Hypoxia-activated oxidative stress mediates SHP2/PI3K signaling pathway to promote hepatocellular carcinoma growth and metastasis

Abstract

Hepatocellular carcinoma is considered to be the fifth most rampant type of cancer in the whole world and has a high death rate. The hypoxic microenvironment is one of the typical features of tumor tissues and has an important impact on the activity of multiple signaling pathways. It is of great significance to study the effects of hypoxia on the pathophysiological processes and molecular mechanisms of HCC. Signalling pathways associated with SHP2 were analysed using bioinformatics. Detection of relevant protein expression using Western blotting. Tube formation assay was used in evalution of the angiogenic potential. The concentrations of MDA and SOD were measured by ELISA. The cell migration and invasion ability were measured with a scratch wound assay and transwell assay in SMMC-7721, HepG2 and Huh-7 cells. The effect of hypoxia on the growth of hepatocellular carcinoma was examined using subcutaneous graft tumors and HE staining experiments in nude mice. Bioinformatics analysis of SHP2 negatively correlates with the PI3K signalling pathway. Hypoxia promotes the concentration of MDA and inhibited the concentration of SOD. Hypoxia may up-regulate NOX2, NOX4 and p-PI3K and down-regulate the treatment of p-SHP2. Compared with NC group, the expression of SHP2 and p-SHP2 was inhibited in SHP2 KD group and the expression of p-PI3K, HIF1α, COX2, FOXM1, β-catenin and MMP9 was promoted. However, the differences of the expression of p-PI3K, HIF1α, COX2, FOXM1, β-catenin and MMP9 between the two groups were abolished after the addition of PI3K inhibitor. The angiogenesis, migration and invasion abilities were significantly increased in SHP2 KD group compared with NC group. Similarly, after the addition of PI3K inhibitor, the difference of these abilities between the two groups was eliminated. Hypoxia can promote the growth of hepatocellular carcinoma. Hypoxia can activate the oxidative stress-mediated SHP2/PI3K signaling pathway to promote angiogenesis, migration, and invasion in hepatocellular carcinoma, thus advancing the development of hepatocellular carcinoma.

Keywords: Angiogenesis; Hepatocellular carcinoma; Oxidative stress; PI3K; SHP2.

Fig. 1

Bioinformatics analysis results. (A) Differential gene volcano plot; (B) SHP2 and ERK correlation scatter plot; (C) SHP2 and JAK2 correlation scatter plot; (D) SHP2 and STAT3 correlation scatter plot; (E) SHP2 and PI3K correlation scatter plot; (F) SHP2 and AKT correlation scatter plot.

Fig. 2

Results of Western blotting to detect NOX2, NOX4, p-SHP2 and p-PI3K. (A) SMMC-7721 cells were subjected to hypoxia and then treated with the antioxidant NAC, the oxidant H₂O₂, and the SHP2 agonist Trichomide A. Corrective experiments were performed to observe the effects of hypoxia on the relevant signalling pathways in SMMC-7721 cells. Protein banding plots of NOX2, NOX4, p-SHP2, t-SHP2, p-PI3K and t-PI3K detected by Western blot assay; (B) Statistical plot of relative protein expression of NOX2, NOX4, p-SHP2, t-SHP2, p-PI3K and t-PI3K detected by Western blot assay. GAPDH was used as a control protein; (C) Plot of results of ELISA assay for MDA and SOD concentrations. Data are expressed as mean ± SEM; N = 3; **P < 0.01; ^nsP > 0.05.

Fig. 3

Results of Western blotting for SHP2, p-SHP2, p-PI3K, HIF1α and COX2. (A) SMMC-7721 cells were subjected to hypoxia treatment before specific knockdown of SHP2 and corrective experiments using the PI3K inhibitor LY294002 were performed to explore the effects of the associated proteins in SMMC-7721 cells. Protein banding plots of t-SHP2, p-SHP2, p-PI3K, t-PI3K, HIF1α and COX2 detected by Western blot assay; (B) Statistical plot of relative protein expression of SHP2, p-SHP2, p-PI3K, t-PI3K, HIF1α and COX2 detected by Western blot assay. GAPDH was used as a control protein; Data are expressed as mean ± SEM; N = 3; **P < 0.01;^nsP > 0.05.

