| Background and objective:Gastric cancer is a common malignant tumor around the world which takes the fifth place of cancer incidence and the third place of mortality.More than half of the new cases ofpatients with gastric cancer were diagnosed in China.Recently,with the development of the screening of gastric cancer and strict control of the risk factors related togastric cancer,the morbidity and mortality have decreased.However,the disease burden of thegastric cancerin China is still severe because of our larger population base and serious aging of population.At early stage,thegastric cancercan be treated effectively by surgery and complementary therapies,but at the late stage,therapies are limited and prognosis is worse.Therefore,it is important to identify the pathogenetic mechanism of gastric cancerand to realize early diagnosis and gene targeted treatment.Polo-like Kinase 1(PLK1)is a serine/threonine kinase,which can interact with various substrates and participate in the regulation of processes of mitosis,cytokinesis,DNA damage response and so on.PLK1 is highly expressed in multiple human malignancies and cell lines are involved in the development including gastric cancer and its up-regulated expression is positively correlated to tumor staging,lymph node metastasis and diffusion.Helicobacter pylori(Hp)infection plays a critical role in the development of gastric carcinoma through a multistep process from chronic gastritis to intestinal metaplasia,dysplasia and finally gastric carcinoma.But,the definite pathogenic mechanism is still unclear.Our prophase research has proved that Hp can induce PTEN phosphorylation inactivation,promote the survival of gastric mucous cells through activation of PI3K/Akt signaling pathways and get involved in the gastric carcinogenesis.Further,studies show that PLK1 can phosphorylate the carbon end of PTEN,which is inactivated by Ser380/Thr382/383 locus and loose the regulatory function for downstream signal pathway,resulting in cell dysfunction and the increased cancer susceptibility.However,there is no relative research on the expression level of PLK1 and Hp infection in China.This paper aims to investigate questions about whether Hp infection can induce the up-regulated expression level of PLK1 and whether cell biological function can be affected through the regulation of PTEN/PI3K/Akt signals,and probable mechanism of gastric cancer induced by Hp.Materials and methods:1.Research on the relationship between the expression of PLK1 on gastric mucosal epithelia and Hp infection in vivo and in vitro.(1)Collect gastric cancerand distal tissues and detect the expression of PLK1 by Western blot;(2)Collect pathological specimens of chronic non-atrophic gastritis,intestinal metaplasia,gastric dysplasia and gastric cancer,detect the expressions of PLK1 on gastric mucosas at different stages by Immunohistochemistry and analyze the relationship between PLK1 and Hp infection;(3)Establish a mongolian gerbil animal model of Hp infection,detect the expression of PLK1 by immunohistochemistry and analyze the relationship between PLK1 and Hp;(4)Establish a co-culture model of Hp live strains and stomach cancer gastric cancer cells(MKN-28?HGC27?SGC-7901?AGS)in vitro,detect the expression of PLK1 in gastric cancercells at various degrees of differentiation by western blot and analyze the relationship between PLK1 and Hp infection;2.Effects on PTEN/PI3K/Akt signaling pathways in gastric mucosal epithelia by Hp infection and functions of PLK1 in vitro;(1)Establish a co-culture model of Hp live strains and MKN-28 cellsin vitro,control the multiplicity of infection and infection time and detect the expressions of PLK1 and relative proteins of PTEN/PI3K/Akt signaling pathways under different infectious conditions by western blot;(2)Preprocess MKN-28 cells by PLK1 inhibitor BI2536,establish a co-culture model with Hp live strains in vitro and detect the expressions of PLK1 and relative proteins of PTEN/PI3K/Akt signaling pathways by western blot after preprocessing;3.Functions of PLK1 for the promotion of the growth of epithelial cell strains in gastric mucosa;(1)Establish a co-culture model of Hp live strains and MKN-28 cellsin vitro,and detect the survival rate of cells by CCK-8;(2)Preprocess MKN-28 cells by PLK1 inhibitor BI2536,establish a co-culture model with Hp live strains in vitro and detect the survival rate of cells by CCK-8.Results:1.Research on the relationship between the expression of PLK1 on gastric mucosal epithelia and Hp infection in vivo and in vitro.Through the detection of the expression of PLK1 in gastric cancerand distal tissues,we found that the expression of PLK1 in cancer tissues was significantly higher than that in the distal tissues.Through the detection of PLK1 expression in specimens of gastric mucosa at different stages,we found that PLK1 played an important role in the development of gastric carcinogenesis.The expression of PLK1 showed an increasing trend gradually and in intestinal metaplasiawith positive Hp was significantly higher than that with negative Hp,while there was no significant difference in chronic non-atrophic gastritis,heterosexual hyperplastic and gastric cancertissue.By establishing the mongolian gerbil animal model of Hp infection,we also found that Hp infection could induce a higher expression level of PLK1 in gastric mucosa epithelial cell of mongolian gerbil which showed a positive correlation with the infection time of Hp infection.In vitro,we further found that Hp infection could induce an increased expression level of PLK1 in gastric cancer,cells at various degrees of differentiation in which the PLK1 expression level in well-differentiated MKN-28 cells was the lowest and Hp-induced PLK1 expression level was significantly up-regulated.The above results indicate that Hp infection in vivo and vitro can induce increased PLK1 expression level in gastric mucosa epithelial cell which might be involved in the gastric cancer.2.Effects on PTEN/PI3K/Akt signaling pathways in gastric mucosal epithelia by Hp infection and functions of PLK1 in vitro.Hp live strains could induce an increased PLK1 expression level when acting on MKN-28 cells after 2 hours with MOI=10 and stimulate PTEN/PI3K/Akt signaling pathways in concentration dependent manner.At the same time,Hp live strains could also induce an increased PLK1 expression level when acting on MKN-28 cells after 15 min with MOI=200 in time dependent manner.Through preprocessing MKN-28 cells by PLK1 inhibitor BI2536,PLK1 and PTEN/PI3K/Ak signaling pathways and relative protein expressions were not significantly different.The above results indicate that Hp infection can activate PTEN/PI3K/Akt signaling pathways by up-regulating PLK1.3.Functions of PLK1 for the promotion of the growth of epithelial cell strains in gastric mucosa.By detecting the cells survival rate by CCK-8,we found that after 15 min Hp live strains action with MOI=10,50,100 and 200 respectively,cells survival rate in every group increased gradually with time in time dependent manner.Besides,after 1 hour,cell survival rate presented an increased trend with the concentration.By preprocessing MKN-28 cells by PLK1 inhibitor BI2536,there was still no significant difference of cells survival after the second detection and the cell survival rate in control group was significantly higher than that pre-treated by BI2536.The above results indicate that Hp in vitro depends on the activation of PTEN/PI3K/Akt signaling pathways by PLK1 to promote the survival of gastric mucosal cell Conclusions:1.Hp infection could up-regulate the expression of PLK1 at very early stage,and phosphorylate the Ser380/Thr382/383 locus of PTEN,subsequently resulting in activation of PI3K/Akt signal pathway and ultimately promoting cell survival.2.Increased PLK1 expression is an important molecular event in gastric carcinogenesis which may work by activating the PTEN/PI3K/Akt signal pathway,and PLK1 maybe a potential molecular target for the chemical prevention and treatment of gastric carcinogenesis. |
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