Objective Construct a stable human alveolar rhabdomyosarcoma(ARMS)cell line(RH30)knocking out PAX3-FOXO1 fusion gene using CRISPR/Cas9 technology,and verify the function of PAX3-FOXO1.Methods Using online websites to design sgRNAs for PAX3 and FOXO1,the group with highest cutting efficiency were selected to construct LV-PAX3-sgRNA,LV-FOXO1-sgRNA,and LV-Cas9 lentiviral vectors;LV-Cas9 virus was infected into RH30,and after screened with hygromycin,the expression of Cas9 in RH30-Cas9 cells were detected by RT-PCR and qRT-PCR;Infecteded RH30-Cas9 cells with LV-PAX3-sgRNA and LV-FOXO1-sgRNA lentiviral vectors,and screened fluorescent positive cells through flow cytometry;Sanger sequencing and PCR were used to verify the expression of PAX3-FOXO1 in cell lines RH30-PAX3-KO and RH30-FOXO1-KO;EDU,TUNEL,and Transwell experiments were conducted to detect functional changes in cell lines RH30-PAX3-KO and RH30-FOXO1-KO.Results Successfully constructed CRISPR/Cas9 lentiviral vectors targeting PAX3 and FOXO1;The construction of RH30 cell line(RH30 Cas9)with stable expression of Cas9 was successfully verified by PCR;PCR and Western Blot were used to verify the successful knockout of the PAX3-FOXO1 fusion gene in RH30 targeting PAX3 or FOXO1;Targeted PAX3 or FOXO1 inhibits the proliferation,invasion,and migration of RH30 cells,promoted apoptosis.Conclusion Successful construction of RH30 cell line knocking out PAX3-FOXO1 using CRISPR/Cas9;Knockout of PAX3-FOXO1 inhibited malignant biological behaviors such as proliferation and migration of RH30 cells.
维普
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