The Antitumor Effect And Mechanism Of Lingo-1 To Glioblastoma Cells

Part I、Purpose:LINGO-1 gene is located on human chromosome 15q24.3 region. LINGO-1 is a component of Nogo receptor containing leucine-rich repeat structure and immunoglobulin domains. REMBRANDT database shows that LINGO-1 expression levels of gliomas were significantly lower than normal brain tissue. Compared to the average level, mean survival of LINGO-1 lower cases was significantly decreased. Through the worldwide Meta-analysis of microarray results, aimed at finding a potential target for glioma, Rheal A T et al. identified five molecules significantly altered in gliomas, comprising:spondinl, Plexin-B2, SLIT3, fibulin-1, and LINGO 1 by immunohistochemical staining. And based on the results of immunohistochemical staining, LINGO 1 was considered to be the most specific target for the center nervous system. Especially when the grade of glioma was higher, its importance increased.This paper aims to study the function of LINGO-1 for glioma, especially tumor inhibition towards glioblastoma. If LINGO-1 was identified as a tumor suppressor, we need to study its mechanism. In summary, we will study this conjecture at this stage of the experiment.Part Ⅱ、methodsFirst, according with the principles of molecular cloning, we recombine lentiviral vectors, carrying LINGO-1 FL (full length) or LINGO-1 Ecto (extracellular domain) sequence. Then, we use two glioma cell lines (U251/U87MG) in the following research. U251/U87MG were infected with recombinant plasmid, which could stably express LINGO-1 FL or LINGO-1 Ecto at high level. After U251/U87MG infected with lentiviral, we extracted the total cellular protein, which was used to verify expression of LINGO-1 FL or LINGO-1 Ecto by Western Blot assay. We examined the migration, invasion, colony forming and tumorigenesis ability of the tumor cell lines infected with lentivirus. In addition, we also use effective agonist to activate LINGO-1, and conducted a series of experiments, including proliferation, migration, invasion, tumor formation and cloning experiments. In the study of tumor suppressive mechanism, we adopted immunofluorescence staining and Western blot assays to verify expression LINGO-1, TrkB /p-TrkB and downstream molecular of Akt/p-Akt.PartⅢ、resultIn our study, when the expression of LINGO-1 FL or LINGO-1 Ecto increased, U251 /U87MG cell ability has been inhibited. Compared with control group, U251/U87MG cell proliferation has been inhibited. Cell migration and invasion of the experimental group were significantly lower than the control group by wound-healing and trans-well assay. In experiment group, the ability of U251/U87MG-derived stem cells to form colonies in suspension also significantly reduced. After expression of LINGO-1 FL and LINGO-1 Ecto increased, nude mice grew smaller tumor. In addition, when LINGO-1 was activated by PHEN, these function also showed varying degrees of inhibition. Immunofluorescence staining and Western blot experiments confirmed that LINGO-1 might inhibit TrkB phosphorylation, and affect the expression level of TrkB-Akt signaling pathway.PartⅣ、conclusionAfter LINGO-1 expression increased or activated in U251/U87MG, cell proliferation, migration, invasion and the potential of stem cells were significantly inhibited. This inhibition of tumor has been further verified by experiments in vivo. This might be explain by inhibition of the TrkB/p-TrkB-Akt/p-Akt signaling pathway experiments.The experiment is divided into seven parts:The first part:Recombination of LINGO-1 FL/LINGO-1 Ecto expression plasmid. The second part:Construction of cell lines overexpressing LINGO-1 FL/LINGO-1 Ecto. Part III:Proliferation, migration and invasion assay of cell lines overexpressing LINGO-1 FL/LINGO-1 Ecto. Part IV:Sphere formation, colony formation and xenograft assays of cell lines overexpressing LINGO-1 FL/LINGO-1 Ecto. Part Ⅴ:Proliferation, migration and invasion assay of cell lines dosed with PHEN. Part VI:Sphere formation, colony formation and xenograft assays of cell lines dosed with PHEN. Part Ⅶ:Change of related signal pathway after LINGO-1 activated by PHEN.Part Ⅰ、Recombination of LINGO-1 FL/LINGO-1 Ecto expression plasmid1、Purpose:Construct lentivirus vector carrying different fragments of LINGO-1 for the following experiment.2、Methods:We used human LINGO-1 FL(full-length) cDNA(BC011057), which was linearized by DNA polymerase NcoⅠ. By means of PCR, target sequence was amplified. Then the product was separated with agarose gel electrophoresis, and recovered by using gel extraction kit. The product was cleaved by enzyme EcoR Ⅰ and Not Ⅰ and cloned onto PCDH1-MSCV-EF1a-GFP-T2A-Puro (EcoRⅠ/NotⅠ) vector. And we detected whether the target sequence has mutation. Finally, we transfected HEK293FT cells with plasmid constructed above, and by using lentiviral packaging systems, virus-containing medium was sub-packaged in clean bench, and frozen at -80℃ low temperature freezer.3、Result:In accordance with the principles of molecular cloning, we received lentiviral vectors carrying the exact sequence of LINGO-1 FL and LINGO-1 Ecto by the linearization, PCR amplification, enzyme digestion and lentivirus packaging. The lentiviral will be used in subsequent experiments to build cell lines stable expressing LINGO-1 FL and LINGO-1 Ecto.4、Conclution:By molecular cloning and lentiviral packaging, we gain the lentivirus which was able to infect and glioma cell lines and stably express LINGO-1 FL and LINGO-1 Ecto.Part