The Effects Of Helicobacter Pylori Infection On DNA Damage Repair Mechanism In Gastric Epithelial Cell

Background and objective: Gastric cancer is one of the global common malignant tumors. The latest epidemiological data indicates that gastric cancer is the fourth leading cause of new cases of cancer in male and fifth in female, and the third leading cause of cancer death in male and fifth in female worldwide. More than half of the new cases of gastric cancer were diagnosed in China. Most of the new cases were already in the advanced stage of gastric cancer when diagnosed, with limited treatment and poor prognosis. It is urgent to elucidate the mechanism of gastric carcinogenesis, and to develop an effective strategy for primary prevention of gastric cancer. DNA double-strand breaks(DSBs) are the most severe type of DNA damage. The precise repairment of DSBs requires the activation of DNA damage signaling pathway and the synergy of various repair proteins. Autophagy also plays an important role in maintaining genome stability. The impairment of autophagy could result in aberrant DNA repair through regulating the activity of the essential proteins in DNA damage repair. The impairment of DNA damage repair mechanism is liable to cause genome instability, which is vital for the occurrence and development of malignant tumours.Materials and methods: I. Study of H. pylori-induced DSBs in gastric mucosa epithelial cells in vitro and in vivo 1. Biopsies of chronic non-atrophic gastritis(CNAG), intestinal metaplasia(IM), dysplasia(Dys) and gastric cancer(GC) were collected. The expression of γH2AX in each stage and its relation to H. pylori infection were detected by immunohistochemical staining. 2. GES-1 was co-cultured with H. pylori. The expression of γH2AX was detected by western blot, and DSBs was detected by neutral single cell gel electrophoresis experiment. 3. The expression of γH2AX in Mongolian gerbils’ gastric mucosa was detected after intragastric administration of H. pylori. II. The effect of H. pylori infection on DNA damage repair signaling pathways and cell autophagy 1. DNA damage repair pathways and autophagy-related proteins expression in gastric mucosa biopsy were detected by immunohistochemical staining at various stages of lesions,and its relation to H. pylori infection was analyzed. 2. The expression of the DNA damage repair pathways and autophagy-related proteins in GES-1 were detected after co-cultured with H. pylori in vitro. 3. Autophagy flux in GES-1 was detected by Ad-m RFP-GFP-LC3 after co-cultured with H. pylori. 4. Cell cycle of GES-1 was detected by flow cytometry after co-cultured with H. pylori. 5. DSBs and its related proteins in GES-1 cells with Rad51 over-expressed or knocked out were detected after co-cultureed with H. pylori. III. The effect of autophagy on H. pylori-induced DSBs damage repair in GES-1 cells 1. GES-1 cells were co-cultured with H. pylori after pretreated with autophagy agoinst of inhibitor to examine DSBs, its related proteins, the expression of Rad51 and cell cycle. 2. The level of autophagy and expression of Rad51 were intervened at the same time, and whether the effect of autophagy DNA damage repair is dependent on the expression of Rad51 was examined.Results: I. H. pylori infection could induce DSBs in gastric mucosa epithelial cells both in vitro and in vivo We found that γH2AX plays an important role in gastric carcinogenesis via detecting its expression in gastric biopsies at different stages of lesions. Moreover, the expression of γH2AX was related to the lesion location of gastric cancer, gross appearance, differentiation, depth of invasion, TNM stage and lymph node metastasis, and was negatively correlated with prognosis of gastric cancer. Furthermore, the expression of γH2AX was higher in the H. pylori-positive IM and Dys compared with the negative group. H. pylori could also induce DSBs in GES-1 cells in vitro. It was further verified that H. pylori infection could up-regulate the expression of γH2AX in Mongolian gerbils’ gastric mucosa. Taken together, our data indicated that H. pylori infection could induce DSBs in gastric mucosa epithelial cells both in vivo and in vitro. II. The effect of H. pylori infection on DNA damage repair signaling pathways and cell autophagy By detecting the expression of critical proteins at different gastric lesions, we found that DNA damage repair pathways and cell autophagy participated in the gastric carcinogenesis, which were also closely related to the clinical pathological parameters. Moreover, the expression of Beclin-1, p62 and BRCA2 was positively correlated with the prognosis of gastric cancer, and the expression of p-ATM(S1981) and Rad51 showed a negative correlations. Further analysis showed that i). The expression of p-ATM(S1981), p-Chk2(T68)and p-p53(S15)was higher in H. pylori-positive CNAG and IM compared with the H. pylori-negative group. ii). In Dys stage, the expression of Rad51 was lower in the H. pylori-positive group compared with the negative group. In cellular studies, we found that the H. pylori could activate the DNA damage response signaling pathways and cell autophagy, thus induce S-phase arrest in GES-1 at early stage. However, after co-culture for 12 h, the expression of Rad51 protein and autophagy had been down-regulated. DSBs in GES-1 could be abrogated by transfection with Rad51 plasmid, while knocking down of Rad51 played the opposite role. Animal studies further demonstrated high p62 expression in the Mongolian gerbils’ gastric mucosa after infected by H. pylori for 12 months. And the expression of Rad51 decreased after infected by H. pylori for 18 months. All of the results above indicated that the DNA damage repair pathway played as anti-cancer barrier in early stage of gastric carcinogenesis. H. pylori infection could activate DNA damage pathway signaling pathways and autophagy in the gastric mucosa epithelial cells. However, H. pylori infection could also down-regulate autophagy and impair DNA homologous recombination repair with persistent infection. III. The effect of autophagy on H. pylori-induced DSBs damage repair in GES-1 cells GES-1 cells were pretreated with two types of autophagy agonists(Rapamycin and Starvation) and autophagy inhibitor(3-MA and Baf A1), respectively before co-cultured with H. pylori. We found that up-regulated autophagy could promote Rad51 expression and reduce DSBs in the GES-1 cells, while down-regulated autophagy showed the opposite phenotype. We also performed the rescue experiment, by raising autophagy and knocking down Rad51, or inhibiting autophagy and over-expressing Rad51 at the same time, respectively. The results showed that autophagy played the protective role for DSBs on a Rad51-dependent manner.Conclusions 1. H. pylori infection could induce DNA double-strand breaks in gastric mucosa epithelial cells and activate the DNA damage response signaling pathways, which play an important role in gastric carcinogenesis. 2. H. pylori infection could down-regulate the expression of Rad51 via inhibiting autophagy, leading to impairment of DNA damage repair in the gastric mucosa epithelial cells.