Alpha-Fetoprotein Regulates the Expression of Immune-Related Proteins through the NF-κB (P65) Pathway in Hepatocellular Carcinoma Cells

Abstract

Background. The prognosis of patients with hepatocellular carcinoma (HCC) is poor, with 60% to 70% of patients developing recurrence and metastasis within five years of radical resection. Alpha-fetoprotein (AFP) plays a significant role in predicting the recurrence and metastasis of HCC after surgery. However, its role in modulating tumor immunity has not been investigated. Our objective was to examine the effect of AFP on the expression of B7 family and activation of the NF-κB (P65) pathway in HCC. Methods. We generated human hepatoma SMMC-7721 cell lines with or without recombinant AFP transfection (AFPup and control groups). Colony formation assay, Transwell invasion assay, and wound healing assay were used to detect the function of AFP. Liver cancer xenografts were made in BALB/c nude male mice (N = 6 per group). After 28 days of inoculation, the expression of immune genes in the HCC tissues, including PD-L (B7-H1), B7-H3, B7-H4, and P65, was evaluated by quantitative real-time PCR (qPCR) and western blot. In addition, immunofluorescence was used to determine the subcellular localization of the P65 protein, a key factor in the NF-κB pathway. An online HCC patients’ dataset was also used to detect the connection between AFP and P65. Results. Overexpression of AFP could enhance proliferation, invasion, and migration of HCC cells. Both qPCR and western blot results demonstrated that the expressions of PD-L1, B7-H4, and P65 were significantly higher in the AFP group compared to the controls (P<0.05). Immunofluorescence results indicated that the majority of the P65 protein was located in the cytoplasm in the control group but was translocated to the nucleus in the AFPup group. The Spearman correlation coefficient confirms that AFP has a positive correlation with P65 in HCC patients (R = 0.33, P=0.05). Conclusion. AFP could enhance proliferation, invasion, and migration in HCC cells. The upregulation of AFP would increase the PD-L1 and B7-H4 mRNA and protein expression in HCC tissues through the upregulation and activation of the P65 protein.