Molecular Functions And Mechanisms Of Leucine-rich Repeats And Immunoglobulin-like Domans 1 Gene In Human Gliomas

Background & Objectives: Leucine-rich Repeat Immunoglobulin-1(LRIG1)gene is a newly identified human gene that displays homologies to the Drosophila Kek-1 gene. The latter encodes a Drosophila cell surface protein, Kekkon-1, which inhibits epidermal growth factor receptor-mediated signalling in the Drosophila melanogaster. Human LRIG1 protein is a transmembrane glycoprotein, of which the extracellular region is organized with the leucine-rich repeats and immunoglobulin-like domains. Previous studies showed that the expression of LRIG1 gene was detected in almost all analyzed human tissues, particularly in cerebral cortex. However, its roles in human tumors, especially in brain tumors, are still unclear. Its interaction with human EGFR and the relevant mechanisms are still unknown. The goals of present study were to study the relationship between LRIG1 gene and human gliomas and determine the interaction between LRIG1 and EGFR. Methods: A triphasic oligonucleotide probe of LRIG1 gene was designed and used to detect the expression of the LRIG1 gene in human astrocytomas and the corresponding tissues around the tumors by in situ hybridization (Part I). Then some primary astrocytoma cells were cultured and the expression of the LRIG1 gene and Proliferating Cell Nuclear Antigen (PCNA) protein among the cultured cells and the tumor tissues were evaluated by in situ hybridization and immunohistochemistry respectively, and the proliferation after the cells were stimulated by epidermal growth factor (EGF) was observed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay (Part II). Two plasmids were constructed containing extracellular and transmembrane regions of LRIG1 (LRIG1-ET) or cytoplasmic and transmembrane regions of LRIG1 (LRIG1-TC). Their sequence and expression were identified by restriction endonuclease analysis, DNA sequencing, and eukaryotic transfection (Part III). The two contructed chimeric eukaryotic expression vectors and a plasmid p3XFLAG-CMV-9-LRIG1 containing full length of the LRIG1 gene (LRIG1-FL) were transfected into human glioma line U251 or primary cultured astrocytoma cells. The number of EGFR on transfected cell's surface and the receptor's capacity of binding epidermal growth factors (EGF) were evaluated by 125I-EGF.