CRISPR/Cas9技术联合脂质体转染子宫内膜癌细胞单质粒基因敲除方法首次编辑免疫相关基因HLA-DRA

Editing immune-related gene HLA-DRA for the first time by CRISPR/Cas9 technology combined with liposome transfection of endometrial cancer cells with single-plasmid gene knockout method

背景:HLA-DR基因异常表达与多种肿瘤的进展及预后相关,采用CRISPR/Cas9技术编辑子宫内膜癌HLA-DRA基因的研究目前国内外尚未见报道。目的:利用CRISPR/Cas9技术对HEC-1-A细胞的HLA-DRA基因靶向敲除,检测其敲除效率,并初步探索HLA-DRA基因的功能。方法:根据HLA-DRA基因序列,针对HLA-DRA外显子设计单链向导RNA(sgRNA),分别将sgRNA、CRISPR/Cas9、增强绿色荧光蛋白和嘌呤霉素抗性克隆至YKO载体中,通过脂质体将sgRNA表达质粒转染到HEC-1-A细胞中。根据荧光表达,观察评估sgRNA敲除效果后,使用表达sgRNA的质粒进行转染,用最佳浓度的嘌呤霉素筛选,获得稳定的绿色荧光蛋白阳性表达细胞后,进行PCR、基因测序、转录组测序。结果与结论:经转染和嘌呤霉素筛选后,获得稳定表达绿色荧光蛋白的HEC-1-A细胞,对其进行Sanger测序分析,结果显示HEC-1-A细胞中HLA-DRA第一号外显子基因被定向敲除,对转录组测序结果进行差异基因筛选,根据筛选所得686个差异表达基因进行GO富集分析和KEGG富集分析,构建蛋白互作网络图,通过对肿瘤增殖分化有关差异基因分析发现COL3A1、BCL2A1、TACSTD2、CXCR2等基因表达上调,GNG11、BMP7、IL1RN、PLEK2、CDH6等基因表达下调。结果可见,HLA-DRA基因参与了多种器官、组织和细胞的功能调控过程。此外,HLA-DRA基因可能通过2种途径(上调/下调)调控子宫内膜腺癌细胞的增殖、分化、迁移及侵袭过程。此研究建立了CRISPR/Cas9技术联合脂质体转染子宫内膜癌细胞的单质粒基因敲除方法,首次敲除子宫内膜癌免疫相关基因HLA-DRA,为子宫内膜癌免疫治疗的研究奠定实验基础。

BACKGROUND:The abnormal expression of HLA-DR gene is related to the progression and prognosis of various tumors.The research on editing the HLA-DRA gene of endometrial cancer using CRISPR/Cas9 technology has not been reported at home and abroad.OBJECTIVE:The HLA-DRA gene of HEC-1-A cells was targeted knocked out by CRISPR/Cas9 genome engineering technology,and the function of HLA-DRA was preliminarily explored.METHODS:According to the HLA-DRA gene sequence,sgRNA was designed for HLA-DRA exons,and sg RNA,CRISPR/Cas9,enhanced green fluorescent protein and puromycin were cloned into YKO vector,respectively.The plasmid expressing sgRNA was transfected into HEC-1-A cells by liposome.After evaluating the knockout efficiency of sgRNA according to fluorescence expression,the plasmid expressing sgRNA was transfected and screened with the best concentration of puromycin to obtain stable green fluorescent protein positive cells,which were verified and analyzed by PCR,gene sequencing,and transcriptome sequencing.RESULTS AND CONCLUSION:After transfection and puromycin screening,HEC-1-A cells stably expressing green fluorescent protein were obtained.Sanger sequencing showed that the HLA-DRA exon gene in HEC-1-A cells was targeted knockout.The transcriptome sequencing results were screened for differential genes.According to the 686 differentially expressed genes screened,GO enrichment analysis and KEGG enrichment analysis were carried out,and the protein interaction network was constructed.Through the analysis of differential genes related to tumor proliferation and differentiation,it was found that the expression of COL3A1,BCL2A1,TACSTD2 and CXCR2 genes was up-regulated,and the expression of GNG11,BMP7,IL1RN,PLEK2 and CDH6 genes was down regulated.It is concluded that the HLA-DRA gene is involved in the functional regulation of a variety of organs,tissues,and cells.In addition,HLA-DRA genes may regulate the proliferation,differentiation,migration,and invasion of endometrial adenocarcinoma cells through two pathways(upregulation/downregulation).To establish a single plasmid gene knockout method using CRISPR/Cas9 technology combined with liposome transfection,HLA-DRA was knocked out for the first time,which laid the experimental foundation for the study of endometrial cancer immunotherapy.