Tumor Cells Talk to Normal Cells Through Exosomes to Rebuild the Tumor Microenvironment

Abstract

BackgroundExosomes play a key role in the growth of normal cells and various diseases such as cancer. Tumor exosomes regulate the connection between normal cells and cancer cells in the tumor microenvironment, thereby promoting the growth and invasion of cancer cells.MethodsWe used HepG2 cells silenced by shRNA targeting GPC3, LO2 and HepG2 cells treated with different concentrations of GPC3. We determined the effects of GPC3 on cell proliferation, apoptosis and invasion using CCK8, flow cytometry and Transwell, and Western blotting Method to determine the expression of GPC3/WNT3A/β-catenin.HepG2 exosomes (Exo) and HepG2 exosomes treated with shRNA targeting GPC3 (sh-GPC3-Exo) were used to treat LO2 and HepG2 cells separately. Cell proliferation was measured by CCK8 experiment.The cell cycle and apoptosis were measured by flow cytometry. The cell invasion ability was analyzed by Transwell. The expression of GPC3/WNT3A/β-catenin signal protein was determined by Western blotting.ResultsThis is the first study to prove the bidirectional regulation of GPC3 between normal cells and liver cancer cells. After treatment of LO2 cells and HepG2 cells with GPC3, the LO2 cell cycle was blocked in the G0/G1 phase, while inhibiting cell proliferation, promoting cell apoptosis and invasion, but for HepG2 cells it appeared to promote proliferation.Silencing GPC3 can inhibit the proliferation and invasion, and promote cell apoptosis of HepG2. Subsequent experiments found that the expression of GPC3 was found in both LO2 and HepG2 exosomes, and the expression of GPC3 in HepG2 exosomes was significantly higher than that in LO2 exosomes. These suggest that GPC3 in exosomes has the potential to become a biomarker of HCC.In addition, HepG2 exosomes (Exo) can inhibit the proliferation of LO2 cells and promote apoptosis and invasion, which is consistent with the effect of GPC3 treatment. We also found that GPC3 is contained in HepG2 exosomes (shGPC3-Exo) that have silenced GPC3, which has the same effect on LO2 cells as HepG2 exosomes (Exo), but the degree of influence is reduced. shGPC3-Exo showed a promoting effect on the proliferation of HepG2 cells, but inhibited cell invasion. Therefore, GPC3 in Exo plays a role in the proliferation of LO2 cells and HepG2 cells. Further studies have shown that GPC3 in liver cancer exosomes regulates the proliferation, apoptosis and invasion of LO2 and HepG2 cells through the Wnt /β-catenin signaling pathway.ConclusionGPC3 in the exosomes of liver cancer cells inhibits the proliferation of normal liver cells and promotes apoptosis by activating the Wnt/β-catenin signaling pathway, promotes the proliferation of liver cancer cells, and assists the occurrence and development of HCC.