Helicobacter pylori CagA Protein Regulating the Biological Characteristics of Gastric Cancer through the miR-155-5p/SMAD2/SP1 axis

Abstract

Helicobacter pylori (Hp) is a grade Ι carcinogen of gastric cancer (GC), and its high infection rate seriously affects human health. Cytotoxin-associated gene A (CagA) plays a key role in the carcinogenesis of Hp as one of its main virulence factors. miR-155-5p is abnormally expressed in patients with GC, associated with the occurrence and development of cancer. However, little is known about the association between CagA and miR-155-5p. (1) Background: This study explored the association and mechanism of CagA and miR-155-5p in GC. (2) Methods: The CagA sequence was obtained from the NCBI. After sequence optimization, it was connected to the pcDNA3.1 vector to construct a CagA eukaryotic expression plasmid (pcDNA-CagA). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to investigate the expression of miR-155-5p and CagA in GC cells. The function of CagA on GC cells was detected by CCK8, wound healing, and Transwell assays. Similarly, the function of miR-155-5p was also studied through the above functional experiments after the miR-155-5p overexpression and knockdown models had successfully been constructed. The associations among CagA, miR-155-5p, and SMAD2/SP1 were evaluated using RNA immunoprecipitation (RIP) and rescue experiments. (3) Results: The expression of miR-155-5p was significantly reduced in GC cells, and the expression of miR-155-5p was further reduced after CagA induction. Both overexpressed CagA and knockdown miR-155-5p cell models enhanced malignant transformation, whereas overexpressed miR-155-5p inhibited malignant transformation in vitro. The function of miR-155-5p on GC cells could be influenced by CagA. We also found that the influence of miR-155-5p on SMAD2 and SP1 could be regulated by CagA. (4) Conclusions: CagA potentially regulates the biological function of GC cells through the miR-155-5p/SMAD2/SP1 axis. miR-155-5p could be a therapeutic target for GC related to CagA.

Keywords: CagA; SMAD2; SP1; gastric cancer; miR-155-5p.

Figure 1

The expression of CagA in AGS cells and the effects of CagA overexpression on the biological characteristics of GC AGS cells. (a) qRT-PCR analysis of CagA mRNA expression after treatment with pcDNA-CagA and pcDNA-NC. (b) Western blot analysis of CagA. (c) Assessment of the proliferation of AGS cells transfected with pcDNA-CagA and pcDNA-NC by CCK-8 assay. (d) Wound healing assay to detect the effect of CagA on cell migration. (e) CagA overexpression regulated the migration and invasion of AGS cells. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 2

The expression of miR-155-5p in AGS cells and the effects of overexpression and knockdown of miR-155-5p on the biological characteristics of AGS. (a,b) qRT-PCR analysis of miR-155-5p mRNA expression after treatment with pcDNA-CagA, miR-155-5p mimics and inhibitor regents. (c,d) Assessment of the proliferation of AGS cells transfected with miR-155-5p mimics and inhibitors by CCK-8 assay. (e) Wound healing assay to detect the effect of miR-155-5p on cell migration. (f) miR-155-5p overexpression and knockdown regulated the migration and invasion of AGS cells. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3

CagA regulates malignant transformation through miR-155-5p in vitro. (a,b) Co-transfection of miR-155-5p mimics and pcDNA-CagA to investigate malignant transformation of cells with CCK-8 and Transwell assays in the AGS cell line. (c,d) Co-transfection of miR-155-5p inhibitor and pcDNA-CagA to investigate malignant transformation of cells with CCK-8 and Transwell assays in the AGS cell line A: pcDNA-CagA + miR-155-5p mimics NC; B: pcDNA-CagA + miR-155-5p mimics; C: pcDNA-NC + miR-155-5p mimics; D: pcDNA-CagA + miR-155-5p inhibitors NC; E: pcDNA-CagA + miR-155-5p inhibitors; F: pcDNA-NC + miR-155-5p inhibitors; * p < 0.05; *** p < 0.001.

Figure 4

SMAD2 and SP1 were targets of miR-155-5p. (a) qRT-PCR of SMAD2 and SP1 expression in AGS cells transfected with miR-155-5p mimics and inhibitor. (b) RIP assays with anti-SMAD2, anti-SP1, and anti-IgG antibodies in AGS cell lines were used to detect miR-155-5p mRNA levels. (c) Western blot analysis of SMAD2 and SP1 transfected with miR-155-5p mimics and inhibitor. * p < 0.05; ** p < 0.01; *** p < 0.001.

Figure 5

CagA regulates malignant transformation through the miR-155-5p/SMAD2/SP1 axis in vitro. (a) Western blot analysis of SMAD2 and SP1 transfected with pcDNA-CagA. (b) Co-transfection of miR-155-5p mimics and pcDNA-CagA to investigate the level of SMAD2 and SP1 expression with Western blots in the AGS cell line. (c). Co-transfection of miR-155-5p inhibitors and pcDNA-CagA to investigate the levels of SMAD2 and SP1 expression with Western blots in the AGS cell line A: pcDNA-CagA + miR-155-5p mimics NC; B: pcDNA-CagA + miR-155-5p mimics; C: pcDNA-NC + miR-155-5p mimics; D: pcDNA-CagA + miR-155-5p inhibitors NC; E: pcDNA-CagA + miR-155-5p inhibitors; F: pcDNA-NC + miR-155-5p inhibitor; * p < 0.05; ** p < 0.01; *** p < 0.001.

Scheme 1

General diagram summarizing the discovery and pathway.