Molecular Mechanism Of USP49 Regulating Proliferation Of HCT16 Cells

BackgroundPosttranslational modifications of histone play important roles in regulating chromatin structure and function.Previous studies have shown that ubiquitination and deubiquitination of histones are very important for the process of transcriptional regulation.The ubiquitination of histone H2 A inhibits gene expression whereas the ubiquitination of histone H2 B promotes gene expression.USP49 is a vital member of the USP family.It has been reported that USP49 can directly bind and deubiquitinate p53 protein,regulate the stability of p53 protein,and thus play essential roles in the development of tumors.At the same time,USP49 is also a specific deubiquitination enzyme for histone H2 B,which may regulate the gene expression by removing of histone H2 B ubiquitination.MDM2 is a ubiquitin-ligase that works upstream of p53.Previous report shows that H2Bub is enriched in MDM2 locus.Whether USP49 can regulate the gene expression of MDM2 through removing of H2 B ubiquitination,and thus regulate P53 and its downstream genes has not been reported.ObjectiveConstruct HCT116 cell line with stable USP49 knockout using CRISPR/Cas9 technology.Study the role and molecular mechanism of USP49 in HCT116 cells proliferation.Methods(1)Construction of USP49 defective cell line in HCT116 cells The USP49 gene of HCT116 cells was knocked out by CRISPR/Cas9 gene editing technology and lentivirus transfection.Monoclonal cell lines were selected and identified by Sanger sequencing,RT-qPCR and Western blot to verify the success of the gene knockout.(2)Study on the growth and biological characteristics of USP49 deficient HCT116 cells Using HCT116 with empty vector monoclonal cells as control,the cytological characteristics of the USP49 deficient HCT116 cells were observed,including cell proliferation,clonal formation,cell scratch,etc.(3)The effect of USP49 deficient cells on the MDM2-P53 signaling pathway Using β-actin as internal reference,the mRNA expression levels of MDM2,P53,P21,Bax and other genes in USP49 knockout cell lines were detected by RT-qPCR assay.(4)Effect of USP49 overexpressing cell line on HCT116 cell proliferation HCT116 cells were transfected with USP49 overexpression vector pc DNA3USP49.The overexpression of HCT116 cells was verified by Western and RT-qPCR.The proliferation of HCT116 cells was verified by clone formation.(5)Effect of USP49 on histone H2Bub The total histones of USP49 deficient cells and USP49 overexpressed cells were extracted,and the changes of the target protein H2Bub were detected by using H3 as internal reference.(6)The effect of USP49 on the level of H2Bub in MDM2-p53 signaling pathway gene Chromatin immunoprecipitation(ChIP)experiment detects the level of H2Bub of MDM2-p53 signaling pathway genes,so as to further clarify how USP49 regulates MDM2-p53 signaling pathway genes during colon cancer process.(7)The target gene regulated by USP49 during colon cancer development ChIP assay was used to detect the binding sites of USP49 in the development of colon cancer by using USP49 antibody.Results(1)The USP49 gene-deficient cell line of HCT116 was successfully constructed.(2)Studies on the growth and biological characteristics of USP49 deficient HCT116 cells showed that the cell proliferation and migration rate were accelerated in USP49 knockout cells,as well as the enhanced formation of clone was significantly.(3)In USP49 knockout cells,RT-qPCR detection showed that the mRNA level of MDM2 was increased,whereas the transcript level of downstream genes P53,P21 and Bax were decreased.(4)In the overexpression of USP49 cells,the protein and mRNA level of USP49 were significantly increased,and the clone formation ability was decreased at the same time.(5)Western blot showed that USP49 knockout led to an increase in histone H2Bub level,while USP49 overexpression led to a decrease in H2 bub level.(6)ChIP-qPCR experiment showed that the H2Bub level of MDM2 sites were elevated in USP49 Knock-out cells,whereas the H2Bub level of P53,P21 and BAX genes were decreased.(7)ChIP experiment showed that USP49 binding on the promoter and gene body of MDM2 locus was elevated in the overexpressing USP49 cells,suggesting that USP49 directly bind to the MDM2 gene.Conclusions1.USP49 acts as a colon cancer suppressor to regulate the gene expression of MDM2-p53 signaling pathway in tumor cell proliferation.2.As an epigenetic modifying enzyme,USP49 binds to the promoter and coding region of MDM2,regulates the gene expression of MDM2 gene by modifying the level of H2Bub.USP49 participates in HCT116 proliferation by regulating the transcript level of MDM2,as well as the downstream of gene P53 and other genes.