The Mechanism And Clinical Research Of RUNX3 Suppress Esophageal Squamous Cancer Cell Growth

Background and ObjectiveEsophageal squamous cell cancer (ESCC) is a malignancy that arises from esophageal epithelial cells, it is a potentially fatal disease with high incidence worldwide, particularly in China. The treatment for ESCC includes surgery, radiotherapy and chemotherapy. Despite technical progress for ESCC, the effect of treatment is still unsatisfactory. ESCC is still a leading cause of cancer-related death. About 90% of esophageal cancer in our country is squamous cell cancer. The structure of the esophagus is different from other digestive tracts, because there are lots of lymph-vessel in mucoderm and muscularis mucosae. Therefore, lymph node metastasis of esophageal cancer can occur even in the early T stage of esophageal cancer. To the patients with complete resection of esophageal cancer, there are still about 27% of them appeared lymphatic metastatic recurrence within 2-3 years.Consequently, it is of great clinical value to find new genes involved in ESCC tumorigenesis and progression, in order to develop early diagnosis and improved disease outcome predication following treatment of this dangerous disease. Many studies have shown that the occurrence and development of ESCC are the result of the multi-factors, multi-steps and multi-genes participation. Inactivation and mutation of tumor suppressor gene is one of mechanisms which involved in ESCC develops through the accumulation of genetic and epigenetic abnormalities, evolving from preinvasive lesions to invasive esophageal cancer, with the malignant cells commonly exhibiting abnormal important genes expression. Antioncogene, named recessive oncogene, played veryimportant negativited regulated function on control of auxesis, cellproliferation and differentiation, in the meantime it could inhibit tumorgrows potentially. If the abnormal phenomenon of antioncogene functiondeactivated,or antioncogene deletion and mutation happened,the vicioustransformation would take place on cells in order to the development ofthe malignant tumor.Consequently, it has a great clinical value to find new genes involved in ESCC tumorigenesis and progression, in order to explore the generation and development of ESCC.Multiple genes participate in the occurrence and development of ESCC. Runt-related transcription factor 3 (RUNX3) is one of the runt-domain family of transcription factors which has been first reported in gastric cancer. RUNX3 was thebasic of runt family of mammal evolution. RUNX3 gene pitched on humanchromosome NO.l galianconism 1p36. All member of Runt family were endowed with RuntDomain which Contained 123 amino acids. Runxprotein was closed to occurrence of human disease by transmit TGF-β/activin signal through forming complexment with Smad 2 andSmad 3. It is regarded as a tumor suppressor gene in many human tumors, and its inactivation is believed to be related with the occurrence and development of tumor.Recently it was reported that RUNX3 gene expression is downregulated in various solid tumors. Previous study demonstrated that the down-regulation of RUNX3 in ESCC tissues is associated with poor prognosis.The aim of this study was to detect the expression of RUNX3 in ESCC, and further investigate the correlation between ESCC cellline and RUNX3 with biological behavior and prognosis of ESCC. In this study, Immunohistochemistry (IHC), Western-blot and quantitative real-time polymerase chain reaction(qRT-PCR) assays were used to detect the expression of RUNX3. The findings of this study will provide theoretical bases for the further exploring of pathogenesis of esophageal cancer and searching for the ideal target for gene therapy of esophageal cancer.Part IOverexpression of RUNX3 inhibits biological behaviour of Eca109 cell in vitro and vivoObjectiveThe aim of this study is to investigate the clinical relevance of RUNX3 in ESCC patient and the effect of RUNX3 gene overexpression on biological behaviour of Eca109 in vitro and vivo.MethodsImmunohistochemistry was performed to detect the clinical relevance of RUNX3 and lymph node metastasis in 80 ESCC tissues and 40 non-cancerous tissues by using SP (streptavidin-perosidase) method. Reverse transcriptase-polymerases chain reaction (RT-PCR) and Western blot were test the RUNX3 mRNA and protein expression in the different groups of Eca109 cells to verify the Eca109 cell line with stablely overexpression of RUNX3 was founded. The localization of RUNX3 proteins was performed by Cell immunofluorescence staining assay. Cell Counting kit (CCK)-8 assay and Scrape motility assay was used to determine the proliferation of Eca109 cells. Terminal deoxynucleotidyl transferase-mediated UTP-biotin nick end labeling assay (TUNEL assay) was used for analyzing cell apoptosis. The invasiveability was detected by cell transwell invasion experiment. By nude mouse tumorigenesis