| Aging of the human body is accompanied by the decline of tissue function.This kind of decline mainly comes from the senescence of tissue stem cells,therefore,stem cells play crucial roles during the aging of the human body.Mesenchymal stem cells(MSCs)have two unique characteristics:self-renewal and pluripotency.Under certain conditions,they can continuously proliferate and differentiate into one or more cell types that constitute human tissues and organs,and can effectively regulate immunity.Compared with other types of MSCs,human umbilical cord-derived mesenchymal stem cell(hUCMSC)is derived from the umbilical cord of full-term infants,which is easy to obtain and has less ethical controversy compare with embryonic stem cells.Compare with other MSCs,hUCMSC is easy to isolate,culture,amplify and purify.Due to the low immunogenicity of hUCMSCs,the matching requirements were not strict,moreover,the rejection after transplantation has not been reported..Recently,hUCMSCs are used in the treatment of various diseases.Studies on senescence of hUCMSCs are expected to provide a new reference for the study of senescence and senescence-related diseases.In this study,by establishing a natural senescence model of hUCMSCs and a D-Galactose(D-Gal)-induced rapid senescence model,the proliferation ability,cell viability,cell cycle,pluripotency and Reactive oxygen species(ROS)of hUCMSCs after senescence were observed.ROS)levels were analysed to explore the biological characteristics of senescent hUCMSCs.Whole transcriptome sequencing,GO gene analysis and KEGG analysis were used toscreen senescence-related Long non-coding RNA(lncRNA)in hUCMSCs.Some differential expressed lncRNAs were selected,and the regulatory mechanism of related lcnRNAs were further explored by gene overexpression and knockdown.To explore roles and molecular mechanisms of these lncRNA during senescence process of hUCMSCs,we established lncRNA overexpression hUNMSCs cell lines and knockdown cell lines,and found the target genes of these senescence related lncRNAs.This study provides a novel idea and reference for the regulation of lncRNAs on the senescence process of hUCMSCs.Key findings were as following:1.The natural senescence model of hUCMSCs was successfully constructed.The senescence of hUCMSCs was determined by morphological observation and senescence-associated βgalactosidase(SA-β-gal)staining.The expression of cell surface markers CD73,CD90 and CD 105 were decreased by flow cytometry.The level of Reactive oxygen species(ROS)was significantly increased.2.D-Gal induced rapid senescence model was successfully constructed.After treatment with 2%(0.2g/100mL)D-Gal for 4 days,the positive rate of SA-β-gal staining in hUCMSCs was significantly increased,the proliferation ability and cell activity were significantly decreased,the cell cycle was arrested,and the level of ROS was significantly increased.The expression of cell surface markers CD90,CD73 and CD 105 did not decrease significantly,while the expression of multipotency related protein OCT4 increased significantly,and the expression of NANOG decreased significantly.The results indicate that 2%D-Gal induced senescence model is similar to biological senescence model and can be used as an early senescence model of hUCMSCs.3.Using the D-Gal senescence model,two lncRNAs of research significance were identified:Overexpression of lncRNA MALAT1-213 and lncRNANEAT1-206 could cause G0/G1 phase cell cycle arrest in hUCMSCs cells,but inhibition of the above lncRNAs had no effect on the cell cycle.GLS1,WNT5A and DKK1 were significantly up-regulated after overexpression of lncRNA MALAT1-213.GLS1,WNT5A and DKK1 genes were significantly down-regulated after silencing the expression of lncRNA MALAT1-213.Overexpression of lncRNA NEAT1-206 significantly increased DKK1,and inhibition of lncrna NEAT1-206 significantly decreased DKK1.The results showed that lncRNA MALAT1-213 may affect hUCMSCs by regulating glutamine metabolism and Wnt classical and non-classical signaling pathways.lncRNA NEAT1-206 affects hUCMSCs through the canonical Wnt pathway involving DKK1.In summary,the results of the present study demonstrated that 2%D-Gal treatment for 4 days induced early senescence of hUCMSCs and significantly upregulated the expression of lncRNA MALAT1-213 and lncRNA NEAT 1-206.Further study found that lncRNA MALAT1-213 and lncRNA NEAT1-206 affected cell proliferation by regulating the cell cycle of hUCMSCs,and were involved in D-Gal-induced hUCMSCs cell senescence,which played an important role in the aging process of hUCMSCs.This study successfully established an aging model of hUCMSCs and preliminarily revealed the function of senesing-related lncRNA MALAT1-213 and lncRNA NEAT1-206 in the aging process,which can provide a target for further study of hUCMSCs aging. |
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