Study On Function Of The NSUN2 Gene Through CRISPR/Cas9-Mediated Gene Knockout

As a widespread modification in RNA,5-methylcytosine(m5C)has got extensive attention in recent years.Increasing studies demonstrated that RNA m5C modification plays important roles in multiple biological processes,such as RNA processing,RNA stability,RNA transport and mRNA translation.NSUN2(NOP2/Sun domain family,member 2)is a well-known RNA m5C methyltransferase,which mainly accounts for methylation of tRNA and mRNA.Recent studies have shown that NSUN2 is involved in regulating the development of organisms such as cell proliferation,stem cell differentiation,brain and testicular development,and cancer.However,the molecular mechanism of NSUN2 in biological development and disease remains unclear.In this study,the NSUN2 gene was knockout in renal epithelia cell line HEK293 and hepatocellular carcinoma cell line HepG2 via CRISPR/Cas9-mediated gene editing method.The alterations of phenotypes,cellular gene expression and RNA m5C modification were observed based on the knockout cell lines to further study the biological function of NSUN2 and its mediated RNA methylation.1.The establishment of RNA m5C methyltransferase NSUN2 deletion HEK293 and HepG2 cell lines by CRISPR/Cas9In this study,CRISPR/Cas9-mediated homologous recombination was used to integrate screening markers(NeoR and PurR)and transcriptional termination signal BGH poly(A)to the first exon of the NSUN2 gene to terminate NSUN2 mRNA transcription.Guide RNA with low off-target effect was designed and screened for efficient targeting the NSUN2 gene.Two donor vectors,NSUN2-PURO-BGH-Donor and NSUN2-NEO-BGH-Donor were constructed for silencing NSUN2 expression.After transfection,screening and identification,we obtained HEK293 and HepG2 cell lines with NSUN2 deletion and the gene silencing efficiencies reached 99%and 90%,respectively.2.The effect of NSUN2 knockout on the phenotypes in HEK293 and HepG2 cellsThis study aims to understand the effects of NSUN2 knockout on the physiological phenotype of the HEK293 and the HepG2 cell lines.We examined the effects of NSUN2 knockout on the proliferation,migration and metastasis of HEK293 cells and HepG2 cells by cell growth curve assay,wound healing assay,transwell invasion assay,tube formation assay and flow cytometry technique.The results showed that NSUN2 was involved in the regulation of cell proliferation and migration.NSUN2 knockout could significantly inhibit the proliferation and migration of both HEK293 and HepG2 cells,and this inhibitory effect could be restored by re-expressing of NSUN2.NSUN2 participated in the regulation of cell cycle process.NSUN2 knockout could affect the distribution of cell cycle in HepG2,decrease the cells of G1 phase,and increase cells in S and G2/M phase.Furthermore,NSUN2 knockout could significantly inhibit the invasion of HepG2 cells.In addition,we first found that NSUN2 may participate in regulating the angiogenesis process of tumor cell.3.The profiling of gene expression in NSUN2-deficient HEK293 and HepG2 cells.The study aimed to understand the effect of NSUN2 knockout on cell gene expression profiles.We detected the expression profiles of mRNAs in NSUN2 knockout HEK293 and HepG2 cells by high-throughput RNA-seq.The total of 967 and 2681 differentially expressed genes were obtained in NSUN2 knockout HEK293 and HepG2 cells.Functional analysis revealed that the differentially expressed genes were involved in regulating cell adhesion,neural development,differentiation and cell surface receptor signaling,cell proliferation through signal pathways such as nerve receptor ligand interactions,protein digestion and absorption,systemic lupus erythematosus,ECM receptor interaction pathways,etc.In HepG2 cells,NSUN2 deletion-induced differentially expressed genes were significantly related to cellular signal transduction,migration,proliferation,angiogenesis,cell adhesion,inflammatory response and nervous system development.The signal pathway is mainly enriched in Hippo signaling pathway,cancer pathway,nerve ligand receptor interaction,ECM-receptor interaction,Wnt pathway and MAPK signaling pathway.Further analysis indicated that ECM-receptor interaction,TGF-beta and PI3K-Akt signaling pathways might be involved in NSUN2 knock-out mediated cell proliferation and migration inhibition,and the TGFB1,THBS1,CD44,TGFA and LOXL2 genes might play key roles in these processes.4.The profiling of RNA differentially methylated genes in NSUN2-deficient HEK293 and HepG2 cellsNSUN2-mediated RNA methylation may be involved in regulating the processing,stabilization and translation of RNAs.In order to figure out the function of NSUN2-mediated RNA methylation,we screened the differentially methylated(DM)genes caused by NSUN2 knockout by RNA-BisSeq technology.The results showed that 4052 and 2192 differentially methylated sites were detected in HEK293 and HepG2 cells,which distributed in 1185 and 707 RNAs,respectively.Through the BSP method,we verified the results of the RNA-BisSeq and confirmed the reliability of the results.Function analysis of differentially methylated genes demonstrated that,in HEK293 cells,the DM genes were mainly involves in biological process such as translation,mRNA processing,mRNA splicing,mRNA nuclear transport and mRNA stability,affecting ribosomes,spliceosome,RNA transport,proteoglycan in cancer,focal adhesion,tight junction and PI3K-Akt signaling pathways etc.,while in HepG2 cells,the DM genes were mainly involved in biological processes such as translation,mRNA metabolism,cell adhesion,protein folding,RNA splicing and cell cycle,affecting pathways including ribosome,protein processing in endoplasmic reticulum,spliceosome,non-alcoholic fatty liver disease,proteoglycans in cancer,focal adhesion,tight junction,PI3K-Akt signaling pathways and cell cycle.These results provided a powerful basis for functional research of NSUN2 and RNA methylation.In conclusion,we established two cell lines with NSUN2 deletion.Konckout of the NSUN2 gene could significantly decrease cell proliferation and migration,affect cell cycle,inhibit angiogenesis of tumor cells.The DE genes induced by NSUN2 deletion were mainly related to the biological processes such as nervous system development,signal transduction,cell differentiation,proliferation,migration and angiogenesis,affected nerve receptor ligand interactions,ECM-receptor interaction,TGF-beta signaling pathway and PI3K-Akt signaling pathway,etc.While,the DM genes mainly involved biological processes such as transcriptional regulation,translation initiation,RNA processing and RNA stability.The regulatory pathways included ribosomes,spliceosome,RNA transport,proteoglycan in cancer,focal adhesion,tight junction and PI3K-Akt signaling pathways.