Construction and identification of a double gene knockout cell model for RNA methyltransferase-like in human bladder cancer cells / 中国职业医学

ABSTRACT

OBJECTIVE: To construct a cell model with simultaneous knockout of RNA methyltransferase-like( METTL) 3 and METTL14 using clustered regularly interspaced palindromic repeats( CRISPR)/CRISPR-associated protein 9( Cas9)system in human bladder cancer T24 cells,in order to provide new tools and means for further studying gene intervention in occupational cancers. METHODS: The small guide RNA( sgRNA) oligonucleotide sequences targeting METTL3 and METTL14 were designed,synthesized and constructed into the Lenti-multi-CRISPR vector. The Lenti-iCas9-neo inducible plasmids were transfected into T24 cells by lentivirus system,and the positive cells were sorted by flow cytometry. Finally,the recombinant plasmid Lenti-multi-METTL3-METTL14 was transfected into positively sorted cells and screened to obtain stable cell lines. T24-Lenti-multi-CRISPR cells and T24-KO-METTL3-METTL14 were used as control group and multigene knockout group. The Western blot was used to examine relative expression of METTL3 and METTL14 protein in the two groups. RESULTS: The targeting sgRNA was successfully inserted into the Lenti-multi-CRISPR plasmid and confirmed by sequencing. The flow cytometry was used to obtain a large amount of the positive cells. The stable cell line inducted of doxycycline with simultaneous knockout of METTL3 and METTL14 gene was obtained. The relative expression of METTL3 and METTL14 protein decreased respectively by 52. 08% and 64. 04% after 96 hours of doxycycline treatment in the multigene knockout group compared with the control group( P < 0. 01). CONCLUSION: The stable T24 bladder cancer cell with simultaneous double gene knockout of METTL3 and METTL14 was obtained for the first time,which will be helpful for providing a foundation for mechanistic study for prevention and treatment of candidate tumor targets and bladder cancer.