Functional And Mechanistic Studies Of Dimt1 In Regulating The Malignant Behavior Of Gastric Cancer

Background Gastric cancer(GC)is one of the most familiar malignant tumors of international community currently,and it is the second leading cause of cancer-related death worldwide,and about 1 of every 4 cancer patients who dies of disease will die of GC.GC with occult onset,high malignancy,high fatality rate,rapid progression and poor prognosis features,there is a greater difficulty in its treatment.China is one of the high incidence areas of GC in the world,the situation is more serious.And report showed that the incidence and mortality of GC ranked second,with about 700,000 new cases,about half of the world's new cases,and more than 490,000 deaths in 2015,nearly three-fifths of global deaths.Current state of GC is low in early diagnosis,much younger in the incidence,lower in total resection rate,high in recurrence and metastasis rate and low in the5-year survival rate in China.The causative mechanism depends on multiple factors,multi-step process and continuous progress.However,the etiologic factors and mechanisms of GC still are not entirely clear.Present study suggests that abnormal expression of oncogene and antioncogene,Helicobacter pylori infection,chronic atrophic gastritis,and environmental pollution were the major causative factors,oncogene activation or antioncogene inactivation were considered to be the most critical factor.Surgery,chemotherapy and radiation are the main means of treatment of GC,but these treatments have certain limitations,and cause the mortality of GC still remains high at present.Facing this dire situation,the exploration of new diagnostic and prognostic biomarkers for GC is urgently needed.The genesis of GC resulted from abnormal expression of many genes,to explore these genes that induce the occurrence and development and prognosis judgement,and goes deep investigate its related mechanism,which has important meaning for the diagnoses,prognosis and treatment of GC.The DIM1 dimethyladenosine transferase 1 homolog gene(DIMT1)is located on chromosome 5q12.1 and encodes a protein with 313 amino acids.The expression of the miRNA210 is decreased significantly and DIMT1 is increased in the chronic gastritis caused by Helicobacter pylori.Suppressed miRNA210 can up-regulate expression of DIMT1 and could promote the proliferation of gastric epithelial cells.Other studies also found that there was a close correlation between expression of DIMT1 and NEDD9,UBC,and RIOK2.Those 3 proteins are closely associated with proliferation,apoptosis,invasion,migration,and other malignant biological behaviors of tumor cells.Furthermore,they can be used to monitor the recurrence ormetastasis of the tumor and also to assess a patient's prognosis.However,the role of DIMT1 in GC has not been reported.This study first to collect GC tissues,paired normal tissues adjacent to carcinoma and normal gastric mucosa tissues,and test DIMT1 expression in tissues by IHC,qPCR and WB technology,further analysis the relationship DIMT1 expression with the clinical pathologic characteristics and prognoses of GC.Next,use gene transfection techniques from the cell level to observe the effects of different DIMT1 expression levels on the proliferation,apoptosis,invasion and migration of GC cell lines in vivo and in vitro.At the end,further researching the molecular mechanism of DIMT1 promoting the development of malignant GC biology.All in all,we regarding the DIMT1 as the target molecule,and investigating and discussing the role of DIMT1 in the malignant biological behavior and its related mechanism in GC cell line in vitro.The research mainly divided into the following three parts:Part ? The expression and significance of DIMT1 in GCObjective1.To detect DIMT1 expression level in GC tissues,paired normal tissue adjacent to carcinoma and normal gastric mucosa tissues;2.To analyze DIMT1 expression and the relationship between the expression of DIMT1 and the clinico-pathological parameters and prognoses in GC;3.To explore potential clinic value of DIMT1 in monitoring and diagnosis and treatment for GC.Methods1.The expression of DIMT1 in GC tissues,paired normal tissues adjacent to carcinoma and normal gastric mucosa tissues were determined with IHC,qPCR and WB;2.The relationship between the expression of DIMT1 and the clinico-pathological parameters were analyzed by a single-factor and multifactor conditional logistic regress analysis;3.The relationship between the expression of DIMT1 and the survival time of GC patients was analyzed by Kaplan-Meier methods and