Synergic effect of diabetes mellitus and H. pylori infection on proliferation of cells via downregulating the expression of PTEN by hypermethylating its promoter region

Background It has been reported that CagA of H. pylori reduced the expression of PTEN by enhancing its promoter methylation. Furthermore, DM may also promote the methylation status of PTEN, a tumor suppressor gene in Gastric cancer. It is intriguing to explore whether DM may strengthen the tumorigenic effect of HP by promoting the methylation of PTEN promoter and whether the administration of metformin may reduce the risk of GC by suppressing the methylation of PTEN promoter. Methods Bisulfite sequencing PCR was performed to measure the DNA methylation of PTEN promoter in GC patients and HGC-27 cells treated under different conditions. Quantitative real-time PCR was carried out to measure the expression of PTEN mRNA. Immunohistochemistry and Western blot were used to evaluate the expression of PTEN protein. Immunofluorescence and flow cytometry were performed to analyze the apoptosis of GC tissue samples and HGC-27 cells treated under different conditions. MTT assay was carried out to examine the proliferation of HGC-27 cells. Results DNA methylation of PTEN promoter was synergetic enhanced by HP infection and Diabetes mellitus in patients with Gastric cancer. Accordingly, the expression of PTEN was suppressed in GC patients with HP infection and DM. Furthermore, cell apoptosis was decreased in GC patients with HP infection and DM. Metformin showed an apparent effect on maintaining CagA induced elevation of PTEN promoter methylation, thus attenuating the PTEN expression, increasing the proliferation and suppressing the apoptosis of HGC-27 cells. Conclusion In this study, we collected GC tumor tissues from GC patients with or without DM/HP to compare their PTEN methylation and expression while testing the effect of Metformin on the methylation of PTEN promoter. Our study provided evidence for the mechanism underlying the therapeutic role of Metformin in GC treatment.


Abstract
Background It has been reported that CagA of H. pylori reduced the expression of PTEN by enhancing its promoter methylation. Furthermore, DM may also promote the methylation status of PTEN, a tumor suppressor gene in Gastric cancer. It is intriguing to explore whether DM may strengthen the Conclusion In this study, we collected GC tumor tissues from GC patients with or without DM/HP to compare their PTEN methylation and expression while testing the effect of Metformin on the methylation of PTEN promoter. Our study provided evidence for the mechanism underlying the therapeutic role of Metformin in GC treatment.

Background
Gastric cancer (GC) is one of highly frequently diagnosed cancers in the world. GC causes the 2nd highest death rate among all types of cancers. The majority of GC cases are actually gastric carcinoma and gastric antrum cancer, although the occurrence of carcinoma at gastroesophageal junction has actually been rising steadily [1][2][3]. Review of the onset of GC showed that the occurrence of GC is slowly increasing among youth adults as well as in children. Also, the occurrence, death rate, as well as metastasis of GC are pretty high, along with low rate of early diagnosis, good prognosis, as well as 5 year survival [4].
The gene of phosphatase and Tensin homologue on chromosome 10 (PTEN) is deemed as a possible tumor suppressor in numerous forms of cancers in human [5]. PTEN is likely to suppress phosphatidylinositol-3-kinase (PI3K) expression through catalyzing the elimination of D3 phosphate from phosphatidylinositol (3,4,5)-triphosphate (PIP3) to dysregulate the production of phosphatidylinositol 4,5-bisphosphate (PIP2) as well as PIP3 [6][7][8]. PTEN is actually downregulated in GC, and the expression of PTEN is also negatively correlated to metastasis in lymph nodes, depth of invasion, as well as the age of GC patients [9][10][11]. PTEN was shown to suppress the cell cycle arrest, apoptosis, as well as metastasis of GC cells [12,13]. Formerly, Tet1 has actually been shown to prevent the metastasis as well as development of GC through PTEN demethylation as well as its expression. Tet1 reduces the migration, development, as well as invasion of GC through the demethylation of CpG islands in the promoter region of PTEN, thus downregulating focal adhesion kinase as well as AKT activity [14].
The role of DM in the risk of cancer has been analyzed in various meta analyses [15]. Epidemiologic research reviewing the relationship between DM and the risk of GC has generated contradictory results [16,17]. It was shown that PTEN promoter hypomethylation is actually quite common among Uyghur patients carrying wild type T2DM, which might contribute to T2DM pathogenesis. The abnormal methylation of CpG sites in the promoter of PTEN might function as a biomarker for T2DM diagnosis.
Helicobacter pylori are a global threat that has infected about 4 billion individuals [18]. Helicobacter pylori are actually the primary reason for peptic ulcer disease, gastritis, as well as GC. Previous research has actually revealed that the infection by helicobacter pylori caused an epithelial-tomesenchymal transition of epithelial cells in the stomach [19]. It was revealed that CagA considerably lowered the PTEN as well as Tet1 expression. Furthermore, it was uncovered that CagA lowered PTEN expression through enhancing its own methylation, which was actually dramatically decreased by Tet1 [20]. Metformin is a compound extracted from Galega officinalis and has actually been utilized for years in medical treatment to type 2 diabetes mellitus (T2DM) [21]. It was shown that metformin dramatically reduces AKT-dependent phosphorylation [22].
CagA of H. pylori reduces the expression of PTEN by enhancing its promoter methylation [20].
Furthermore, DM may also promote PTEN methylation [23]. Giving the fact that DM and HP infection may synergistically elevate the risk of GC as well as the ability of metformin to suppress the methylation of PTEN, we hypothesized that DM may strengthen the tumorigenic effect of HP by promoting PTEN promoter methylation, and the administration of metformin may reduce the risk of GC by suppressing PTEN promoter methylation [23][24][25]. In this study, we collected GC tumor tissues from GC patients with or without DM/HP to compare their PTEN methylation and expression while testing the effect of Metformin on the methylation of PTEN promoter.

