The Role Of CDR1as In Proliferation And Differentiation Of Human Umbilical Cord Derived Mesenchymal Stem Cells

Objective: Human umbilical cord mesenchymal stem cells(hucMSCs)are widely used for tissue damage repair.Understanding the regulation and differentiation of hucMSCs is beneficial to improve the application of hucMSCs.Circular RNA(circRNA)are a new type of non-coding RNAs.It is reported that circRNA are abundantly in eukaryotic cells and invovled in multiple biological processes as cell proliferation,apoptosis and senescence.The aim of this study was to investigate the circRNAs expressed in hucMSCs and study the regulation of proliferation and differentiation of highly expressed CDR1 as in hucMSCs.Methods: The circBase database was searched and five circRNAs expressed in mesenchymal stem cells(MSCs)were selected.The relative expression of circRNAs in hucMSCs were determined by qRT-PCR,and confirmed the most abundant circRNA in hucMSCs.The adipogenic solution was used to induce the differentiation of hucMSCs,and different time points of RNA were collected.The expression of stemness transcription factors(STFs),adiponectin and circRNAs including CDR1 as were measured by qRT-PCR.Treatment of hucMSCs with 3,3'-diindolylmethane(DIM)and detection the expression levels of STFs and CDR1 as.Using siRNA to knockdown the expression of CDR1 as in hucMSCs and 293 T,and cell colony formation assay,CCK-8 assay and cell cycle analysis were used to evaluate the effect of CDR1 as on cell proliferation.Western Blot and flow cytometry were applied to detect the apoptosis associated proteins and the proportion of apoptotic cells,respectively.The changes of STFs such as Oct4,Sox2 and Nanog were tested by qRT-PCR and Western Blot.Both adipogenesis and osteogenesis assays were used to judge the differentiation potential of hucMSCs.Results: All five circRNAs we selected were found to be expressed in hucMSCs and CDR1 as was the most highly abundant.With induce time extended,the adiponectin gradually increased while the STFs such as Oct4,Nanog and Sox2 gradually decreased.Surprisingly,CDR1 as expression level also decreased with hucMSCsadipogenesis.After DIM treatment of hucMSCs,the expression levels of CDR1 as as well as Oct4,Nanog,Sox2 and Sall4 were significantly up-regulated.After CDR1 as knockdown,the proliferation of hucMSCs was remarkably retarded;the percentage of cells at G0/1 phase was increased while the cells at S and G2 phases were decreased;the protein level of PCNA was significantly decreased.In CDR1 as knockdown hucMSCs,the rate of apoptotic cells was significantly increased;the protein level of Bcl-2 decreased while the levels of Bax,actived caspase 3 and caspase 9 increased.The expression levels of STFs such as Oct4,Sox2,Nanog and Lin28 were down-regulated in cells with CDR1 as knockdown,while Sall4 did not change significantly.The differentiation potential of hucMSCs with CDR1 as knockdown was significantly impaired.Conclusion: CDR1 as was highly expressed in hucMSCs.CDR1 as knockdown induced cell cycle arrest,cell apoptosis and suppressed hucMSCs differentiation,which providing clues for MSCs modification and further for stem cell therapy and tissue regeneration.