Log-rank test.Results1.The protein of DIMT1 location was mainly in the GC cell nucleus,and little expression in normal gastric mucosa tissues;the mRNA and protein of DIMT1 in GC tissues was significantly higher than that in paired normal tissues adjacent to carcinoma and normal gastric mucosa tissues;2.The high expression of DIMT1 is closely correlated with differentiation,primary focus tissue invasion,lymph node metastasis,distant metastasis and TNM stage;but there were no significant correlation between DIMT1 expression and gender,age and tumor size;3.Postoperative survial time of GC patients with high express level ofDIMT1 were significantly lower than GC patients with low level expression of DIMT1;Kaplan-Meier survival analysis showed that high DIMT1 expression exhibited a negative correlation with poor prognosis for GC;4.The DIMT1 was one of the independent prognostic factors for GC patients.Part ? Influence of DIMT1 Towards to GC Cells Malignant Biological BehaviorObjective1.Observe the expression of DIMT1 in BGC-823,MKN-45,SGC-790 l,MKN-28 and GES-1;2.Study the effects of DIMT1 gene silencing and over-expression on the proliferation,invasion,migration and apoptosis of GC cells.Methods1.The expression of DIMT1 in BGC-823,MKN-45,SGC-790 l,MKN-28 and GES-1 were determined with IF,qPCR and WB;2.Constructing DIMT1 gene silencing,over-expression and negative control lentivirus,and establishing the stable transfected cell lines of DIMT1 gene silencing and over-expression;3.The effect of DIMT1 on the GC cells proliferation were investigated by EdU cell proliferation and cell colony assay;4.The effect of DIMT1 on the GC cells apoptosis and cell cycle wereinvestigated by flow cytometry;5.The effect of DIMT1 on the GC cells migration and invasion were investigated by wound healing and Transwell assay;6.Animal experiment: the effect of DIMT1 on the GC cells proliferation,migration,invasion and tumor neovascularization in mice were investigated by subcutaneous tumor model,tail intravenous injection model and implantation model of human in situ.Results1.The results of IF showed that the protein of DIMT1 location was mainly in the nucleus of GC cells;2.The results of qPCR and WB showed that the expression of DIMT1 positively correlates with the tumor cell malignant.It was highly expressed in BGC-823 and MKN-45,low expressed in SGC-7901 and MKN-28,and rarely expressed in GES-1;3.We succeeded in packing the DIMT1 gene over-expression,silencing and negative control lentivirus;Then SGC-7901 and MKN-45 cell lines were transfected and selected by puromycin(SGC-7901-D and shMKN-45);4.EdU cell proliferation assay and cell colony forming assay showed that the ability of proliferation and growth increased significantly in SGC-7901-D and reduced remarkably in shMKN-45 compared with the blank control group;5.The flow cytometry revealed that GC cell apoptosis resistance and cells of S phase increased in over-expression of SGC-7901-D,and opposite results found in shMKN-45;6.Wound healing and Transwell assay suggested that the ability of migration and invasive increased significantly in SGC-7901-D and reduced remarkably in shMKN-45;7.Nude mice assays revealed that faster proliferation and metastasis,net weight loss in SGC-7901-D compared with the blank control group;The opposite result in the shMKN-45.Part ? The Research of DIMT1 Change GC Cells Malignant Biological Behavior MechanismObjective1.To explore the molecular mechanisms of DIMT1 in regulating proliferation,apoptosis,invasion,migration and angiogenesis of GC cells;2.Analysis the potential targets of DIMT1.Methods1.Detected the ability of proliferation,apoptosis,invasion,migration after joined genes inhibitors by the methods of part two;2.The expression of genes related to tumor proliferation,apoptosis,migration and invasion was determined with qPCR and WB.Results1.DIMT1 can rised significantly the expression of AKT,p-AKT,MMP2,MMP9 and Cyclin D1;2.CDK and MMP2/9 inhibitors can inhibited significantly GC cells proliferation,invasion and migration;3.AKT inhibitor decreased significantly the expression level of MMP2,MMP9 and Cyclin D1.Conclusion1.There was a significantly negative correlation between the expression level of DIMT1 and prognosis of GC patients,and it was one of the independent prognostic factors with GC patients.2.DIMT1 promoted proliferation,invasion,migration and angiogenesis of GC cells through the AKT signaling pathway.3.DIMT1 plays a potential tumor promoter in GC,it will provide new perspectives for molecular targeted therapies of GC,and can be a marker of judging prognosis for GC patients.