Candidate Tumor Suppressor Gene ECRG4 Inhibits Glioma Proliferation And Invasion

Glioma is the most common tumor in the central nervous system, it possesses the following features: plentiful microvessels, invasive growth and prone to recurrence after operation, et al. The pathogenesis of glioma is still uncertain until now. Thus , it is urgent to new biological markers for glioma early diagnosis and evaluation of the prognosis. Esophageal cancer related gene-4 was first cloned and identified by Bi. et al. Their research showed that the expression of ECRG4 in esophageal carcinoma tissues and esophageal carcinoma cell lines were significantly decreased comparing to the normal esophageal tissue epithelium. Recently, many studied indicated that ECRG4 were highly expressed in plentiful normal tissues, however, decreased in tumor tissues. Therefore, ECRG4 is considered as newly candidate tumor suppressor gene.In this research ,we first detected the ECRG4 mRNA expression by real time Polymerase Chain Reaction in gliomas and their matched brain tissues. then establish the ECGR4 overexpression glioma cell line model by gene transfection method. The effect of ECRG4 expression on cell proliferation, invasion, and migration was investigated in human U251 glioma cells. The results were as followed:1.The expression of ECRG4 mRNA were detected in gliomas and their matched brain tissues by real time PCR .Of the 10 paired samples analyzed, except 1 hair cell type astrocytoma, 9 glioma tissues displayed the decreased expression of ECRG4 mRNA compared to matched normal brain tissues.2.Stable transfection of ECRG4 in U251 cell line and selecting and identification.The eukaryotic expression vector carring whole length gene of ECRG4 were stably transfected into U251 cells using liposome-mediated transfection. G418 was used to select the positive cell clones. The positive cells which expressedECRG4 were identified by real time RT-PCR and Western blotting.3.The effect of ECRG4 on the proliferation of U251 cells were detected by MTT method and clony formation assays.It showed that cells transfected with ECRG4 significantly inhibited the cell proliferation ability as evaluated by MTT . The numbers of cellular colonies significantly decreased by colony formation assay as comparing to control groups (P < 0.05).4.The effect of ECRG4 on the migration and invasion of transfected U251 cells.The polycarbonate membrane on the upper layer of transwell chamber were firstly coated by fibronectin, RPMI 1652 with 10% newborn calf serum was added in the lower layer as the chemotatic factor. After incubating for 12 hours, the migrating cells in the lower layer were numbered, the result showed that the migrating cells significantly decreased in the transfected group comparing the control groups (P < 0.05). Similarly, The polycarbonate membrane was coated with matrigel to detect the invasion ability, overeexpression of ECRG4 significantly decreased the invasion ability of U251 cells(P < 0.05).. In conclusion, it provides a new experimental prove to take ECRG4 as a newly tumor suppressor gene. It also provides a new target for the treatment of gliomas.