Knockdown And Overexpression Of The DNA Methyltransferase Gene N6AMT1 And Preliminary Study For Its Function

As an component of epigenetic regulations,DNA methylation plays an important role in cell function maintenance,gene expression,embryonic development and tumorigenesis.N6-methyladenine(6mA)is one of the DNA methylation modifications,which regulated by methyltransferase N6AMT1 and demethylase ALKBH1.Recent studies have found that the 6mA and N6AMT1 levels were frequently downregulated in tumor tissues,while the ALKBH1 levels were upregulated.So far,there have been few reports about the N6AMT1 gene research,so there is a long way to study its specific biological functions.To explore the function of the N6AMT1 gene,the study identified the expression levels of the N6AMT1 gene in several cell lines.Subsequently,we selected HEK293 cells with a higher N6AMT1 expression level and Hela cells with a lower N6AMT1 expression level for research.The HEK293 N6AMT1 knockdown cell lines was constructed by CRISPR/Cas9 system.At the same time,N6AMT1 gene was overexpressed in Hela cells.In this study,the effects of N6AMT1 gene editing on cell phenotype were analyzed to further understand the biological function of the N6AMT1 gene.The experimental results are as follows:1.The expression levels of the N6AMT1 gene in different cell lines.We explored the difference of N6AMT1 gene expression in different cell lines by detecting the expression levels of N6AMT1 gene in different cell lines,especially in cancer cells.The existing cell lines are divided into two categories,one is the human normal tissue cell lines including HEK293 and L02,and the other is the tumor tissue cell lines including SMMC-7721,MCF7HepG2,BGC823,MDA-MB-231,MCF7,AGS,HCT116 and KYSE-150.The results of RT-qPCR shows that the expression level of the N6AMT1 gene in HEK293 was higher than that in cancer cells.The expression level of the N6AMT1 gene in normal hepatocyte line L02 was significantly higher than that in hepatocellular carcinoma cell lines HepG2 and SMMCC-7721.Among the cell lines detected,the expression level of the N6AMT1 gene was the highest in HEK293 cells,while the expression level of Hela cells was relatively low.Therefore,HEK293 cells were selected for knockdown of the N6AMT1 gene,and Hela cells were used for overexpression of the N6AMT1 gene.2.The construction of N6AMT1 knockdown HEK293 cell line and the effect of N6AMT1 knockdown on cell phenotypesFirst,the sgRNA expression vector with high gene editing efficiency was designed to target the first exon of N6AMT1.Then,the homologous recombination vector was constructed,and 40bp sequences were selected as homology arms ligated to both ends of the screening gene and the transcription termination sequence BGH ploy(A).In this experiment,two combinations of screening genes were used,one containing PuroR and NeoR,the other containing PuroR and eGFP.The former used Puromycin and Neomycin to screen for transfected cells,while the latter used Puromycin and fluorescent expression to screen cells.Next the donor sequences was knocked into the first exon of the N6AMT1 gene through specific targeted homologous recombination mediated by CRISPR/Cas9 system,and HEK293 cells were screened by the expression of insertion tag gene.Finally,the knockdown of the N6AMT1 gene was identified from DNA,RNA and protein levels.The results showed that the N6AMT1 gene was successfully edited,both RNA and protein levels of the N6AMT1 gene were significantly reduced.It was finally confirmed that the N6AMT1 knockdown HEK293 cell line(designated:HEK293-N6AMT1-Knowdown,HEK293-NK)was obtained.Subsequently,The effect of the N6AMT1 gene knockdown on the phenotypes of HEK293 cells was studied.The cells were examined in terms of cell cycle,cell proliferation,cell migration,and cell clone formation,respectively.The results showed that knockdown of the N6AMT1 gene significantly enhanced the proliferation,migration and clonality of HEK293 cells.3.Effect of overexpression of the N6AMT1 gene on Hela phenotypesWe firstly constructed and identified the N6AMT1 overexpression vector pcDNA3.1-N6AMT1.Then,the plasmid was transiently transfected into Hela cells.After the overexpression of the N6AMT1 gene was identified by RT-qPCR,we performed proliferation and migration experiments in Hela cells.The results showed that the proliferation and migration of Hela cells were inhibited after overexpression of N6AMT1.The experiment proved once again that the N6AMT1 gene did affect cell proliferation and migration.Therefore,it is speculated that the N6AMT1 gene may play an important role in tumorigenesis and metastasis.