| BackgroundGastric cancer is one of the most common malignant tumors,which accounts for the third leading cause of death caused by cancer,and seriously endangers people's life and health.In China,the morbidity and mortality rate of stomach cancer is the second highest malignant tumor.Clinical data shows that invasion and metastasis is the leading cause of death in patients with gastric cancer,which represents more than 40%of patients with stomach cancer,and the five-year survival rate is only 30%.However,the biological and molecular mechanism of gastric cancer metastasis remains unclear.It is of great significance to explore the molecular mechanism of gastric cancer metastasis in depth.Extracellular matrix protein 1(ECM1)is a secreted glycoprotein that stimulates the proliferation of endothelial cells and promotes the formation of blood vessels.ECM1 is highly expressed in many malignant tumors such as liver cancer,invasive breast catheter cancer,throat cancer,bile duct cancer,and plays as an oncogene in tumor metastasis,which is closely related to prognosis.The expression level of ECM1 in stomach cancer tissue with lymph node metastasis was significantly higher than that of paracarcinoma tissues and the unmet stomach cancer ousise of lymph nodes,and ECM1 was associated with tumorigenesis and lymph node metastasis in the epithelial cells of the stomach tissue.In addition,ECM1 expression was associated with the expression of the vascular endothelial growth factor(VEGF)in gastric cancer tissue.These studies suggest that ECM1 is closely related to gastric cancer metastasis,but the specific function and mechanism of ECM1 in gastric cancer metastasis is still unclear.MiRNA-92a is a kind of miRNA that is highly expressed in many tumors such as breast cancer,cervical cancer,colorectal cancer and osteosarcoma,but the relative expression in stomach cancer tissues is significantly lower than that of paracarcinoma tissues.We performed a prediction search for miRNA binding sites and found a putative binding site of miRNA-92a in the ECM1 gene,and speculated that ECM1 might be the target gene for miRNA-92a.However,the function of miRNA-92a in stomach cancer and the molecular mechanism of regulating ECM1 expression are still unclear.This study aims to explore the role of ECM1 and miRNA-92a in gastric cancer metastasis and elucidate the interaction between ECM1 and miRNA-92a,which might provide new treatment for the clinical treatment of stomach cancer.ObjectiveThe main objectives of this study:(1)To determine the expression of ECM1 in gastric cancer tissue and paracarcinoma tissue and its correlation with clinical pathological parameters.(2)To elucidate the effect of ECM1 on the metastasis of gastric cancer cells.(3)To reveal the regulation mechanism of miRNA-92a on ECM1 expression and its role in the metastasis of gastric cancer.Methods1)ECM1 gene expression data and overall survival data were retrieved from the Cancer Genome Atlas(TCGA)and Kaplan Meier-plotter database,and the relationship between ECM1 gene expression level and survival data were analyzed.ECM1 expression levels in the gastric cancer tissues and paracarcinoma tissues were determined by immunohistochemical staining,and the relationship between ECM1 expression levels and clinical pathological parameters were also analyzed.2)Stable gastric tumor cell lines for low or high ECM1 expression were generated by transducing with lentivirus which express a specific shRNA against ECM1 or a protein encoded ECM1.Transwell assay,scratch test,anti-nest apoptosis,soft agar clone formation,adhesion assay and naked mice model for abdominal cavity tumor were employed to investigate the expression change of ECM1 on the biological phenotypes of gastric cancer cells in vitro and in vivo.3)A binding site of miRNA-92a was predicted to locate in the 3'UTR region of ECM1 gene and a luciferase reporter vector with mutant miR-92a site was construct by site-directed mutagenesis.Dual luciferase reporter assay was employed to evaluate the role of miRNA-92a in the transcriptional regulation of ECM1 and ECM1 expression levels were investigated in the gastric cancer cells transfected with miRNA-92a mimic or inhibitors,and metasis as well.Results1)From the data of ECM1 expression and the overall survival retrieving from TCGA and Kaplan Meier-plotter database,it was found that ECM1 was highly expressed in the gastric tumor tissues and it had a poor prognostic in the patients with high ECM1 expression.High expression level of ECM1 was also confirmed in the stomach cancer tissues,which were collected from the First Hospital affiliated with China University of Science and Technology.There was a significant positive correlation between the expression of ECM1 with tumor size(p=0.013),TNM staging(p=0.039)and lymphatic leaching(p=0.006).ECM1 has a positive correlation with tumor pathological phenotypes and negative correlation with survival and prognosis.2)The MGC-803 gastric cancer cell strain with low expression ECM1 and the BGC-823 gastric cancer cell strain with high expression ECM1 were established.Compared with the control cells,cells with high ECM1 expression level exihibited an increased level of ECM 1 protein and mRNA,a significantly increased scratch healing ability,increased aggression ability.The percentage of anti-nest apoptosis gastric cancer cells and the adhesion ability to the extracellular matrix were also significantly increased.The expression levels of EMT-related markers Vimentin and Snail proteins were significantly increased along with a decreased expression level of E-cadherin.In vivo study showed that ECM1 overexpression led to an increase in the number of abdominal tumor nodules and the volume of abdominal water in the mice subjecttotas,and the overall survival rate of the mice with lotus tumors as well.In the MGC-803 cell line with lower ECM1 expression,the completely opposite results were found.The expression level of ECM1 protein and mRNA,scratch healing ability,tumor cell invasion ability,percentage of anti-nest apoptosis gastric cancer cells,adhesion ability to the extracellular matrix,non-anchor-dependent growth ability were decreased significantly.The number of abdominal tumor nodules and the volume of abdominal water were decreased significantly in the mice with the tumor,while the total survival rate of the mice increased significantly.3)By using bioinformatics software it was found a putative miRNA-92a binding site located in the 3'UTR region of ECM1 gene and the transfection of miRNA-92a mimic or inhibitor into gastric cancer cells could cause a decreased and increased expression level of ECM1,respectively.It has been confirmed that miRNA-92a had a regulatory effect on ECM1 in a dual luciferase reporter assay.The anti-nest apoptosis resistance of gastric cancer cells after transfection of miRNA-92a mimics,matrigel adhesive adhesion and migration ability were significantly reduced compared to the control group,while transfection of miR-92a inhibitor had an opposite effect and ECM1 overexpression could inhibit the above effects induced by miRNA-92a mimics.ConclusionOur study showed that ECM1 was highly expressed in gastric cancer and it has poor prognosis in gastric cancer patients with high ECM1 expression.It was found ECM1 enhanced the metastasis of gastric cancer by affecting the expression of EMT-related proteins.miRNA-92a was found for the negatively regulate the expression of ECM1 by targeting ECM1 3'UTR,affecting metastasis of stomach cancer.miRNA-92a could promote the metastasis of gastric cancer via regulating the expression of ECM1 in gastric cancer cells,providing new research targets for the treatment of gastric cancer. |
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