Construction Of HMGA2 Knockout Hepatocellular Carcinoma Cell Lines And Rabbit Models Using The CRISPR/Cas9 System

Background and Object:High-mobility group AT-hook 2(HMGA2)is a member of high mobility group protein family(HMG family)and belongs to non-histone chromatin binding protein.It contains three AT-hook domain that can interact with AT-rich region in genome to remodel the chromatin topological conformation and adjust target gene expression.HMGA2 is highly expressed during embryonic development and little in adult tissue.The animals with HMGA2 mutation or deletion showed severe fetal development retardation and obvious dwarf phenotype,indicating that HMGA2 was related to the embryonic development of animals and involved in the growth and development regulation of animals.In addition,it is also highly expressed in various cancer compared with adjacent mucosa,involved in tumorigenic processes such as proliferation,migration and invasion of tumor cells,and also associated with poor prognosis of patients,implying that HMGA2 maybe as an importance oncogene.Based on the above information,this project firstly investigated the effect of HMGA2 on the HepG2,one of the hepatoma carcinoma cell line,tested the effect of the inactivation of HMGA2 on cell invasion,migration,growth and proliferation,and explored the potential role of HMGA2 in the development of liver cancer.Secondly,HMGA2 knockout pygmy rabbit model was constructed by CRISPR/Cas9 system,so as to provide some strategies for the miniaturization of experimental animals.Methods:(1)The sgRNA targeting exon1 or exon2 in human HMGA2 gene was designed respectively,and the recombinant vector of sgRNA was constructed.HepG2 cells were transfected with the recombinant vectors and large-fragment knockout cell lines were obtained by clonal screening.The effect of HMGA2 knockout on growth and proliferation of tumor cell was investigated via CCK8 and clone formation assay,the ability of migration and invasion was tested by Transwell assay.(2)Two sgRNAs targeting the second exon of HMGA2 gene in rabbit were designed and the recombinant vector of sgRNA was constructed.In vitro transcription of sgRNA vector and Cas9 into sgRNA and Cas9 mRNA.HMGA2 knockout pygmy rabbit model was prepared by intracellular microinjection and embryo transferResults:(1)Two sgRNAs targeting the first and second exon of HMGA2 were successfully designed by online sgRNA design software,and the recombinant vector PX458-sgRNA was successfully constructed.The vectors were transfected into HepG2 cells,and the large-fragment knockout cell line was obtained by clonal screening.Compared with the wild-type,the knockout cells showed decrease in the ability of proliferation,clone formation(121.83 ± 21.68 vs 59.50 ± 20.68,P<0.01),invasion(251.33 ± 43.43 vs47.00±10.00,P<0.05)and migration(359.67±32.53 vs 245.61 ± 24.23,P<0.01)(2)Two sgRNAs targeting rabbit HMGA2 exon 2 were successfully designed by online sgRNA design software.There was high editing efficiency and no offtarget in the level of embryos.Eventually,two HMGA2-/-rabbits were obtainedConclusion:(1)Consistent with previous studies,HMGA2 also has a strong oncogenic effect in liver cancer.This suggests that HMGA2 may also be a potential target molecule for the treatment and prognosis evaluation of liver cancer.(2)The efficient editing of HMGA2 gene in rabbits can be achieved at the level of embryos.Two HMGA2-/-rabbits were obtained in the individual levels.However,the knockout rabbits obtained in this study did not show dwarf phenotype,which was suspected that the locus of HMGA2 was important in controlling mammal growth.