The "Global Cancer Report 2020" published by the WHO pointed out that there are more than 900,000 liver cancer patients worldwide and 830,000 deaths. The leading cause of death in patients with liver cancer is tumor progression and metastasis. The tumor microenvironment determines the type of liver cancer, because tumor-specific markers carried by tumor exosomes participate in cell communication in the tumor microenvironment, and are related to the growth and invasion ability of tumor cells. In recent years, researchers have found that the expression rate of Glypican-3 in liver cancer tissues has gradually and significantly increased with the increase of clinical stages. We also discovered increased levels of Glypican-3 in the blood of liver cancer patients. Huixia Di et al. also found that compared with healthy people and hepatitis B patients, the expression of GPC3 in serum exosomes of HCC patients can be detected and the content is significantly increased, indicating that liver cancer cells can secrete GPC3 into the blood circulation. We noticed in vitro experiments that GPC3 was expressed in normal liver cells, HCC cells and their exosomes, and the GPC3 content in exosomes of HCC cell was significantly higher than that in normal liver cell exosomes. It showed that when GPC3 promotes the metastasis of liver cancer cells, it may also carry out cell communication with normal liver cells, affecting the growth of normal liver cells, thereby helping the metastasis of tumor cells. Therefore, elucidating the role of GPC3 in hepatocytes and signal transduction pathways in exosomes may provide a theoretical basis for revealing the development process of liver cancer and determining new early diagnosis methods.
Shanshan W et al. found that GPC3 showed a significant promoting effect in the growth and migration of HCC cells , which is consistent with our experimental results. In this research, we discovered that the number of apoptosis of HCC cells remains unchanged after GPC3 is added, but the number of apoptosis of HCC cells increased after GPC3 gene knockdown. Therefore, GPC3 in HCC cells may inhibit cell apoptosis. This is the first time we have discovered that GPC3 inhibits the growth of normal hepatocytes by blocking the cell cycle and significantly promotes their apoptosis. Therefore, GPC3 may help HCC cell growth by inducing normal liver cell apoptosis and growth, and further promote the development of liver cancer. GPC3 was found stimulating the growth of HCC cells by increasing the autocrine/paracrine pathway and activating the canonical Wnt signaling pathway. We noticed that for normal liver cells, GPC3 also activates the classic wnt / β-catenin signaling pathway, which further promotes the occurrence of HCC.
Exosomes transmit information through the contents of vesicles. Exosomes secreted by tumor cells can regulate surrounding or distant non-tumor cells to form a pre-metastasis microenvironment[27, 28]to promote tumor cell metastasis. We next tested whether GPC3 in liver cancer exosomes can perform its function in normal liver cells. The optimal concentration of liver cancer exosomes (100 µg/mL) to promote growth acts on liver cell lines. In normal liver cells, liver cancer exosomes can significantly inhibit growth, block cell cycle, and promote apoptosis. After the decrease of GPC3 content in liver cancer exosomes, the effect on normal cells is reduced, which proves that the exosomes of liver cancer GPC3 can inhibit the growth of normal liver cells and promote apoptosis. The above results indicate that the content of GPC3 in liver cancer cell exosomes is significantly higher than that of normal liver cell exosomes, while GPC3 in liver cancer cell exosomes inhibits the growth of normal liver cells, promotes apoptosis, which can contribute to the development of HCC.
The activation of the Wnt signaling pathway is one of the most common molecular events related to the progression of HCC. Further studies have shown that the Wnt signaling pathway is essential for GPC3-mediated cell growth. However, the regulatory mechanism of GPC3 in HCC exosomes involved in HCC cell growth is still unclear. In this study, we proved that GPC3 and wnt / β-catenin signaling protein exist in the exosomes of normal hepatocytes and HCC cells, and the content of wnt / β-catenin signaling protein is significantly lower than that in cells. GPC3 in the exosomes of HCC cells can activate the Wnt/β-catenin pathway of normal hepatocytes, and the effect is reduced after adding HepG2-shGPC3-exo, which is consistent with the trend of adding GPC3. This further proves that GPC3 in HCC cell exosomes activates the Wnt /β-catenin pathway in normal liver cells, thereby promoting the occurrence of HCC.