Expression Of MCM7 And Its Effect And Mechanism On Proliferation,Invasion And Metastasis Of Gastric Cancer

BackgroundAt the beginning of the 21st century,the morbidity and mortality of gastric cancer in China remains high.In recent years,as the development of the comprehensive treatment such as new adjuvant chemotherapy,targeted therapy and surgery,the prognosis of patients with gastric cancer has preliminary improved.But most patients’prognosis is still poor,especially in advanced gastric cancer patients.Therefore,it is of great significance to make the early diagnosis and to perform early treatment in order to improve the prognosis of these patients.However,there is still a lack of accurate pathological diagnostic criteria for early gastric cancer.Multiple statistical data show that the detection rate of early gastric cancer in China is less than 10%,which indicates that the;is still a lot of room for improvement in the diagnosis and treatment of early gastric cancer.The occurrence of gastric cancer is a multi-step and multi-stage heterogeneity.The complex process could be divided into several phases including normal gastric mucosal,intestinal metaplasia,intraepithelial neoplasia and gastric cancer.The evolution is affected by many factors and the pathological morphologymay be not typical.Besides,pathological diagnoses of the early gastric cancer are mainly based on small specimens acquired by gastroscopic biopsy.Sometimes it’s hard to be accurate.Therefore,differentpathologistsusually makedifferentpathological diagnoses.Most cases of gastric cancerareintestinal type.According to Lauren classification,gastric cancer is classified into three categories:intestinal type,diffuse type and mixed type.The early diagnosis and differentiation between gastric intraepithelial neoplasia and gastric intramucosal carcinomaplays an important role for treatment and prognosis of gastric cancer patients.However,for a long time,the differentiations betweenlow grade neoplasia(LGN)and intestinal metaplasia(IM),as well as that between high grade neoplasia(HGN)and gastricintramucosal earcinoma(GIC)are of all great difficulties.If pathological diagnosis is difficult to make based on morphology,it tends to resort to related molecular detection to make diagnosis.So,we have to deepen the research of mechanism and related signaling pathways in gastric cancer.Minichromosome maintenance proteins(MCMs)are a set of protein family which is widely exists and highly conserved in eukaryote.They are closely associated with DNA replication and mainly involved in DNA replication licensing and control the cell cycle progression.Theprotein levels of the MCMs can be sensitive and specific to reflect the level of cell proliferation.Minichromosome maintenance protein 7(MCM7)is a member of the MCMs family.It is part of the MCMs protein complex and is a necessary helicase for the initiation of DNA replication in eukaryotic cells.Many reports indicate that MCM7 is closely related to cell cycle regulation,transcription,cell proliferation and the tumor formation.lt is highly expressed in a variety of malignant tumor tissues such as liver cancer,oral squamous carcinoma,colon cancer and prostate cancer.It can promote proliferation and inhibit the apoptosis of tumor cell,and is significantly associated with tumor metastasis.The high expression of MCM7 means poor prognosis.However,expression and clinical significance of MCM7 in gastric cancer is rarely mentioned.Its function and mechanism are still unclear.The main purpose of this study is:1,to detect the expression of MCM7 in gastric cancer and gastric mucosal diseases and to analyze its relationship with patients,clinicopathological characteristics and prognosis;2,to illuminate the effects of MCM7 onthe proliferation,invasion and migration of gastrie caneer eell;3,to explore the regulating mechanism of MCM7 on the invasion and migration ingastric cancer cell.Chapter 1 Expression of MCM7 in early gastric cancer and its diagnosticsignificanceObjective:The aim of this study was to investigate the expression of minichromosome maintenance complex component 7(MCM7)in gastric mucosal lesions,further to find its potential effect as a biomarker to distinguish intraepithelial neoplasia from gastric mucosal lesions,finally find out whether its expression is related to the multi-step process of gastric cancer.Methods:MCM7 and Ki67 were detected in 93 cases of gastric mucosal lesions byHE staining and immunohistochemistry.Patients were divided into 6 groups:18 were gastritis with Helicobacter pylori infection(GT)(19.35%),16 were IM(17.20%),11 were LGN(11.83%),8 were HGN(8.60%),13 were GIC(13.98%),and 27 were gastric submucosal carcinoma/beyond(GSC)(29.03%).The MCM7 and Ki67 staining in nuclear were scored and the results were used for statistical analysis.Results:MCM7 and Ki67 expression in GT were lowest compared with other groups(P<0.001),meanwhile there were significant differences compared with Group IM and other groups in MCM7 and Ki67 expression(P<0.001).MCM7 and Ki67 expression in GSC were highest(P<0.05).Groups of LGN,HGN and GIC had no significant differences in MCM7 expression(P>0.05),but there was significant difference compared with Group LGN and Group GIC in Ki67 expression(P<0.05).MCM7 expression elevated with tumor grade increasing and had positive correlation with Ki67 significantly(r=0.940,P<0.001).Furthermore,in some cases,some tumor cells were immunoreactive to MCM7 but negative to Ki67.Conclusions:MCM7 is helpful to make differential diagnosis in pathological grade,MCM7 combination of Ki67 may serve as more sensitive proliferation