Helicobacter pylori-Induced Progranulin Promotes the Progression of the Gastric Epithelial Cell Cycle by Regulating CDK4

Abstract

Helicobacter pylori, a group 1 carcinogen, colonizes the stomach and affects the development of stomach diseases. Progranulin (PGRN) is an autocrine growth factor that regulates multiple cellular processes and plays a tumorigenic role in many tissues. Nevertheless, the mechanism of action of PGRN in gastric cancer caused by H. pylori infection remains unclear. Here, we investigated the role of PGRN in cell cycle progression and the cell proliferation induced by H. pylori infection. We found that the increased PGRN was positively associated with CDK4 expression in gastric cancer tissue. PGRN was upregulated by H. pylori infection, thereby promoting cell proliferation, and that enhanced level of proliferation was reduced by PGRN inhibitor. CDK4, a target gene of PGRN, is a cyclin-dependent kinase that binds to cyclin D to promote cell cycle progression, which was upregulated by H. pylori infection. We also showed that knockdown of CDK4 reduced the higher cell cycle progression caused by upregulated PGRN. Moreover, when the PI3K/Akt signaling pathway (which is promoted by PGRN) was blocked, the upregulation of CDK4 mediated by PGRN was reduced. These results reveal the potential mechanism by which PGRN plays a major role through CDK4 in the pathological mechanism of H. pylori infection.

Keywords: CDK4; Helicobacter pylori; PGRN; cell cycle; gastric epithelial cells.

Fig. 1. Differences of PGRN and CDK4 protein expression between gastric cancer tissue and adjacent normal tissue.

(A, B) Expression levels of PGRN (A) and CDK4 (B) in both gastric cancer and adjacent normal tissues as measured by immunohistochemistry. The results represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001, paired t-test. All data are mean values of three biological replicates.

Fig. 2. H. pylori infection promotes cell cycle progression and cell proliferation.

(A) BGC-823 cells cocultured with H. pylori at a multiplicity of infection (MOI) 50:1 for 3 h, and their clonogenic potential were then assessed. (B) Flow cytometry results of BGC-823 cells infected with H. pylori 26695 at a MOI of 50:1 for 6, 12, and 24 h. (C) Flow cytometric results of H. pylori 26695 infected BGC-823 cells at different MOIs (10:1, 20:1, 50:1, 100:1, and 200:1). (D) Flow cytometry results of BGC-823 cells pre-treated with BAY11-7082 (5 μM), LY294002 (10 μM) and UO126 (10 μM) for 2 h before incubation with or without H. pylori at a MOI of 50:1 for 12 h. The results represent the mean ± SD of three independent experiments. ns, not significant, HP, Helicobacter pylori, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3. PGRN promotes cell cycle progression and cell proliferation in gastric cancer cells with or without H. pylori infection.

(A) qPCR analysis of the expression of PGRN after transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus. (B, C) Colony formation assays (B) and cell cycle assays (C) of BGC-823 cells transfected with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN and their negative control lentivirus and cocultured with H. pylori at a MOI of 50: 1 for 3 h. The results represent the mean ± SD of three independent experiments. SI, PGRN knockdown group, NS, the control group of PGRN knockdown, GFP, the control group of PGRN overexpressing, HP, Helicobacter pylori, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4. PGRN positively regulates CDK4 to promote cell cycle progression.

(A, B) qPCR and western blot analysis of the expression of CDK4 in BGC-823 cells infected with H. pylori 26695 at a MOI of 100: 1. (C, D) qPCR and western blot analysis of the expression of CDK4 after transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus. (E, F) CDK4 expression after transfection with CDK4-RNAi-13, CDK4-RNAi-14, CDK4-RNAi-15 of CDK4-knockdown lentivirus. (G) Flow cytometry analysis of the cell cycle changes of CDK4 knockdown and coculture with H. pylori in 50:1 MOI in BGC-823 cells. (H) Flow cytometry analysis of the cell cycle changes of CDK4 knockdown cell lines infected with PGRN-knockdown /overexpressed lentivirus and cocultured with H. pylori at a MOI of 50: 1. The results represent the mean ± SD of three independent experiments. SI, PGRN knockdown group, NS, the control group of PGRN knockdown, GFP, the control group of PGRN overexpressing, ns, not significant, HP, Helicobacter pylori, *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 5. PGRN regulates CDK4 through the PI3K/Akt signaling pathway and promotes progression of the gastric mucosal epithelial cell cycle.

(A) qPCR analysis of the expression of PGRN after transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus and cocultured with H. pylori at a MOI of 100: 1. (B) Western blot analysis of CDK4 protein expression in cells pretreated with a PI3K signal pathway inhibitor (LY294002) for 2 h before transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus. (C) qPCR analysis of the expression of CDK4 after transfection with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus and cocultured with H. pylori at a MOI of 100: 1. (D) Western blot analysis of CDK4, Akt, and p-Akt protein expression in cells transfected with pLKO.1-PGRN shRNA-GFP and Plenti6/V5-PGRN lentivirus and cocultured with H. pylori at a MOI of 100: 1. The results represent the mean ± SD of three independent experiments. SI, PGRN knockdown group, NS, the control group of PGRN knockdown, GFP, the control group of PGRN overexpressing, HP, Helicobacter pylori, *p < 0.05, **p < 0.01, ***p < 0.001.