Materials And Methods Human subjects sample collection
In this study, we enrolled 107 patients with gastric cancer and divided them into three groups

Rna Isolation And Real-time Pcr
Real time PCR was done to measure the expression of PTEN and CagA mRNA in collected samples. In brief, total RNA in each sample was isolated by utilizing a PureLink RNA Mini assay kit (Thermo Fisher Scientific, Waltham, MA) in accordance with the routine assay protocol provided by the kit manufacturer. Then, cDNA was generated from isolated total RNA by making use of a PrimeScript RT Reagent assay kit (Thermo Fisher Scientific, Waltham, MA) in accordance with the routine assay protocol provided by the kit manufacturer. In the next step, the real time PCR was carried out on an

MTT cell proliferation assay
The proliferation status of treated cells was measured by utilizing a CellTiter-Glo MTT assay (Promega, Madison, WI) in accordance with the routine assay protocol provided by the kit manufacturer.

Western Blot Analysis
Collected tissue and cell samples were lysed by using a Trizol reagent (Invitrogen, Carlsbad, CA) in accordance with the routine assay protocol provided by the reagent manufacturer to obtain protein lysate, which was then resolved via electrophoresis by making use of a 10% SDS-PAGE gel. After transferring the resolved protein samples onto PVDF membranes, the membranes were blocked by making use of 5% skim milk and then incubated overnight at 4 ° C with anti-PTEN primary antibody (R&D systems, Minneapolis, MN). In the next step, the membranes were washed and further incubated at room temperature for 1 h with horseradish peroxidase-labeled secondary antibody.
Finally, the relative expression of PTEN protein in each sample was analyzed after the protein blot was developed by making use of an Immobilon Western Chemiluminescent HRP Substrate assay kit (EMD Millipore, Billerica, MA) in accordance with the routine assay protocol provided by the kit manufacturer.

Apoptosis Analysis
The status of apoptosis in each collected cell and tissue sample was analyzed by using a propidium iodide/annexin V-FITC apoptosis assay kit (Sigma Aldrich, St. Louis, MO) in accordance with the routine assay protocol provided by the kit manufacturer. The detection of apoptosis was carried out on a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA) at a 488 nm wavelength.

