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.
Highlights
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most frequent cause of cancer-related deaths in the world
Cell Line and Cell Culture. e human HCC cell line SMMC-7721 was obtained from the Chinese Center for Type Culture Collection (CCTCC, Wuhan, China). e cell line was cultured in DMEM (Life Technologies, Beijing, China) supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and maintained at 37°C in a humidified atmosphere with 5% CO2
The B7 family is associated with HCC aggressiveness, leading to a poor prognosis [15,16,17,18,19]
Summary
Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most frequent cause of cancer-related deaths in the world. Alpha-fetoprotein (AFP) is one of the major proteins in the serum of vertebrate embryos and is highly expressed in HCC. According to the National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines in Oncology 2019, patients are suspected to develop HCC when they have a serum AFP level > 100 ng/ mL or AFP level increases by ≥ 7 ng/mL/month in at least 3 tests. Using the AFP level detection, HCC is the only solid tumor that can be diagnosed by a blood test. AFP has been used as a tumor marker of HCC and plays an essential role in liver cancer screening, diagnosis, monitoring, and prognosis prediction, but its biological functions are still poorly understood [5, 6]
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