Construction Of Immortalized Umbilical Cord Mesenchymal Stem Cell Line Using CRISPR/Cas9 Technique And Preliminary Study On The Antitumor Function Of MYC-AS1 Transgenic Engineered Exosomes

Human umbilical cord mesenchymal stem cells(hUCMSCs)are pluripotent stem cells with multiple differentiation potential,which play an important role in tissue repair and clinical application of some diseases treatment.The number of generations of hUCMSCs in vitro is limited,and the activity and differentiation ability of hUCMSCs are decreased obviously after 8 generations.With the increase of the number of generations,the cell loses its biological characteristics,and it is not convenient to be used for gene editing.SV40 LT is a functional protein from simian virus,also known as SV40 LT antigen.The SV40 LT gene is usually randomLy inserted into the genome to construct an immortalized cell line.It is of great significance to construct an immortalized cord mesenchymal stem cell line for its function research,gene editing and clinical application.Long non-coding RNA(IncRNA)is a kind of non-coding RNA,which lacks open reading frame and has fewer exons and is longer than 200 nt.Antisense RNA is a kind of RNA that is partially or completely complementary to the transcripts in sense chain.It can block and inhibit the expression of sense genes by pairing specific RNA with complementary bases.The newly-identified long noncoding antisense RNA MYC-AS1 of the MYC gene in our laboratory can inhibit the expression of proto-oncogene MYC and has a significant anti-tumor effect.In this study,the SV40 LT gene with PGK promoter and SV40-polyA was inserted into the first exon of the AAVS1 gene by CRISPR/Cas9 system,which made MSC cells achieve immortalization.A long antisense long non-coding RNA MYC AS1 transgenic MSC line was constructed by using lentiviral system,and exosomes were isolated from the culture medium of the cell line.The effects of the exosomes expressing MYC-AS1 on HepG2 phenotype were studied by treating HepG2 cells.It provides the basis for the basic research and clinical application of MSCs and their exosome.The main results are as follows:1.Establishment of cord mesenchymal stem cell line with SV40 LT gene knockinIn this study,the SV40 LT gene with the PGK promoter and SV40-polyA was integrated into the first exon of the AAVS1 gene through homologous recombination mediated by CRISPR/Cas9 technology,so that MSC could achieve immortalization.First,the first exon of the AAVS1 gene was designed and screened out with high editing efficiency,and AAVS1-Cas9-sgRNA and homologous recombinant plasmid AAVS1-Neo-SV40 LT were constructed.Through co-transfection,G418 screening and identification.The results showed that compared with wild type MSC,the proliferation of MSC-SV40 LT cells was significantly accelerated,but the differentiation characteristics of the cells were not changed.β-galactosidase staining showed that MSC-SV40 LT cells did not show aging and could be stably passed on for more than 40 generations in vitro.Their proliferation ability was enhanced,but they were not found to be tumorigenic in nude mice.2.The effect of immortalization of MSC cells on the gene expression profileTo understand the effect of immortalization on gene expression profile of MSC cells,we analized the transcriptome of MSC-SV40 cells.The results showed that 3088 differentially expressed genes(DEGs)were screened between MSC and MSC-SV40 LT cells after the SV40 LT gene was knocked in.Compared with MSCs,1675 upregulated genes and 1413 downregulated genes were found in MSC-SV40 LT cells.The results of GO enrichment show that the DEGs are mainly concentrated in the biological processes of cell cycle,cell adhesion,cell division,DNA repair,extracellular matrix tissue and so on.KEGG analysis showed that the DEGs were mainly concentrated in PI3K-Akt signaling,cancer,mitochondria,human papillomavirus infection,cytokine receptor interaction,cell aging,cell cycle,autophagy and other pathways.Three tumor related genes FOS,SET and MYC were randomLy selected from these DEGs to verify the results by RT-qPCR.The results were consistent with the RNA-seq.The expression levels of three tumor-related genes in HepG2 cells were significantly higher than these of both wild-type MSCs and immortalized MSCs.The increased proliferation of MSC-SV40 LT cells may be related the rise of expression of these tumor-related genes.However,the difference of expression in these upregulated genes in MSC-SV40 LT cells is not significant(p>0.05).Combined with the results without tumorigenicity in nude mice,it can be preliminarily considered that the immortalized cells are safe.3.Effects of exosomes of MYC-AS1 transgenic MSCs on the phenotype of tumor cells HepG2In this study,we used lentivirus system to construct an antisense long non-coding RNA MYC-AS1 transgenic mesenchymal stem cell line based on immortalized cell line.The effects of exosomes containing MYC-AS1 on the phenotype of tumor cells HepG2 were observed by cell migration,cell proliferation and cell apoptosis tests.The results showed that MYC-AS1 transgenic MSC line was successfully constructed,and high level of MYC-AS1 expression was detected in its exosome.Exosome MYC-AS1 could significantly inhibit the migration and proliferation and promote apoptosis of HepG2 cells,In conclusion,we successfully constructed an immortalized hUCMSC line by using CRISPR/Cas9 system and a MYC-AS1 transgenic cell line by lentiviral system.It was found that the exosomes of the transgenic cell line had high level of MYC-AS1,and had obvious inhibitory effect on HepG2 tumor cells.The results of this study have important significance for the basic research and clinical application of hMSCs and its exosomes.