Immunofluorescence And Immunohistochemistry
Immunofluorescence and immunohistochemistry assays were used to detect the expression of PTEN and the status of apoptosis of collected tissue samples, respectively. In brief, for immunohistochemistry assays, the tissue samples were fixed by using 4% of formaldehyde (Sigma  Table 1. Student's t test was utilized to perform the statistical comparison, and revealed that there was no obvious difference in all above characteristics among the three groups.  It is well known that DNA methylation in the promoter region can repress gene expression. Therefore, we further performed quantitative real time PCR and immunohistochemistry assay to examine the PTEN expression in gastric cancer patients in the above three groups. As expected, the expression of PTEN mRNA (Fig.2) and protein (Fig.3) (Fig.8B).

Discussion
Metformin is well known for its anti-diabetic role [26]. It was also shown that metformin reduced the risk of cancer in patients with T2DM [27,28]. The role of metformin in suppressing the proliferation of GC cells is related to its role in blocking cell cycles, which could explain why metformin decreased tumour size in mice with xenograft GC tissues [29,30]. It was shown that the use of metformin significantly decreased the risk of infection by helicobacter pylori dose dependently [31]. In this study, we recruited 107 gastric cancer patients and divided them into three groups according to their status of HP infection and DM to explore the molecular mechanism underlying gastric cancer pathogenesis. We found that HP infection and DM effectively increased the DNA methylation of PTEN promoter in GC patients.
PTEN acts as a crucial mediator in the carcinogenesis of a wide array of cancers [32]. PTEN can degrade the derivatives of PI3K through dephosphorylating position 30 of PIP3 as well as PIP2, subsequently boosting AKT phosphorylation and reducing apoptosis [33,34]. It was shown that the methylation of PTEN promoter can be used as a prognostic marker of survival [35]. It was illustrated Helicobacter pylori infection is one of the most significant dangers for stomach cancers [36]. In a research on 114 GC patients, gastric adenocarcinoma was shown as accounting for 90% in all patients. In helicobacter pylori, the expression of a 120 kDa protein called cytotoxin associated gene A (CagA) was shown to be related to some of the essential features of helicobacter pylori to interact with kinases in host cells to trigger tyrosine phosphorylation [37][38][39] [42][43][44]. The activation of Akt is actually a common event in chemotherapy treatment of GC, indicating a significant role of Akt in causing apoptosis resistance [45,46]. Previous results presented that etoposide boosted the phosphorylation of Akt while minimizing cell survival via the induction of GC apoptosis [47].
It was actually shown that the abnormal methylation in PTEN promoter was substantially reduced in Uyghur subjects suffering from mild cases of T2DM. It was further shown that the hypomethylation in PTEN promoter was actually pretty common in T2DM subjects, indicating that PTEN might contribute to T2DM pathogenesis among Uyghur people [23]. A previous research illustrated that the enhanced PTEN expression in adipose tissues as well as muscular tissues of T2DM rodents might exert a significant effect on insulin resistance in T2DM [48]. Zhu et al. proposed that PTEN controls the production of extracellular matrix in kidneys through activating Akt while enhancing CTGF in T2DM [49,50]. In this study, we tested the therapeutic effect of Metformin on HGC-27 cells stimulated by CagA. We found that Metformin treatment could effectively maintain CagA-induced dysregulation of PTEN promoter methylation, PTEN mRNA and protein expression, as well as the proliferation and apoptosis of HGC-27 cells.

Conclusion
In summary, these findings suggest that the hypermethylation of PTEN promoter is a common event in GC patients with DM and metformin treatment. DM may strengthen the tumorigenic effect of HP by promoting the methylation of PTEN promoter, while the administration of metformin may reduce the risk of GC by suppressing the methylation of PTEN promoter.

Ethics approval and consent to participate
The Human Research Ethics Committees of The First Affiliated Hospital of Zhengzhou University has approved this research and all methods were performed in accordance with the last vision of the Declaration of Helsinki. Written informed consent was obtained from all patients before the study.

Consent for publication
Not applicable.

Availability of data and material
The data that support the findings of this study are available from the corresponding author upon reasonable request.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
HL planned the study, HL, XH, JR and KR collected the data, ZL and QZ collected the literature, XH and JR analyzed the data, HL and QZ composed the manuscript, and all authors approved the final manuscript.
associated with more severe insulin resistance in women with gestational diabetes mellitus. Med Sci     Bisulfite sequencing PCR analysis showed that metformin treatment reversed CagA-induced hypermethylation of PTEN promoter.