Exosomes isolation and verification
Previous experimental results found that the expression level of GPC3 in liver cancer tissues was significantly higher than that of normal controls. In order to prove the expression of GPC3 in HCC cell lines, we selected normal liver cells LO2 and representative HCC cells HepG2.
Nanoparticle tracking analysis (NTA) was performed with ZetaViewPMX 110 to observe the particle size of exosomes in the cell culture medium. The sample showed a vesicle structure with a diameter of about 50-150 nm, which was in line with the morphological characteristics of exosomes in the literature(Figure 1A). Western blot analysis showed the presence of CD63 and TSG101, which were reported to be exosomal markers(Figure 1B). The results indicate that exosomes have been successfully isolated from LO2 and HepG2 cell lines. In this study, we verified the expression of GPC3 in exosomes of LO2 and HepG2 cell lines by Western blot and ELISA. The expression of GPC3 in liver cancer cell line HepG2 exosomes was more than that in normal liver cell line LO2 exosomes, which was consistent with the comparison of GPC3 expression in the two cells (Figure 1C,D).
The effect of GPC3 on the proliferation and apoptosis of normal cells
Studies have shown that GPC3 promotes the development of liver cancer, but due to the low content of GPC3 in healthy human tissues, there is no research on the effect of GPC3 on normal cells. In order to explore whether the effect of GPC3 on the proliferation and invasion of normal liver cells is different from that of liver cancer cells, we added GPC3 to LO2 and HepG2 cells to study the effects of different concentrations of GPC3 on cell proliferation. After adding different concentrations of GPC3, the growth of LO2 cells was inhibited, and the growth of HepG2 cells was promoted. The effect of GPC3 increased first and then decreased with the increase of concentration. GPC3 at a dose of 1 mg/ml had the most obvious effect on the two types of cells (Figure 2A). Flow cytometry analysis of Annexin V/PI staining also showed that apoptotic cells increased in LO2 cells after the addition of GPC3 at a concentration of 1 mg/ml, while there was no difference in the level of apoptosis between GPC3 treated and untreated HepG2 cells (Figure 2B) . Subsequently, we tested whether GPC3 affects cell growth through cell cycle arrest. As shown in Figure 2C, the addition of GPC3 resulted in an increase in the number percentage of the G0/G1 population of LO2 cells, resulting in G0/G1 block; on the contrary, the number percentage of the G0/G1 population decreased in HepG2 cells. Later, in the transwell analysis of cell invasion ability, the invasion ability of LO2 cells and HepG2 cells increased after adding GPC3 (Figure 2D). These data indicated that GPC3 can inhibit the proliferation of normal cells by enhancing cell apoptosis and cell cycle arrest, while for HCC cells it can reduce cell cycle arrest to promote the proliferation of HCC cells, and not effect on cell apoptosis. GPC3 has the ability to promote the invasion of normal liver cells and HCC cells.
In order to further explore the role of GPC3 in the growth of HCC cells, shRNA targeting GPC3 was transfected into HepG2 cells. After transfection, the expression of GPC3 in HepG2 cells decreased in mRNA and protein levels (Figure 3A–C). The growth of HepG2 cells was inhibited after knockdown (Figure 3D). Flow cytometry analysis of Annexin V/PI staining also showed an increase in apoptotic cells in HepG2 cells after GPC3 knockdown (Figure 3E). Subsequently, we tested whether cell cycle arrest affected the growth of GPC3 knockdown HepG2 cells. As shown in Figure 3F, knockdown of GPC3 led to an increase in the percentage of the G0/G1 population of HepG2 cells, showing a G0/G1 blocking trend. Next, transwell analysis was performed to evaluate the effect of GPC3 on HCC cell invasion. The results showed that the invasive ability of the HCC cell line knocked down by GPC3 was reduced (Figure 3G). These data indicated that GPC3 gene silencing in HCC cells can promote cell apoptosis and cell cycle arrest, thereby inhibiting the proliferation of HCC cells and inhibiting the invasion of HCC cells.
Exosomal GPC3 stimulated the proliferation and migration of HCC cells
The previous research found that GPC3 protein was detected in serum exosomes of HCC patients, which was consistent with the expression of GPC3 in HCC tissues. Next, we will observe the effect of GPC3 in liver cancer exosomes on normal liver cells. LO2 and HepG2 cells were treated with HepG2 exosomes (Exo) and GPC3 knockdown HepG2 exosomes (shGPC3-Exo), respectively. The GPC3 content in exosomes of HepG2 cells decreased after GPC3 was knocked down (Figure 4A).
