RGS4 promotes the progression of gastric cancer through the focal adhesion kinase/phosphatidyl-inositol-3-kinase/protein kinase B pathway and epithelial-mesenchymal transition

Figure 1 High expression of regulator of G protein signaling 4 was associated with poor prognosis in patients with gastric cancer. A: Differential expression of regulator of G protein signaling 4 (RGS4) in The Cancer Genome Atlas Stomach Cancer dataset (TCGA-STAD); B: Differential expression of RGS4 in the integrated analysis of TCGA-STAD and the Genotype-Tissue Expression dataset; C: High expression of RGS4 was associated with lower overall survival in TCGA-STAD dataset; D: The correlation between RGS4 and T stage, N stage, M stage, tumor-node-metastasis stage and tumor grade of gastric cancer (GC); E: Differential expression of RGS4 in GC and para-carcinoma normal tissues of our center’s patients; F: Representative immunohistochemistry images showing the expression of RGS4 in GC and para-carcinoma normal tissues (strong staining: +++; moderate staining: ++; weak staining: +; negative staining: -); G: Differences in protein expression levels of RGS4 between GC and para-carcinoma normal tissues; H: High expression of RGS4 was associated with lower overall survival in GC patients in our center. ^bP < 0.01, ^cP < 0.001. RGS4: Regulator of G protein signaling 4; GC: Gastric cancer.

Figure 2 Regulator of G protein signaling 4 knockdown inhibited the proliferation, migration, and invasion of gastric cancer cells in vitro. A: The expression levels of regulator of G protein signaling 4 (RGS4) in different gastric cancer (GC) cell lines were detected by real-time quantitative polymerase chain reaction; B: The interference efficiency of the lentiviral human RGS4-targeting short hairpin RNA was validated by real-time quantitative polymerase chain reaction (left) and western blot (right) analysis; C: The proliferation of MGC-803 cells with RGS4 knockdown was determined by cell counting kit-8 assays; D: The effects of RGS4 knockdown on the colony formation of MGC-803 cells; E: The migration ability of MGC-803 cells was measured by wound healing; F: The invasion of MGC-803 cells with RGS4 knockdown was determined by transwell assays. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, NS: No significance. RGS4: Regulator of G protein signaling 4.

Figure 3 Overexpression of regulator of G protein signaling 4 promoted the proliferation, migration, and invasion of gastric cancer cells in vitro. A: The interference efficiency of the lentiviral carrying regulator of G protein signaling 4 (RGS4) was validated by real-time quantitative polymerase chain reaction (left) and western blot (right) analysis (overexpressing RGS4); B: The proliferation of AGS cells with RGS4 overexpression was determined by cell counting kit-8 assays; C: The effects of RGS4 overexpression on the colony formation of AGS cells; D: The migration ability of AGS cells was measured by wound healing; E: The invasion of AGS cells with RGS4 overexpression was determined by transwell assays. ^aP < 0.05, ^bP < 0.01, ^dP < 0.0001, NS: No significance. RGS4: Regulator of G protein signaling 4.

Figure 4 The expression level of regulator of G protein signaling 4 affected the focal adhesion kinase/phosphatidyl-inositol-3-kinase/protein kinase B pathway and epithelial mesenchymal transition in gastric cancer cells. A: Gene Set Enrichment Analysis results showed the relevant pathways that regulator of G protein signaling 4 may affected in The Cancer Genome Atlas Stomach Cancer dataset; B-E: The expression levels of focal adhesion kinase signaling pathway and epithelial mesenchymal transition related protein of MGC-803 cells and AGS cells were determined by western blot analysis. β-actin was used as a loading control. ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. FAK: Focal adhesion kinase; PI3K: Phosphatidyl-inositol-3-kinase; AKT: Protein kinase B.

Figure 5 Knocking down regulator of G protein signaling 4 in gastric cancer cells inhibited tumor growth in vivo. A-F: Tumors from MGC-803 cells with regulator of G protein signaling 4 (RGS4) knockdown and AGS cells with RGS4 overexpression and their controls (n = 5/groups) (A and D), tumor growth curves (B and E), tumor weight of each group (C and F); G: The expression of focal adhesion kinase/phosphatidyl-inositol-3-kinase/protein kinase B pathway and epithelial mesenchymal transition related proteins in MGC-803 cells and AGS cells in vivo were validated by western blot. β-actin was used as a loading control; H: The expression of RGS4, Ki67 and p-AKT in these xenografts via immunohistochemistry staining; I: Schematic diagram of the mechanism of RGS4 promoting gastric cancer progression. ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. FAK: Focal adhesion kinase; PI3K: Phosphatidyl-inositol-3-kinase; AKT: Protein kinase B; RGS4: Regulator of G protein signaling 4; PIP: Prolactin-induced protein; mTOR: Mechanistic target of rapamycin.