基于CRISPR/Cas9技术构建PAX3-FOXO1融合基因敲除腺泡状横纹肌肉瘤细胞模型  

Construction of the PAX3-FOXO1 fusion gene knockout cell model of alveolar rhabdomyosarcoma based on CRISPR/Cas9 technology

作　　者：张乾儒 田娇 孟莲 史奇 李亚 夏慧欣 李锋[1,3] ZHANG Qianru;TIAN Jiao;MENG Lian;SHI Qi;LI Ya;XIA Huixin;LI Feng(Department of Pathology,School of Medicine,Shihezi University,Shihezi,Xinjiang 832000,China;Department of Pathology,the First Affiliated Hospital of Shihezi University,Shihezi,Xinjiang 832000,China;Medical Research Center,Beijing ChaoYang Hospital,Capital Medical University,Beijing 100020,China)

机构地区：[1]石河子大学医学院病理系,新疆石河子832000 [2]石河子大学第一附属医院病理科,新疆石河子832000 [3]首都医科大学附属北京朝阳医院医学研究中心,北京100020

出　　处：《石河子大学学报（自然科学版）》2024年第5期621-630,共10页Journal of Shihezi University(Natural Science)

基　　金：国家自然科学基金项目(82173057,82060487)。

摘　　要：目的使用CRISPR/Cas9技术构建稳定敲除PAX3-FOXO1融合基因的人腺泡状横纹肌肉瘤(ARMS)细胞系(RH30),并验证PAX3-FOXO1功能。方法运用在线网站针对PAX3、FOXO1设计sgRNA,选择切割效率最高的一组构建LV-PAX3-sgRNA、LV-FOXO1-sgRNA及LV-Cas9慢病毒载体;LV-Cas9病毒感染RH30细胞,潮霉素筛选后,通过RT-PCR及qRT-PCR检测RH30-Cas9细胞中Cas9基因表达;LV-PAX3-sgRNA及LV-FOXO1-sgRNA慢病毒载体分别感染RH30-Cas9细胞,流式分选筛选荧光阳性细胞;Sanger测序、PCR等分别验证RH30-PAX3-KO细胞及RH30-FOXO1-KO细胞中PAX3-FOXO1表达;EDU、TUNEL、Transwell实验检测敲除RH30-PAX3-KO细胞及RH30-FOXO1-KO细胞功能改变。结果成功构建靶向PAX3、FOXO1的CRISPR/Cas9慢病毒载体;PCR验证稳定表达Cas9的RH30细胞系(RH30-Cas9)构建成功;PCR、Western Blot验证靶向PAX3或FOXO1均成功敲除RH30中PAX3-FOXO1融合基因;靶向PAX3或FOXO1抑制RH30细胞增殖、侵袭、迁移,促进凋亡。结论使用CRISPR/Cas9成功构建敲除PAX3-FOXO1的RH30细胞系;敲除PAX3-FOXO1抑制了RH30细胞增殖、迁移等恶性生物学行为。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 LVFOXO1-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.

关 键 词：横纹肌肉瘤 PAX3-FOXO1 CRISPR/Cas9 基因编辑 

分 类 号：R738.6[医药卫生—肿瘤]