ABSTRACT
ObjectiveTo explore the function of DANCR during the differentiation of human embryonic stem cells （hESC） toward definitive endoderm （DE）. MethodsThe 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. ResultsThe 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 TGFβ pathway effectors SMAD2/3 and p-SMAD2 were statistically insignificant in shDANCR and control groups （P > 0.05）. Forced activation of β-CATENIN rescued DANCR knock down-induced deficiency in DE differentiation. ConclusionsThe expression of DANCR decreases during DE differentiation. DANCR may promote DE differentiation through modulating the activity of the WNT pathway.
ABSTRACT
@#【Objective】Using the CRISPR/Cas9（CRISPR/crispr-associated（Cas）9 method，a dual-target lentiviral vector containing single- guide RNAs（sgRNAs）targeting both the 5’and 3’ends of the anti- differentiation noncoding RNA（DANCR）gene was constructed. Stable knockout of DANCR gene in mesenchymal stem cells（MSC）would be helpful for the future study of the biological function of DANCR.【Methods】Designed sgRNAs targeting either the 5’or 3’ end of DANCR and cloned into two CRISPR vectors. The vector was transfected into 293FT cells，and the genomic DNA of 293FT cells was extracted to verify the efficiency of individual sequence. Two functional sgRNAs targeting either the 5’ or 3’end were incorporated into a same lentiviral CRISPR vector through gateway and enzymatic ligation. 293FT was used for lentiviral packaging，after which the virus was harvested to infect MSC，and the knockout efficiency of DANCR in MSC was detected.【Results】All four sgRNA sequences targeting DANCR successfully guided Cas9 to cleave the gene. sgRNAs targeting either the 5’and 3’end were combined to establish a dual-target lentiviral vector for stable knockout of DANCR. The vector was packaged into lentivirus and infected MSC. Finally，we successfully obtained mesenchymal stem cell lines with DANCR gene knockout.【Conclusions】Using the CRISPR method，a dual-target lentiviral vector can efficiently and stably knock out DANCR gene in MSC.
ABSTRACT
Long non-coding RNA( LncRNA) is an RNA molecule that is longer than 200 nucleotides and is not translated into a protein.LncRNA DANCR has been identified in hepatocellular carcinoma ( HCC) and markedly increased stemness features of HCC cells to promote tumorigenesis.Recent studies show that DANCR contributes to the differentiation and proliferation of synovium-derived mesenchymal stem cell ( SMSCs) and may be as a key point for this process.This article reviews the role of long non-coding RNA DANCR in enhancing chondrogenic differentiation and proliferation of human SMSCs.