NUSAP1 Promotes Immunity and Apoptosis by the SHCBP1/JAK2/STAT3 Phosphorylation Pathway to Induce Dendritic Cell Generation in Hepatocellular Carcinoma

Abstract

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and is associated with high morbidity and mortality rates. The aims of this study were to investigate the immune-promoting action of nucleolar and spindle-associated protein 1 (NUSAP1) and identify an immunotherapy target for HCC. The Cancer Genome Atlas (TCGA) was used to analyze interaction molecules and immune correlation. The interaction between NUSAP1 and SHC binding and spindle associated 1 (SHCBP1) was examined. The role of the SHCBP1/Janus kinase 2/signal transducer and activator of transcription 3 (SHCBP1/JAK2/STAT3) pathway in this process was explored. After co-culture with HCC cell lines, the differentiation of peripheral blood mononuclear cells (PBMCs) into dendritic cells (DC) was evaluated by measuring the expression of surface factors CD1a and CD86. Pathological tissues from 50 patients with HCC were collected to validate the results of cell experiments. The expression levels of CD1a and CD86 in tissues were also determined. The results show that NUSAP1 interacted with SHCBP1 and was positively correlated with DC. In HCC cell lines, an interaction was observed between NUSAP1 and SHCBP1. It was verified that NUSAP1 inhibited the JAK2/STAT3 phosphorylation pathway by blocking SHCBP1. After co-culture, the levels of CD1a and CD86 in PBMC were elevated. In the clinical specimens, CD1a and CD86 expression levels were significantly higher in the high-NUSAP1 group versus the low-NUSAP1 group. In Summary, NUSAP1 enhanced immunity by inhibiting the SHCBP1/JAK2/STAT3 phosphorylation pathway and promoted DC generation and HCC apoptosis. NUSAP1 may be a target of immunotherapy for HCC.

FIGURE 1

Expression profile characteristics of the TCGA-LIHC data sets NUSAP1 and SHCBP1. Volcano plot of the differentially expressed genes. Red dots indicate upregulated genes; black dots indicate genes with no significant expression changes; and Green dots indicate downregulated genes. The threshold was | log2 fold change |>0.565, P<0.05; (B) Univariate Cox analysis identified prognosis-related differentially expressed genes.High-risk gene: HR>1, P<0.05; Low-risk genes: HR <1, P<0.05; (C) PPI networks of prognosis-related differentially expressed genes. The cytohubba was used to find the network hub genes (top 20); (D, G) NUSAP1 and SHCBP1 expression in 50 normal tissues and 374 tumor tissues from the TCGA-LIHC data set; (E, H) NUSAP1 and SHCBP1 expression in 50 paired tissues from the TCGA-LIHC data set; (F, I) ROC curves for distinguishing the NUSAP1 and SHCBP1 expression of 50 normal tissues and 374 tumor groups in the TCGA-LIHC data set; (J) survival analysis of NUSAP1 and SHCBP1 from TCGA-LIHC database; (K) NUSAP1 and SHCBP1 expression correlation analysis from the TCGA-LIHC data set; (L) Association between SHCBP1 expression and immune cell infiltration from the TCGA-LIHC database.

FIGURE 2

NUSAP1 inhibits the malignance of the human HCC cell lines. (A) Expression of NUSAP1 in HCC and normal hepatocytes cell line; (B) The transfection efficiency of NUSAP1 overexpressing in HCC; (C and D) NUSAP1 overexpressing HCC cells proliferation and colony formation; (E and F) The migratory and invasive properties of NUSAP1 overexpressing HCC cells were determined through Transwell experiments; (G) Sphere formation assays investigated the NUSAP1 overexpressing HCC cells stemness; (H) TUNEL experiments investigated the NUSAP1 overexpressing HCC cells level of apoptosis (***P<0.001).

FIGURE 3

NUSAP1 role in the JAK/STAT3 pathway in human HCC cell lines. (A) GSEA was used to analyze the KEGG_JAK_STAT_SIGNALING_PATHWAY in TGCA (n=374); (B) CO-IP experiments detect NUSAP1 and SHCBP1 binding; (C) WB detect NUSAP1 and SHCBP1 correlation; (D) WB detect NUSAP1 and JAK2/STAT3 pathway correlation; (E) WB detect SHCBP1 role in NUSAP1 and JAK/STAT3 pathway correlation; (F) Resuce experiment validate SHCBP1 role in NUSAP1 and JAK2/STAT3 pathway correlation.

FIGURE 4

NUSAP1 intervene in the differentiation of PBMCs around HCC into DC. (A) Elisa assay test IL-6 levels in the cell supernatant; (B and C) After co-culture, the expression levels of the surface factors CD1a and CD86 of PBMCs were determined by flow cytometry; (D and E) After LMT-28 intervene, the expression levels of the surface factors CD1a and CD86 of PBMCs were determined by flow cytometry; (F and G) After Coumermycin A1 intervene, the expression levels of the surface factors CD1a and CD86 of PBMCs were determined by flow cytometry; (H) After co-culture, WB detect Apoptosis-related proteins in HCC.

FIGURE 5

NUSAP1 expression levels in HCC patient tissues. (A) Expression of NUSAP1 in tumor and normal group in HCC patient tissues; (B) Expression of NUSAP1, CD1a, CD86, Bax, and Bcl-2 in HCC patient tissues.

FIGURE 6

Mechanism diagram.