endoderm(DE).【Methods】The in vitro DE differentiation system was established and its efficiency was verified.The correlation between the expression level of DANCR and DE differentiation process was detected.Using lentivirus system,we stably knocked down DANCR in hESC.The shDANCR hESC line was applied to DE differentiation,using qPCR and Western blot to detect the expression of DE marker genes SOX17 and FOXA2,and that of primitive streak marker genes Brachyury(T),EOMES,MIXL1 and GSC.Dual luciferase reporter assay and qPCR were used to confirm the interaction between DANCR and the WNT pathway during DE differentiation.【Results】The in vitro differentiation system mimicked DE differentiation efficiently.And the expression of DANCR was gradually downregulated during differentiation.DANCR was efficiently knocked down in the shDANCR hESC line(P<0.001).Compared with those in the control group,the expression levels of primitive markers Brachyury(T),EOMES,MIXL1 and GSC,as well as DE markers SOX17 and FOXA2,were significantly decreased in shDANCR groups(P<0.05).Furthermore,the transcriptional activity of the WNT pathway in shDANCR groups was lower than that in the control group(P<0.05).And RNA levels of downstream genes of the WNT pathway,FZD5,FZD8,SFRP1,FRZB and ANKRD6,were significantly decreased in shDANCR groups(P<0.05).However,differences in protein levels of the TGFβpathway effectors SMAD2/3 and p-SMAD2 were statistically insignificant in shDANCR and control groups(P>0.05).Forced activation ofβ-CATENIN rescued DANCR knock down-induced deficiency in DE differentiation.【Conclusions】The expression of DANCR decreases during DE differentiation.DANCR may promote DE differentiation through modulating the activity of the WNT pathway.