After adding Exo, the growth of LO2 cells was significantly inhibited, which was concentration-dependent (Figure 4B). shGPC3-Exo has the same inhibitory effect on the proliferation of LO2 cells, but the inhibitory effect is weakened, compared with the addition of Exo. shGPC3-Exo also promoted the growth of HepG2 cells in a dose-dependent manner (Figure 4B). This indicated that GPC3 in HCC exosomes had an inhibitory effect on the growth of normal hepatocytes.
Flow cytometry analysis of Annexin V/PI staining showed that after treatment with 100 μg/mL Exo or shGPC3-Exo, the number of apoptosis of LO2 cells increased, and the effect of Exo group was more obvious than that of shGPC3-Exo group. The addition of shGPC3-Exo to HepG2 cells did not affect the number of apoptosis (Figure 4C). Later, in the detection of the effect of exosomes on the liver cell cycle, it was found that the percentage of the number of LO2 cells G0/G1 population increased after the addition of the two groups of exosomes, showing G0/G1 blockade. The percentage of the number of HepG2 cells in the G0/G1 population decreased after shGPC3-Exo was added (Figure 4D). This indicated that GPC3 in HepG2 cell exosomes can promote the apoptosis of LO2 cells, and may promote apoptosis by inhibiting the cell cycle of LO2 cells.
Then we used Transwell experiment to analyze the influence of liver cancer exosomes on the migration ability of normal liver cells and HCC cells. After adding Exo, the number of migrating cells in LO2 cells remained unchanged, while the number of migrating cells increased significantly after adding shGPC3-Exo. shGPC3-Exo significantly reduced the number of HepG2 cell migration. The results showed that the migration effect of HepG2 exosomes on normal cells was related to the dose of GPC3 (Figure 4E).
These data indicate that HCC cells can promote the apoptosis of normal hepatocytes, inhibit cell cycle and cell proliferation, and affect the invasion of normal hepatocytes through GPC3 in their exosomes; while GPC3 in HCC exosomes can increase cell cycle by promoting cell cycle. Proliferation of HCC cells.
GPC3 in liver cancer exosomes affects Wnt/β-catenin signaling pathway in HCC cells
Wnt/β-catenin signaling pathway plays an important role in promoting the proliferation and migration of HepG2 cells, and plays an important role in the occurrence, development and metastasis of liver cancer. It is an important molecular mechanism of liver cancer. Studies have shown that in liver cancer, GPC3 plays a role through the Wnt/β-catenin signaling pathway. The above experiments also showed that GPC3 in liver cancer exosomes affected the proliferation of normal liver cells.In the follow-up, we will investigate whether GPC3 in liver cancer exosomes also affects the proliferation of normal liver cells through the Wnt/β-catenin signaling pathway.
First, compare the GPC3 and Wnt/β-catenin signaling pathways in hepatocytes and hepatocyte exosomes. Western blot results showed that the GPC3 content of LO2 cell exosomes was significantly less than that in the cells, and the expression of Wnt3a and β-catenin protein was uniformly reduced; and the GPC3, Wnt3a and β-catenin protein content of HepG2 exosomes were also less than that of HepG2 The content in cells (Figure 5A, B).
Later, it was found in further studies that the GPC3 content in LO2 cells increased significantly after GPC3 treatment, while Wnt3a and β-catenin protein increased; after Exo was added, the GPC3, Wnt3a and β-catenin protein content in LO2 cells also increased significantly. After shGPC3-Exo, the contents of GPC3, Wnt3a and β-catenin were increased compared with the control group LO2 cells, but the protein content was lower than that of Exo (Figure 5C, D). It showed that GPC3 in HepG2 cell exosomes increased GPC3, Wnt3a and β-catenin protein content in LO2 cells. In the study of HepG2 cells, it was found that after knocking down the GPC3 in the cells, as the GPC3 in the cells decreased significantly, the content of Wnt3a and β-catenin protein decreased;After adding GPC3 to HepG2 cell culture, it was found that the content of GPC3 and β-catenin protein increased significantly, and the content of Wnt3a also increased (Figure 5C, D). After adding shGPC3-Exo, the contents of GPC3, Wnt3a and β-catenin in HepG2 cells increased compared with the control group.
The results of these studies indicate that the levels of GPC3 and Wnt/β-catenin signaling protein in normal liver cells are significantly lower than those in liver cancer cells. GPC3 in liver cancer cell exosomes can activate Wnt/β-catenin signaling pathway by increasing GPC3 in normal liver cells.