Integration of Single-Cell RNA Sequencing and Bulk RNA Sequencing to Identify an Immunogenic Cell Death-Related 5-Gene Prognostic Signature in Hepatocellular Carcinoma

Abstract

Liqun Peng,1,2 Shaohua Xu,3 Jian-Liang Xu4 1Department of Hepatobiliary and Pancreatic Surgery, Zhongnan Hospital of Wuhan University, Wuhan, People’s Republic of China; 2Clinical Medicine Research Center for Minimally Invasive Procedure of Hepatobiliary & Pancreatic Diseases of Hubei Province, Wuhan, People’s Republic of China; 3Department of Clinical Laboratory, Center for Gene Diagnosis & Program of Clinical Laboratory, Zhongnan Hospital of Wuhan University, Wuhan, People’s Republic of China; 4Department of Hepatobiliary Surgery, the Third Affiliated Hospital of Sun Yat-sen University, Guangzhou, People’s Republic of ChinaCorrespondence: Jian-Liang Xu, Department of Hepatobiliary Surgery, the Third Affiliated Hospital of Sun Yat-sen University, 600 Tianhe Road, Guangzhou, 510630, People’s Republic of China, Tel [phone], Fax [phone], Email [email protected] Shaohua Xu, Department of Clinical Laboratory, Center for Gene Diagnosis & Program of Clinical Laboratory, Zhongnan Hospital of Wuhan University, 169 Donghu Road, Wuhan, 430071, People’s Republic of China, Email [email protected]: Immunogenic cell death (ICD) can enhance the potency of immunotherapy in cancer treatment. Nevertheless, it is ambiguous how ICD-related genes (ICDRGs) contribute to hepatocellular carcinoma (HCC).Methods: Single-cell RNA sequencing (scRNA-seq) data were used to distinguish malignant cells from normal cells in the HCC tumor microenvironment(TME). Bulk RNA sequencing data was employed to acquire the landscape of the 33 ICDRGs. Unsupervised clustering identified two ICD molecular subtypes. The cellular infiltration characteristics and biological behavior in different subtypes were analyzed by ssGSEA. Subsequently, differentially expressed genes (DEGs) between the two subtypes were determined, based on which patients were classified into three gene clusters. Then, the prognostic model was constructed by Lasso-Cox analysis. Finally, we investigated the expression of risk genes in cancer cell line encyclopedia (CCLE) and validated the function of NKX3-2 in vitro experiments.Results: ICD scores and ICDRGs expression in malignant cells were significantly lower than in normal cells by scRNA-seq analysis. ICD-high subtype was characterized by ICD-related gene overexpression and high levels of immune infiltration abundance and immune checkpoints; Three DEGs-related gene clusters were likewise strongly linked to stromal and immunological activation. In the ICD-related prognostic model consisting of NKX3-2, CHODL, MMP1, NR0B1, and CTSV, the low-risk group patients had a better endpoint and displayed increased susceptibility to immunotherapy and chemotherapeutic drugs like 5-Fluorouracil, afatinib, bortezomib, cediratinib, lapatinib, dasatinib, gefitinib and crizotinib. Moreover, NKX3-2 amplification in HCC samples has been verified by experiments, and its disruption suppressed the proliferation and invasion of tumor cells.Conclusion: Our study highlighted the potential of the ICDRGs risk score as a prognostic indicator to aid in the accurate diagnosis and immunotherapy sensitivity of HCC.Keywords: Hepatocellular carcinoma, prognostic model, immunotherapy, immunogenic cell death

Keywords

• hepatocellular carcinoma
• prognostic model
• immunotherapy
• immunogenic cell death