Fig. 4

The number of angiogenesis was measured by tube formation assay. (A) SMMC-7721 cells were subjected to hypoxia treatment before specific knockdown of SHP2, and corrective experiments using the PI3K inhibitor LY294002 were performed to explore the effects on the angiogenic capacity of SMMC-7721 cells. Diagram of angiogenesis experimental results; (B) Statistical graph of the number of blood vessels formed; Data are expressed as mean ± SEM; N = 3; **P < 0.01;^nsP > 0.05.

Fig. 5

Results of Western blotting for FOXM1, β-catenin and MMP9. (A) SMMC-7721 cells were subjected to hypoxia treatment before specific knockdown of SHP2 and corrective experiments using the PI3K inhibitor LY294002 were performed to explore the effects of proteins related to the migratory capacity of SMMC-7721 cells. Protein banding plots of FOXM1, β-catenin and MMP9 detected by Western blot assay; (B) Statistical plot of relative protein expression of FOXM1, β-catenin and MMP9 detected by Western blot assay. GAPDH was used as a control protein; Data are expressed as mean ± SEM; N = 3; **P < 0.01; ^nsP > 0.05.

Fig. 6

Scratch wound assay was used to detect the ability of cell migration. (A) SMMC-7721, HepG2 and Huh-7 cells were subjected to hypoxia treatment before specifically knocking down SHP2, and corrective experiments using the PI3K inhibitor LY294002 were performed to explore the effects on the migratory capacity of SMMC-7721, HepG2 and Huh-7 cells. Plot of scratch wound assay results of SMMC-7721, HepG2 and Huh-7 cells; (B) Statistics of the scratch spacing for SMMC-7721, HepG2 and Huh-7 cells. Data are expressed as mean ± SEM; N = 3; **P < 0.01; ^nsP > 0.05.

Fig. 7

Transwell assay detected the ability of cell migration and invasion. (A) SMMC-7721, HepG2 and Huh-7 cells were subjected to hypoxia treatment before specifically knocking down SHP2, and corrective experiments using the PI3K inhibitor LY294002 were performed to explore the effects of migratory and invasive capacities of SMMC-7721, HepG2 and Huh-7 cells. Transwell results of SMMC-7721, HepG2 and Huh-7 cells; (B) Statistics of migration and invasion of SMMC-7721, HepG2 and Huh-7 cells. Data are expressed as mean ± SEM; N = 3; **P<0.01; ^nsP>0.05.

Fig. 8

Nude mouse tumor-bearing experiments to detect the effect of hypoxia on hepatocellular carcinoma. (A) SMMC-7721 cells were placed in a hypoxic environment, and then the effects of hypoxia on the growth and metastasis of hepatocellular carcinoma as well as the tumor volume and statistical graphs of the tumor experiments were detected using the nude mice subcutaneous xenograft assay; (B) Graphs of the results of HE staining experiments as well as graphs of the statistical results of the area of tumor sections as well as the number of tumor blood vessels. Data are expressed as mean ± SEM; N = 8; **P < 0.01; ^nsP > 0.05.

Fig. 9

EGb761 mediated SHP2/PI3K signaling pathway inhibits migration and invasion in hepatocellular carcinoma. (A) Western blot protein bands of p-SHP2, t-SHP2,p-PI3K and t-PI3K and relative protein expression statistics; (B) Transwell results of migration and invasion ability of SMMC-7721 cells; (C) Scratch wound assay to detect the migration ability of SMMC-7721 cells and cell spacing statistics. **P < 0.01; *P < 0.05; ^nsP > 0.05.

Fig. 10

Hypoxia can activate oxidative stress-mediated SHP2/PI3K signaling pathway to promote angiogenesis, migration and invasion of hepatocellular carcinoma, thus promoting the growth and metastasis of hepatocellular carcinoma.