Chinese Journal of Tissue Engineering Research ›› 2023, Vol. 27 ›› Issue (15): 2356-2362.doi: 10.12307/2023.622

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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

Cai Bo1, 2, Li Xiaoxiao1, 2, Zhang Linghan1, 2, Liu Yanxing1, 2, Mao Genhong1, 2   

  1. 1Academy of Medical Sciences, Zhengzhou University, Zhengzhou 450014, Henan Province, China; 2Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450014, Henan Province, China
  • Received:2022-05-23 Accepted:2022-08-03 Online:2023-05-28 Published:2022-10-17
  • Contact: Mao Genhong, MD, Chief physician, Doctoral supervisor, Academy of Medical Sciences, Zhengzhou University, Zhengzhou 450014, Henan Province, China; Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450014, Henan Province, China
  • About author:Cai Bo, Master candidate, Academy of Medical Sciences, Zhengzhou University, Zhengzhou 450014, Henan Province, China; Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450014, Henan Province, China
  • Supported by:
    National Natural Science Foundation of China (General Program), No. 81971454 (to MGH)

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Abstract: 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.

Key words: CRISPR/Cas9, HLA-DRA, gene knockout, HEC-1-A, transcriptome sequencing

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