Effects Of The Full Length And Ectodomain Of LRIG2Gene On The Biological Characteristics Of Glioblastoma Cell Lines And The Underlying Molecular Mechanisms

Part I:Construction of lentiviral vectors over-expressing full length and ectodomain of LRIG2and the subcellular locationsObjective To transduce glioblastoma cells with constructed lentivral vectors over-expressing full length (LRIG2) and ectodomain of LRIG2(LRIG2ecto), and to investigate their subcellular locations.Methods The full length and ectodomain of LRIG2genes were amplified and inserted into lentiviral vectors pLVX-Puro-3×Flag respectively, and then the constructed vectors (pLVX-Puro-3xFlag-LRIG2and pLVX-Puro-3×Flag-LRIG2ecto) were transfected into glioblastoma cell lines. The expression levels of proteins and mRNA were determined by western blotting and real time RT-PCR, respectively. The sub-cellular locations of proteins were detected by confocal immunofluorescence laser microscopy.Results The DNA sequencing of recombinant plasmids containing full length and ectodomain of LRIG2were correct. Compared with the control group, the flag fusion proteins were all successfully expressed in LRIG2and LRIG2ecto over-expressing cells. The mRNA expression levels of LRIG2and LRIG2ecto increased by (8.42±0.50) and (29.58±0.98) in U87cells respectively and by (65.83±4.14),(126.34±0.98) in U251cells, respectively. Confocal immunofluorescence laser microscopy revealed that the LRIG and LRIG2ecto proteins both localized to the cytoplasm and no nuclear staining was found. Conclusion LRIG2and LRIG2ecto over-expressing glioblastoma cell lines were successfully established. The proteins of LRIG2full length and LRIG2ectodomain were both localized to the cytoplasm. Part Ⅱ:Effects of the full length and ectodomain of LRIG2gene on the proliferation and apoptosis of Glioblastoma cell lines and the underlying Molecular MechanismsObjective To investigate the effects of over-expressing full length (LRIG2) and ectodomain of LRIG2gene (LRIG2ecto) on the proliferation and apoptosis of glioblastoma cell lines and the underlying mechanisms.Methods The proliferation rates and survival rates of cells were determined by cell counting kit-8(CCK8). The expression level of Ki67was tested by immunofluorescence. The cell cycle distribution and the cyclins proteins were determined by flow cytometry after PI staining and Western Blot, respectively. PDGFRP, cMet and EGFR signaling pathway associated proteins were detected by Western Blot. Apoptosis and mitochondria membrane potential of cells were determined by flow cytometry after Annexin V-FITC/PI and JC-1staining, respectively.Results Compared with the corresponding control group, the proliferation rates, the percentage of Ki67positive cells, and the proliferation index of LRIG2and LRIG2ecto over-expressing cells were all increased significantly. The apoptotic rates of LRIG2and LRIG2ecto over-expressing U87cells were (2.86±0.30)%and (4.04±0.59)%, respectively, decreased compared to control group (5.9±0.45)%. The apoptotic rates of LRIG2and LRIG2ecto over-expressing U251cells were (4.12±0.26)%and (3.62±0.24)%, respectively, also decreased compared to that of control group(5.48±0.65)%. Cultured in normal medium, the expression levels of EGFR, phospho-EGFR and the downstream phospho-Akt and-Erk of LRIG2and LRIG2ecto over-expressing cells were all markedly increased; and when stimulated with EGF in medium without serum the levels of phospho-EGFR and-Akt were also significantly elevated. Treated with gefitinib, LRIG2and LRIG2ecto over-expression significantly enhanced the cell survival rates, elevated the mitochondrial membrane potential and reduced the apoptotic rates. The PDGFRβ and cMet proteins levels were both increased in LRIG2and LRIG2ecto over-expression cells. Conclusion Overexpressions of LRIG2and LRIG2ecto both promoted the proliferation, inhibited the apoptosis, enhanced the expressions of EGFR, PDGFRβ and cMet, activated EGFR singaling pathway and enhanced the resistance of treatment with EGFR inhibitor of glioblastoma cells. LRIG2may represent a particularly attractive therapeutic target in the treatment of glioblastoma. Part III:Effects of the full length and ectodomain of LRIG2gene on the migration and invasion of Glioblastoma cell linesObjective To investigate the effects of over-expressing full length (LRIG2) and ectodomain of LRIG2gene (LRIG2ecto) on the migration and invasion of glioblastoma cell lines and the underlying mechanisms.Methods The migration and invasion abilities of glioblastoma cells were determined by Scratch-wound migration assay and transwell invasion assay, respectively. Gelatin zymography was used to detect the expression levels of MMP2in the medium, and real-time RT-PCR was used to determine the levels of MMP2mRNA.Results The migrated pixels of LRIG2and LRIG2ecto over-expressing U251cells for48h were(7.61451±0.31619)x105and (8.34314±0.41888)×105, respectively, significantly decreased compared to the control group (10.17098±0.30520)×105. The invaded cells of LRIG2and LRIG2ecto over-expressing U87cells were (49.38±4.21)%and (59.49±6.02)%, respectively, significantly decreased compared to the control group(100±9.11)%. In U251cells, the invaded cells of LRIG2and LRIG2ecto groups were (36.7±4.09)%and (49.54±5.81)%, respectively, much lower than that of control cells(100±10.01)%. The MMP2expression levels in the medium and MMP2mRNA levels of LRIG2and LRIG2ecto over-expressing cells were decreased compared to that of control group.Conclusion Up-regulations of LRIG2and LRIG2ecto both inhibited the migration and invasion abilities and inhibited the expression of MMP2in human glioblastoma cells.