Helicobacter pylori upregulates circPGD and promotes development of gastric cancer

Abstract

Purpose: Helicobacter pylori (H. pylori) has unique biochemical traits and pathogenic mechanisms, which make it a substantial cause of gastrointestinal cancers. Circular RNAs (circRNAs) have concurrently been identified as an important participating factor in the pathophysiology of several different cancers. However, the underlying processes and putative interactions between H. pylori and circRNAs have received very little attention. To address this issue, we explored the interaction between H. pylori and circRNAs to investigate how they might jointly contribute to the occurrence and development of gastric cancer.

Methods: Changes in circPGD expression in H. pylori were detected using qRT-PCR. Cell proliferation and migration changes were assayed by colony formation, the CCK-8 assay and the transwell assay. Apoptosis was measured by flow cytometry. Western blot was conducted to detect changes in cell migration, apoptosis, proliferation and inflammation-associated proteins. QRT-PCR was used to measure changes in circPGD and inflammation-associated factors.

Results: We found that H. pylori induced increased circPGD expression in infected human cells and facilitated gastric cancer progression in three ways by promoting cell proliferation and migration, enhancing the inflammatory response, and inhibiting apoptosis.

Conclusions: CircPGD appears to play a role in H. pylori-related gastric cancer and may thus be a viable, novel target for therapeutic intervention.

Keywords: Apoptosis; Gastric cancer; Helicobacter pylori; Inflammation; circRNAs.

Fig. 1

Helicobacter pylori upregulated circPGD expression in gastric cancer-associated cells and tissues. A The expression levels of circPGD were evaluated by qRT-PCR after H. pylori infection of cells. B The expression of circPGD in gastric tissues taken from healthy individuals, those with H. pylori infection, and patients with gastric cancer was examined using qRT-PCR. C FISH investigation circPGD expression and localization. D The colony morphology of H. pylori standard strain 26695 on Columbia blood plates. E Gram staining results of H. pylori strain 26695. F Urease test results of H. pylori strain 26695. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 2

Helicobacter pylori reversed the reduced cell migratory capacity and the EMT process caused by circPGD knockdown. A The transwell assay results show that circPGD knockdown inhibited cell migration, while it was increased after H. pylori infection. B Western blotting results show that H. pylori altered the changes of protein expression levels of vimentin, N-cadherin, MMP2 and E-cadherin caused by circPGD knockdown. C The qRT-PCR results validated the efficiency of circPGD knockdown and indicate that circPGD expression was increased after H. pylori infection. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 3

Helicobacter pylori further enhanced the migratory ability of cells and increased the EMT process due to circPGD overexpression. A Vimentin, N-cadherin, MMP2, and E-cadherin levels changed in response to H. pylori, as detected by Western blotting. B The transwell assay showed that cell migration was improved by circPGD overexpression and was also enhanced after H. pylori infection (Fig. 3B). C The effective overexpression of circPGD was validated by qRT-PCR. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 4

Helicobacter pylori affected cell apoptosis and proliferation by modulating the expression of circPGD. A Flow cytometry analysis of the impact of H. pylori infection and the silencing of intracellular circPGD on apoptosis. B Western blotting results showing that H. pylori infection increased PCNA expression in cells transfected with the negative control (NCs), but lowered its expression in the circPGD knockdown group. C Clone-formation experiments and D the CCK-8 assay results in contrast to the siRNA control group, showing that H. pylori increased cell proliferation, whereas circPGD knockdown reduced it. E PCNA expression was determined by Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 5

Helicobacter pylori exacerbated the inhibitory effect of circPGD overexpression on apoptosis and promoted cell proliferation. A Flow cytometry analysis of the impact of intracellular circPGD overexpression and H. pylori infection on apoptosis. B Western blotting was used to examine changes in the expression of proteins linked to apoptosis. C Clone-formation tests and D CCK-8 assay results, showing cell proliferation further facilitated by H. pylori infection. E PCNA expression was elevated by Western blotting. *P < 0.05, **P < 0.01, ***P < 0.001

Fig. 6

Helicobacter pylori enhanced the inflammatory response in cells with circPGD knockdown. A P-p65/p65 alterations were examined by Western blotting. B According to qRT-PCR, circPGD knockdown decreased the expression of markers linked to cellular inflammation, whereas H. pylori infection enhanced their expression

Fig. 7

Helicobacter pylori further amplified the increased inflammatory response of cells caused by circPGD overexpression. A Western blotting was used to examine the changes in p-p65/p65 following circPGD overexpression and H. pylori infection. B According to qRT-PCR, circPGD overexpression increaseed the expression of markers associated with cellular inflammation, while H. pylori infection strengthened this effect