Title: GPC3 in the Exosomes from Hepatocellular Carcinoma HepG2 Cells

Abstract Background Exosomes play an important role in regulating the growth in normal and abnormal cells. Exosomes secreted from tumor cells are also involved in regulating the growth behaviors of normal cells and tumor cells. Methods HepG2 cells, LO2 and HepG2 cells with GPC3 knocked down using shRNA (HepG2-shGPC3), were treated with different concentrations of GPC3. The effects of different concentrations of GPC3 on cell growth and apoptosis were determined using CCK8 and flow cytometry. HepG2 exosomes (Exo) and exosomes of HepG2 cells with GPC3 knocked down using shRNA (shGPC3-Exo) were used to treat LO2 and HepG2 cells separately. Cell growth was measured by CCK8 kit. The cell cycle and apoptosis were measured by flow cytometry. The expression of GPC3/WNT3A/β-catenin signal protein was determined by Western blotting. Results We found GPC3 has a two-way regulation between normal cells and HCC cells, which is the innovation of this research. After treating LO2 cells and HepG2 cells with GPC3, the LO2 cell cycle was blocked in the G0/G1 phase, while cell growth was inhibited and apoptosis was promoted; however, it appeared to promote the growth of HepG2 cells. Knocking down GPC3 can inhibit the growth and promote cell apoptosis of HepG2. In subsequent experiments, we found that GPC3 was expressed in both LO2 and HepG2 exosomes, and the expression of GPC3 in HepG2 exosomes is significantly higher than that of LO2 exosomes. These results suggested that GPC3 in exosomes has the potential to become a biomarker of HCC. In addition, HepG2 exosomes (Exo) can inhibit the growth of LO2 cells and promote apoptosis, which is consistent with the effect of GPC3 treatment. Further, we found that GPC3 in shGPC3-Exo had the same effect on LO2 cells as HepG2 exosomes (Exo), but the degree of influence was reduced. shGPC3-Exo showed a promoting effect on the growth of HepG2 cells. Therefore, GPC3 in exosomes plays a role in the growth of LO2 cells and HepG2 cells. Further studies have shown that GPC3 in liver cancer exosomes regulates the proliferation, apoptosis of LO2 and HepG2 cells through the Wnt /β-catenin signaling pathway. Conclusion GPC3 in the exosomes of liver cancer cells inhibits the growth of normal liver cells and promotes apoptosis by activating the Wnt/β-catenin signaling pathway, and assists the occurrence and development of HCC.