Disulfidptosis-related genes RPN1 inhibits the progression of hepatocellular carcinoma by regulating cell cycle, may be a new therapeutic targets.

backgroundHepatocellular carcinoma (HCC) is the predominant type of liver cancer with a poor prognosis. Treatment methods include surgery, ablation, liver transplantation, and immunotherapy. Programmed cell death (PCD) plays a significant role in the occurrence and treatment of HCC, and disulfidoptosis, as a novel type of PCD, is associated with tumor prognosis and anti-tumor immunity. The purpose of this study is to explore the role and molecular mechanisms of disulfidoptosis-related genes (DRGs) in the occurrence and development of HCC.

methodsWe developed an HCC prognostic signature comprising three DRGs: RPN1, SLC7A11, and GYS1, using the TCGA database. The mRNA expression levels of the signature genes in peripheral blood mononuclear cells (PBMCs) of 196 patients were detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), and the expression of RPN1 in 23 pairs of HCC tissues and adjacent non-tumor tissues were validated by RT-qPCR and immunohistochemistry (IHC). Additionally, the role of RPN1 in HCC was investigated through EdU Assay, CCK8 Assay, wound Healing Test, transwell experiments. The changes of cell cycle were detected by flow cytometry, and the changes of Cyclin (CDK1, CDK2, Cyclin D1, Cyclin E1) were detected by Western blot. We carried out in vivo experiments in a BALB/c nude mice model of HCC established through subcutaneous injection.

resultsRPN1 was significantly upregulated in paired HCC tissues (p < 0.001). HCC group was also significantly higher in PBMCs than healthy group (p < 0.001). Interestingly, RPN1 expression were higher in the HBV group and HBV-LC group than in HBV-HCC group (p < 0.001). IHC experiments confirmed that RPN1 was also up-regulated in HCC tissues (p < 0.05). In vitro experiments showed that knockdown of RPN1 promoted the proliferation and migration of HCC cells, while overexpression of RPN1 inhibited these functions. Flow cytometry and Western blot confirmed that knockdown of RPN1 in HCCLM3 and Huh7 cells resulted in a decrease in the proportion of G0/G1 phase cells and an increase in the proportion of G2/M phase cells. Meanwhile, the expression levels of cell cycle proteins (CDK1, CDK2, Cyclin D1, and Cyclin E1) were significantly elevated. In vivo, overexpression of RPN1 in Hep3B cells can inhibit tumor growth.

conclusionsIn vitro and in vivo experiments confirmed that the overexpression of RPN1 can significantly suppresses the progression of HCC by regulating the cell cycle. RPN1 could potentially serve as a new therapeutic target for HCC.