The Effect Of Lysine(K) Specific Demethylase1A(KDM1A) On The Invasion And Metastasis Of Gastric Cancer And Its Associated Mechanism

Background:The incidence of gastric cancer ranks forth in tumor diseases worldwide. Gastric cancer is also one of the most common malignant tumor in China. Metastasis and recurrence are the main factors of the survival and prognosis for patients with gastric cancer, fifty to seventy percent of patients with advanced gastric cancer have been found metastasis and recurrence after two years, which makes patients lose the opportunity for second surgery. It is very difficult for clinical treatment.Lysine (K)-specific demethylaselA is the first defined histone specific demethylation enzymes. It can remove the methyl and dimethyl from the histone H3at lysine4and9in specificity.KDM1A mRNA and protein have higher expression in a variety of tumor tissues and cells, such as prostate cancer, breast cancer,bladder cancer,colorectal cancer, lung cancer and central nervous system tumors.KDM1A may participate in multiple signaling pathways,and affect the tumorigenesis,growth, proliferation, invasion, metastasis and resistance.MiR-137located in human chromosome1(1p22), some experiments prove that it can be used as a tumor suppressor gene in some kind of cancers. On the other hand, miR-137also has been shown to participate in the stem cell differentiation and development, neurodegenerative disorders, schizophrenia, and the pathophysiology of ischemic diseases. MiR-137and miR-234have the similar structure and function with a common evolutionary origin, and both belong to the family of miR-137.Purpose:To explore the correlation between KDM1A/miR-137expression and the invasion and metastasis of gastric cancer in tissue and cell levels; silencing a KDM1A expression by the RNA interference technique and then testing its impact on the proliferation, invasion and apoptosis of gastric cancer; and the dual luciferase report system detect if KDM1A is targeted by miR-137.Methods:1. Using western blot technique to detect KDM1A protein expression in gastric cancer and its adjacent normal tissue, using the qPCR to detect the expression of KDM1A mRNA and miR-137from24gastric cancer tissues and its adjacent normal tissue, and to analyze the relationship between clinical pathological features of gastric cancer and biochemistry features.2. Using transwell invasion assay to detect the invasive ability of gastric cancer cell lines (AGS, SGC7901, BGC823) and normal gastric mucosa cell line (GES1), at the same time using western blot and qPCR technique to detect KDM1A protein, KDM1A mRNA, miR-137in various cell lines, and then to analyze its relationship between the invasive ability of gastric cancer cell lines and biochemistry features.3. Using RNA interference technique to down-regulate KDM1A expression in gastric cancer cells BGC823, and then using MTT, transwell invasion assay, FCM to detect cell proliferation, invasion, and apoptosis.4. Using the dual luciferase report system to detect whether miR-137targets KDM1A mRNA3-’UTR in gastric cancer cells BGC823.Results:KDM1A protein values, KDM1A mRNA value, miR-137value are regarded as the dependent variables, and classify variable T stage, N stage, M stage, G stage, gender, age (It can be divided into two groups by critical point of50years old), HER2immunohistochemical staining, positive rate of removed lymph node in the surgery and pathologic diagnosis are regarded as the independent variables. A linear regression analysis and one-way analysis are made based on this situation. The regression coefficients, t value, p value are calculated. The results show that cancer tissues from the late T stage have higher KDM1A protein expression and lower miR-137expression.2. There are higher KDM1A protein expression, KDM1A mRNA expression and lower miR-137expression in gastric cancer cells BGC823 than other cell lines, such as AGS, SGC7901, GES1. Correlation analysis shows that positive relationship between KDMIA protein/mRNA and invasiveness of gastric cancer cells, negative relationship between miR-137and invasiveness, positive relationship between KDMIA mRNA and KDM1A protein, negative relationship between miR-137and KDM1A protein/miR137.3. Three independent siRNAs targeting KDM1A are transfected to gastric cancer cell line BGC823and then siRNA-705is selected as the most efficient one. The proliferation, invasion and apoptosis of gastric cancer cell of BGC823are detected by transwell invasion assay, cell proliferation assay and apoptosis assay after its interference in vitro.Our data shows that siRNA can suppress the invasion and growth, as well as, induce cell apoptosis.4. MiR-137can target KDMIA mRNA3-’UTR specifically, KDMIA expression changes after the epigenetic change of miR-137expression.Conclusion:1. It shows that KDMIA has higher expression and miR-137has lower expression in gastric cancer with late T stage.They may affect the invasion and metastasis of gastric cancer. And miR-137may target KDM1A.2. The expression level of KDM1A is positively correlated with the invasive ability in gastric cancer cells in vitro, and miR-137expression level is negatively correlated with the invasive ability in gastric cancer cells in vitro.3. The siRNA for KDM1A can effectively inhibit KDM1A expression in vitro, and inhibit proliferation and invasion, as well as, induce apoptosis of gastric cancer cell.4. MiR-137can target KDMIA mRNA3’-UTR, suppresses epigenetic expression of KDM1A gene.