The Experimental Study Of Evi1 Gene On Proliferation And Invasion Of Glioma Cell Line U87/U251

Background and Objective:As a basic component of the human brain,glial cells account for a large proportion.Glioma tumors in the central nervous system is the highest incidence of the brain,especially grade IV tumors glioblastoma,its average survival is about 15-18 months.With high incidence,high invasive,high lethality and disability,as well as surgical removal of the tumor,the functional protection requirements,tolerance to chemotherapy,glioma research at all levels of tumor research institutions are popular.Through our previous reading a lot of literature,some scholars have pointed out Evi1 gene in human leukemia,has been studied as an important oncogene.It can stimulate the proliferation of leukemia cells.And such as colon cancer,ovarian cancer,lung cancer and other solid tumors.the rapid malignant growth of tumor cells,which can result in the patient’s "cachexia",also has a relatively thorough study.Invasion and metastasis of malignant tumors is another extremely important biological characteristics.It is one of the causes of high-risk malignant tumors,but also a key factor in clinical treatment failure.One of the current research hotspots of matrix metalloproteinases MMP,especially MMP2 plays an important role in the invasion of tumor cells.But these in human glioma cells has little research,so the study in glioma tissue and cell lines presence great significance.We first detected the expression of Evi1 in glioma and glioma cell lines,and then further explored its role in the proliferation and invasion of human glioma and its mechanism.This experiment is mainly divided into two parts as follows:Part Ⅰ: Expression of Evi1 gene in human glioma and glioma cell linesObjective: To study the expression of Evi1 gene in normal human brain and human glioma tissures and glioma cell line in vitro and explore its significance for the growth and development of glioma cells which lays a foundation for further study of its function in gliomas.Methods: We collected 40 cases of different grades of glioma,and 7 cases of normal brain tissue in the traumatic brain injury and intracranial aneurysm,arteriovenous malformations(AVM)and cultured U87 / U251 cell line in the neurosurgery department of the First Affiliated Hospital of Soochow University in recent years.The total RNA was extracted from cells and cryopreserved in liquid nitrogen using Trizol method.The expression level of Evi1 gene in glioma tissues and cell lines was detected by real-time quantitative PCR.Results: There was difference in the expression of Evi1 gene between different grades of glioma tissues,and there was significant difference between normal brain tissue and high grade glioma tissue(grade III and IV)(P <0.05).There was a significant difference between low grade gliomas(grade II)and high grade gliomas(grade III and IV)(P <0.05).With the increasing degree of malignancy in glioma,Evi1 gene expression level is getting higher and higher.In glioma cell line U87 / U251,the expression level was significantly higher than that in normal brain tissue(P <0.05).Conclusion: Evi1 gene is highly expressed in glioma tissues,which may play a role in the growth,proliferation and invasion of glioma cells,and it will provide a basis for further study of its mechanism.Part Ⅱ: Effects of Evi1 gene on the proliferation and invasion of glioma cell line U87 / U251Objective: To study the effect of Evi1 gene on the proliferation and invasion of glioma cell lines,and to provide a theoretical basis for the treatment of glioma.Methods: U87 / U251 cells were transfected with liposome transfection reagent and si RNA.The transfection efficiency was detected by inverted fluorescence microscope and flow cytometry.The three interference chains provided by Gene Pharma were screened,and a chain with the best interference effect was selected for subsequent experiments.CCK8 assay was used to detect the proliferation ability of the cells.Flow cytometry was used to analyze the cell cycle.The migration ability of the cells was examined by scratch test.The invasive ability of the cells was detected by transwell assay.The changes of matrix metalloproteinase(MMP2),which is involved in cell invasion,were detected after tranfected,compared with the negative control group and blank group.Results: U87 and U251 cells transfected with si RNA,the cell proliferation,migration and invasion ability were significantly inhibited(P <0.05).Cell cycle was arrested in G1 phase,and S phase cells decreased.We verified that after transfection,the expression of matrix metalloproteinases2(MMP2)significantly decreased(P <0.01).Conclusion: After si RNA transfection,the cell proliferation,migration and invasion ability were significantly decreased,the cell cycle was blocked,and MMP2 expression was decreased,suggesting that it might be the target protein of Evi1 gene.The Evi1 gene may be regulate the physiological activity of cells by targeting MMP2.