Molecular Mechanisms Of Leucine-rich Repeats And Immunoglobulin-like Domains1Gene Inhibits Proliferation And Invasion In Human Glioma

Part â… Molecular Mechanisms of Leucine-rich Repeats and Immunoglobulin-like Domains1Gene inhibits proliferation and invasion in Human Glioma via EGFR Signaling PathwayObjectives:The molecular mechanisms that drive the development and aggressive progression of malignant astrocytic tumors remain obscure. Recently, in a search after endogenous negative regulator of epidermal growth factor receptor, we cloned and characterized LRIG1as a putative tumor suppressor gene often downregulated in various tumor, including glioma. However, the biological significance of such downregulation is not currently clear. The goals of present study were to uncover the relationship between LRIG1gene and human gliomas proliferation and invasion, to discuss the inner mechanisms between them. Methods:Western blot and immunohistochemistry were used to detect the relationship between LRIG1expression and various grade human gliomas. We used plasmid-based shRNA vector to knowdown expression of LRIG1and full-length expression vector to over-express LRIG1expression in human malignant glioma cell U251MG. To test downregulation and overexpression LRIG1affect glioma proliferation by MTT, soft agar clone assay, PCNA immunohistochemistry, cell cycle detection, Tunel and subcutaneous tumor xenograft model. The cell cycle was synchronized at G2/M phase after the treatment with nocodazole. We test dynamic cell cycle change in synchronized clonal cell line after exogenous LRIG1overexpression in normal culture condition and detect various cyclin proteins. We investigated whether silencing and restoration of LRIG1expression could affect cell invasion and migration in U251MG by transwell and wound scratch assay. Gelatin zymography was used to test MMPs activities and MMP1, MMP2, MMP9were tested by realtime PCR. To test whether downregulation and overexpression LRIG1affect EGFR and its down signaling pathway MAPK-ERK, PI3K-AKT under basal and EGF stimulated culture condition. We tested the effect of LRIG1down-regualtion on the transcription factor c-Myc expression. Results:LRIG1expression inversely correlates with Glioma WHO grade. Loss of the expression of endogenous LRIG1would result in proliferation and exogenous LRIG1introduction could inhibit glioma cell growth, over-expressing LRIG1formed substantially smaller tumors an played vital roles in the tumor incidence in vivo test. LRIG1delays cell cycle progression by regulating time-course expression of cyclin Dl and E protein. LRIG1attenuated invasion of glioma cells by decreasing expression of MMP2, MMP9. LRIG1can inhibit the basal stimulated degradation of PI3K/AKT pathway and MAPK/ERK1/2pathway. However, we found that LRIG1exerted more effect on PI3K/AKT pathway than MAPK/ERK1/2pathway under EGF-stimulated condition. Downregulation LRIG1increased c-Myc expression while LRIG1over-expression decreased the expression of c-Myc. Conclusions:LRIG1restricts the glioma aggressiveness by inhibiting cell proliferation and invasion. Restoration of LRIG1to glioma cells could offer a novel therapeutic strategy. Part â…¡Construction and identification of expressed lentiviral vector of LRIG1geneObjective To construct a lentiviral expression vector of LRIG1gene, and examine its expression by transfected cells, in order to lay the foundation for experiments on LRIG1gene function. Methods The full length of the LRIG1were cutted from plasmid Zeroblunt-topo-LRIG1by BamH I and Ecor I, subjected to agarose gel electrophoresis and then extracted with DNA Gel Extraction Kit. The lentivirus vector PlvxDsred-monomer-n1were cutted by BamH I and Ecor I, prior to insert the full length of the LRIG1using T4DNA Ligase. Each vector and the Lenti-X HTX Packaging Mix are cotransfected into293T cells. Cultured cells were infected by two lentivirus. Results The results of gel electrophoresis showed that the LRIG1gene was cloned into the lentiviral vector. Real Time PCR and Western blot showed that LRIG1gene was expressed. Conclusion The lentiviral expression vector of LRIGlgene was constructed successfully, and lentivirus can tranfect cells and exnous gene was expressed stably.