Metformin attenuates synergic effect of diabetes mellitus and Helicobacter pylori infection on gastric cancer cells proliferation by suppressing PTEN expression

Abstract It has been reported that CagA of Helicobacter pylori reduced PTEN expression by enhancing its promoter methylation. Furthermore, diabetes mellitus (DM) may also promote the methylation status of PTEN, a tumour suppressor gene in gastric cancer (GC). It is intriguing to explore whether DM may strengthen the tumorigenic effect of H pylori (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. In this study, we enrolled 107 GC patients and grouped them as HP(−)DM(−) group, HP(+)DM(−) group and HP(+)DM(+) group. Bisulphite sequencing PCR evaluated methylation of PTEN promoter. Quantitative real‐time PCR, immunohistochemistry and Western blot, immunofluorescence, flow cytometry and MTT assay were performed accordingly. DNA methylation of PTEN promoter was synergistically enhanced in HP(+)DM(+) patients, and the expression of PTEN was suppressed in HP(+)DM(+) patients. Cell apoptosis was decreased in HP(+)DM(+) group. Metformin showed an apparent effect on restoring CagA‐induced elevation of PTEN promoter methylation, thus attenuating the PTEN expression. The reduced PTEN level led to increased proliferation and inhibited apoptosis of HGC‐27 cells. In this study, we collected GC tumour 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. In summary, our study suggested that DM could strengthen the tumorigenic effect of HP by promoting the PTEN promoter methylation, while metformin reduces GC risk by suppressing PTEN promoter methylation.


| INTRODUC TI ON
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 gastric carcinoma and gastric antrum cancer, although the occurrence of carcinoma at gastroesophageal junction has been rising steadily. [1][2][3] Review of the onset of GC showed that the occurrence of GC is slowly increasing among youth adults and in children. Also, the occurrence, death rate and metastasis of GC are pretty high, along with low rate of early diagnosis, good prognosis and 5-year survival. 4 The gene of phosphatase and tensin homologue on chromosome 10 (PTEN) is deemed as a tumour suppressor in numerous forms of cancers in human. 5 PTEN suppresses phosphatidylinositol-3-kinase (PI3K) expression through catalysing the elimination of D3 phosphate from phosphatidylinositol (3,4,5)-triphosphate (PIP3) to dysregulate the production of phosphatidylinositol 4,5-bisphosphate (PIP2) and PIP3. [6][7][8] PTEN is down-regulated in GC, and the expression of PTEN is also negatively correlated with metastasis in lymph nodes, depth of invasion and the age of GC patients. [9][10][11] PTEN was shown to promote the cell cycle arrest, apoptosis and metastasis of GC cells. 12,13 Formerly, Tet1 has been shown to prevent the metastasis and development of GC through PTEN demethylation and its expression. Tet1 reduces the migration, development and invasion of GC through the demethylation of CpG islands in the promoter region of PTEN, thus down-regulating focal adhesion kinase and AKT activity. 14 The role of DM in the risk of cancer has been analysed 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 quite common among Uyghur patients carrying wild-type 2 diabetes mellitus (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 (H pylori) are a global threat that has infected about 4 billion individuals. 18 Helicobacter pylori are the primary reason for peptic ulcer disease, gastritis and GC. Previous research has revealed that the infection by H pylori caused an epithelial-to-mesenchymal transition of epithelial cells in the stomach. 19 It was revealed that CagA considerably lowered the PTEN and Tet1 expression.
Furthermore, it was uncovered that CagA lowered PTEN expression through enhancing its own methylation, which was dramatically decreased by Tet1. 20 Metformin is a compound extracted from Galega officinalis and has 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 also promotes PTEN methylation. 23 Giving the fact that DM and HP infection synergistically elevate the risk of GC and 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 tumour 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.

| Human subjects sample collection
In this study, we enrolled 107 patients with gastric cancer in the Endocrinologists. 26 Those who were diagnosed with other type of cancers, renal dysfunction, liver failure, heart failure or those who take metformin to treat DM were excluded from this study.

| Bisulphite sequencing
We used bisulphite sequencing to determine the level of DNA methylation in the promoter of the PTEN gene. In brief, genomic DNA in

| RNA isolation and real-time PCR
Real-time PCR was done to measure the expression of PTEN mRNA in collected samples. In brief, total RNA in each sample was isolated by utilizing a PureLink RNA Mini assay kit (Thermo Fisher Scientific) 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) 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 iCycler real-time PCR instrument (Bio-Rad Laboratories) by utilizing an iQ SYBR Green master kit (Bio-Rad Laboratories) in accordance with the routine assay protocol provided by the kit manufacturer.

| MTT cell proliferation assay
The proliferation status of treated cells was measured by utilizing a CellTiter-Glo MTT assay (Promega) 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) 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%  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 analysed by using a propidium iodide/annexin V-FITC apoptosis assay kit (Sigma Aldrich) 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) at a 488 nm wavelength.

| Immunofluorescence and immunohistochemistry
Immunofluorescence and immunohistochemistry assays were used

| Statistical analysis
All statistical evaluations were conducted by making use of GraphPad  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.

| HP infection and Diabetes mellitus synergistically increased the DNA methylation level of PTEN promoter in gastric cancer patients
As PTEN expression was reported to be closely related to the pathogenesis of gastric cancer, and DNA methylation level of PTEN promoter is reversely correlated with PTEN expression, we per-

| HP infection and Diabetes mellitus suppressed the expression of PTEN mRNA and protein in gastric cancer patients in a synergistic manner
It is well known that DNA methylation in the promoter region can repress gene expression. Therefore, we further performed

| D ISCUSS I ON
Metformin is well known for its anti-diabetic role. 28 It was also shown that metformin reduced the risk of cancer in patients with T2DM. 29,30 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. 31,32 It was shown that the use of metformin significantly decreased the risk of infection by H pylori dose dependently. 33 50 Zhu et al proposed that PTEN controls the production of extracellular matrix in kidneys through activating Akt while enhancing CTGF in T2DM. 51,52 In this study, we tested the therapeutic effect of Metformin on HGC-27 cells stimulated by CagA. We found that Metformin treatment could effectively restore CagA-induced dysregulation of PTEN promoter methylation, PTEN mRNA and protein expression, as well as the proliferation and apoptosis of HGC-27 cells.

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

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.