test, the tumorigenesis ability in vivo was respectively identified.ResultsResult shows that RUNX3 protein expression is lower in ESCC as compared with that in the surrounding normal tissue. The expression of RUNX3 in esophageal tissue is significantly related to lymph node metastasis (LNM) (P<0.01). In addition, the recombinant lentiviral vector (Lentivirus-CMV-GFP/RUNX3) was constructed and transfected into human ESCC cell line Eca109 successfully. We found that overexpression of RUNX3 can inhibit cell proliferation, migration and invasion, induce apoptosis in Eca109 cell. The in vivo experiment on mice showed the tumorigenicity and growth invasiveness of Eca109 had been significantly inhibited.ConclusionOur studies indicate that expression of RUNX3 in human ESCC tissue is significantly correlated with ESCC progression. The tumor inhibition function of RUNX3 depends on its nuclear localization. Restoration of RUNX3 expression significantly inhibits ESCC cells proliferation, migration, invasion, and tumorigenesis.ConclusionOur studies indicate that expression of RUNX3 in human ESCC tissue is significantly correlated with ESCC progression. The tumor inhibition function of RUNX3 depends on its nuclear localization. Restoration of RUNX3 expression significantly inhibits ESCC cells proliferation, migration, invasion, and tumorigenesis.Part II Effect and mechanism of RUNX3 gene on Biological characteristics of Human esophageal squamous cell carcinoma (ESCC)ObjectiveThe aim of this study was to investigate the role of RUNX3 in ESCC cells biological behavior and the relationship between the expression of RUNX3 and MMP-9, TIMP-1, ICAM-1.MethodsRUNX3 levels in 90 esophageal squamous cell carcinoma specimens using immunohistochemical staining to examine the correlation between RUNX3 expression and clinical stage of ESCC. Furthermore, the role of RUNX3 in ESCC progression was evaluated in vitro by siRNA-mediated knockdown of RUNX3 or lentivirus-mediated over-expression of RUNX3 in ESCC cell lines. The expression and activities of MMP-9, TIMP-1 and ICAM-1 were analyzed.ResultsWe found decreased expression of RUNX3 in ESCC tissue to be significantly related to T-stage of tumor (P<0.01). In vitro, knockdown of RUNX3 in Eca9706 cells resulted in promote cell growth, migration, and invasion. Additionally, MMP-9 and ICAM-1 were upregulated in RUNX3-knockdown cells. Notably, RUNX3 over-expression in Kyse150 cells could significantly decrease MMP-9 and ICAM-1. Tumorigenesis in vivo was significantly determined.ConclusionThe study indicates that low-expression of RUNX3 in human ESCC tissue is significantly correlated with progression. Restoration of RUNX3 expression significantly inhibits ESCC cells migration, invasion and tumorigenesis, which may be caused by RUNX3’s interaction with MMP-9 and ICAM-1, RUNX3 may be a potential therapeutic target for ESCC. significantly determined.ConclusionThe study indicates that low-expression of RUNX3 in human ESCC tissue is significantly correlated with progression. Restoration of RUNX3 expression significantly inhibits ESCC cells migration, invasion and tumorigenesis, which may be caused by RUNX3’s interaction with MMP-9 and ICAM-1, RUNX3 may be a potential therapeutic target for ESCC.Part Ⅲ Effect and mechanism of 5-azacytidine on the biologic behavior of esophageal carcinoma cellObjectiveInvestigate the effects of 5-azacytidine up-regulating RUNX3gene expression on the biologic behavior of esophageal carcinoma cell.MethodsEsophageal squamous carcinoma Eca109cells were cultured invitro with intervention of 5-azac at 10,20and 50μmol/L for 72h.The expression level of RUNX3gene was deteced by RT-PCR and western blotting.Methylation specific PCR was used to determine the degree of methylation of RUNX3gene. The abilities of migration and invasion of Eca109cells were detected by scratch wound and transwell assay.ResultsAfter treatmentwith 5-azac for 72hat 10,20,50μmol/L,The mRNA expression level of RUNX3was increased by (2.18±0.23),(4.57±0.26),(6.90±0.36)times,and the relative coefficient of RUNX3 protien expression were 0.196±0.004,0.278±0.013,0.396±0.025, respectively.The RUNX3gene was demethylated partialy in Eca109cells pretreated with5-azac.After treatment with 5-azac for 72hat 0,20and 50∴mol/L, the brightness value ratio of unmethylated tape were0.125±0.003,1.791±0.112,2.987±0.236, respectively. Pretreated with 0,10,20 and 50μmol/L 5-azac for 72h, thenumber of Eca109cells migrating to the scrape area were 715±20.1,398±19.3,302±16.4,124±13.8, respectively. Compared with the control group,the ratio of migrating cells in 10,20,50μmol/L group were 62%,46%,12%,and theratio of invasing cells were 60%,45%,8%, respectively.ConclusionThe expression level of RUNX3gene in Eca109 cells can be up-regulated by low concentration of 5-azac via demethylation, which inhibits the abilities of proliferation, migration and invasion of Eca109 cells...