markers for evaluation of gastric carcinoma and precancerous lesions.MCM7 expression is positively correlated with the occurrence of gastric cancer,suggesting that MCM7 expression may promote the development of gastric cancer.Chapter2 Effect of MCM7 on proliferation,invasion and migration of gastriccancer cellsObjective:To study the relationship of expression of MCM7 in gastric cancer tissues with clinicopathological characteristics and overall survival rate of patients.Meanwhile,RNA interference technology was applied to construct siRNA targeting MCM7 gene and transfect it into human gastric cancer cells to observe the effect of MCM7 knockout on proliferation,invasion and migration of gastric cancer cells.Methods:1.Immunohistochemicalstainingwas used to detect MCM7 protein expression in thespecimens of 58 patients with gastric cancer.According to the percentage ofMCM7 positive staining in nuclear of tumor cells,two groups were divided:group of "MCM7 low expression"(positive cells percentage<50%),group of "MCM7 high expression"(positive cells percentage≥ 50%).Then we analyzed thecorrelation between the expression of MCM7 and clinical pathological characteristics and the correlation between the expression of MCM7 and overall survival rate2.We chose human gastric cancer cell line MGC-803 and human normal gastric epithelial cell line GES-1 as subjects.On the one hand,we reduced the expression of MCM7 by siRNA interference in gastric cancer cells(siMCM7 group and the control group,NC).On the other hand,we overexpressed MCM7 gene in gastric epithelial cells(dsMCM7 group and the control group,NC).The CCK-8,Transwell chamber and cell scratches experiments were applied to observe the effects of MCM7 on proliferation,invasion and migration of normal gastric epithelial cell and gastric cancer cellResults:1.MCM7 is mainly expressed in gastric cancer cell nucleus.Among 58 patients with gastric cancer,MCM7 expression is defined as "low expression" in 17 cases(29.3%)and as "high expression"in 41 cases(70.7%).It shows thatmost cases highly express of MCM7 in invasive gastric cancer.The expression of MCM7 in gastric cancer is significantly related to lymph node metastasis and TNM stages.The expression of MCM7in cases with node metastasis is significantly higher than those in cases without lymph node metastasis(P<0.001).Meanwhile the higher the TNM stage,the higher the expression of MCM7(P=0.05).The overall survival rate of patients with high MCM7 expression is significantly lower than that of patients with low MCM7 expression(P=0.018).Patients with high MCM7 expression had a poor prognosis2.The results of CCK-8 shows that cell proliferation in siMCM7 group significantly decreases compared with that in control group at 48 hours(P<0.0001).Similarly,in the cell cloning experiments,the numbers of cell clone in siMCM7 group decrease significantly than that in control group,the difference 1s statistically significant(p<0.0001).And results of Transwell assay and scratch experiment also show that ability ofmigration and invasion in siMCM7 group are significantly suppressed compared with that in control group.By contrast,in the normal gastric cells,with the overexpression of MCM7,the results of CCK-8 show that cell proliferation of dsMCM7 group is higher than that in control group.The differences in 48 and 72 hours are significant(p=0.0028,p<0.0001).A significant increase in the numbers of clones is observed in the cell plate cloning experiment with a statistically significant difference(P<0.0001).Meanwhile,results of both cell scratch and Transwell chamber show that overexpression of MCM7 could significantly promote the nigration and invasion of gastric cells.Conclusions:The expression of MCM7 in gastric cancer is related to tumor TNM stage and lymph node metastasis.Patients with high MCM7 expression have a poorer overall survival than those with low MCM7 expression.Down-regulationof MCM7 expression can significantly inhibit the proliferation,invasion and metastasis of gastric cancer cells.Chapter3 Effect and mechanism of MCM7 on EMT in gastric cancer cellsObjective:The purpose of this part is to study the effect and the possible molecular mechanism of MCM7 on the process of epithelial-mesenchymal transformation(EMT)in gastric cancer cells.Methods:We chose human gastric cancer cell line MGC-803 and human normal gastric epithelial cell line GES-1 as subjects.The expression of MCM7 was reduced by siRNA interference in gastric cancer cells(siMCM7 group)while MCM7 was overexpressed in gastric epithelial cells(dsMCM7 group).The EMT-related expression of E-cadherin,N-cadherin and Vimentin were detected by Western Blot.The expression of PI3K,Akt and mTOR in the PI3K/Akt pathway were further examined.Results:1.The expression of epithelial marker,E-cadherin,increased while mesenchymal markers,N-cadherin and Vimentin,decreased in gastric cancer cells in siMCM7 group.However,the expression of E-cadherin decreased while N-cadherin and Vimentin increased in dsMCM7 group.2.The expression of PI3K decreased,the expression of Akt and mTOR do not significantly changed and the expression of p-Akt and p-mTOR decreased in siMCM7 group.On the contrary,in the dsMCM7 group,the expression of PI3K increased,the expression of Akt and mTOR do not significantly changed and the expression of p-Akt and p-mTOR are up-regulated.Conclusions:The expression of MCM7 is related to the EMT process of gastric cancer cells.MCM7 may regulate the expression of EMT-related genes through the PI3K/Akt/mTOR signaling pathway to ultimately affect the invasion and mig:tion of gastric cancer cells in vitro.