Ⅱ、Construction of cell lines expressing LINGO-1 FL/ LINGO-1 Ecto 1、Purpose:Build cell lines stable expressing LINGO-1 FL/LINGO-1 Ecto.2、Methods:In order to determine whether LINGO-1 possesses anti-tumor effect, we infected glioma cell lines with lentivirus packaged above. By the use of lentivirus carrying LINGO-1 FL and LINGO-1 Ecto sequences (expressed sequence with green fluorescent protein), LINGO-1 FL and LINGO-1 Ecto was cloned into glioma cells U251 and U87MG genome. The expression level of LINGO-1 FL and LINGO-1 Ecto was artificially increased in U251 and U87MG, which was confirmed by the western blot experiments. Then the cell lines were used to detect whether LINGO-1 FL and LINGO-1 Ecto had anti-tumor effect during the next part of the experiment.3、Result:Lentivirus carrying LINGO-1 FL and LINGO-1 Ecto sequence was used to infect U251 and U87MG cells. After 48h, we observed that more than 95% of the cells had visible green fluorescence under fluorescence microscopy. Western blot test results also proved that LINGO-1 FL and LINGO-1 Ecto expression levels in U251 and U87MG cells were significantly increased.4、Conclution:The packaged lentivirus carrying LINGO-1 FL and LINGO-1 Ecto sequence could infect glioma cell lines U251 and U87MG, and stably express LINGO-1 FL and LINGO-1 Ecto.Part Ⅲ、Proliferation, migration and invasion assay of cell lines overexpressing LINGO-1 FL/LINGO-1 Ecto1、Purpose:To verify the impact of LINGO-1 on proliferation, migration, invasion, and other functions of glioma cells, the constructed cell lines with lentiviral infection were used in a series of function experiment.2、Methods:Based on cell lines expressing LINGO-1 FL and LINGO-1 Ecto, they were screened by puromycin, and then 100% of the cells showed visible green fluorescence under a fluorescence microscope. WST-1 was used to detect the inhibition of LINGO-1 FL and LINGO-1 Ecto on U251 and U87MG proliferation. Wound-healing experimental was used to verify cell migration in 24 hours, while Transwell kit was used to detect changes in the invasive ability of cells stable expressing LINGO-1 FL and LINGO-1 Ecto. Finally, the experimental data recorded was dealt with Graphpad Prism 5.0 and Photoshop CS3.3、Result:Compared with the control group, LINGO-1 FL and LINGO-1 Ecto could significantly inhibit proliferation, migration and invasion of glioma cell lines.4、Conclution:Full-length or extracellular domain of LINGO-1 can play an anti-tumor effect in glioma cell lines in vitro.Part Ⅳ、Sphere formation, colony formation and xenograft assays of cell lines overexpressing LINGO-1 FL/LINGO-1 Ecto1、Purpose:For further verification of whether LINGO-1 has antitumor effect in glioma cells.2、Methods:First, we carried out in vitro experiments. By the use of the cell line of U251 and U87MG expressing LINGO-1 FL and LINGO-1, adherent cultured cells (serum-containing medium) were digested into single cells, and conduct cell counting, and planted in the ultra-low attachment dish with serum-free Neurobasal medium. After eight days suspension culture, the number and size of the spheres were detected. We use soft agar colony formation assay to detect the impact of LINGO-1 FL and LINGO-1 Ecto on clones forming of U251 cells. Then, we performed in vivo experiments. By using 4-week-old nude mice, the left and right sides were subcutaneous injected with U87-control and U87-LINGO-1 FL/LINGO- Ecto cells. Tumor size was measured every two days until 26 days feeding. The tumor was gained by surgical resection after the mice were killed. The tumor tissue to be fixed, and then conducted Ki67 immunofluorescence staining and HE staining.3、Result:We found that LINGO-1 FL and LINGO-1 Ecto could inhibit glioma stem cell potential and the ability of clone formation in vitro. It was also demonstrated that LINGO-1 FL and LINGO-1 Ecto could inhibit tumor formation compared to the control group in vivo. ki67 expression levels were significantly decreased in the two experimental groups. HE staining reflected looser for the organization and less angiogenesis in experimental groups.4、Conclution:LINGO-1 FL and LINGO-1 Ecto glioma cells can produce anti-tumor formation effect.PartV、Proliferation, migration and invasion assay of cell lines dosed with PHEN1、Purpose:To further verify the effect of PHEN on proliferation, migration, invasion, and other functions for glioma cell.2、Methods:Based on the third part of the experiment, LINGO-1 FL and LINGO-1 Ecto can significantly inhibit the proliferation, migration and invasion of glioma cell lines. We suspect that these functions may also change after LINGO-1 activated by PHEN. First, U251 cells were dosed with lOμM and other concentration of PHEN. WST-1 was used to detect the inhibition of PHEN on U251 and U87MG proliferation. Wound-healing experimental was used to verify cell migration in 24 hours, while Transwell kit was used to detect changes in the invasive ability of cells dosed with PHEN. Finally, the experimental data recorded was dealt with Graphpad Prism 5.0 and Photoshop CS3.3、Result:Compared with the control group, PHEN could significantly inhibit proliferation, migration and invasion of glioma cell lines.4、Conclution:PHEN can play an anti-tumor effect in glioma cell lines in vitro.Part Ⅵ、Sphere formation, colony formation and xenograft assays of cell lines dosed with PHEN1、Purpose:We suppose to verify whether PHEN has antitumor effect in glioma cells.2、Methods:First, we carried out in vitro experiments. By the use of the cell line of U251 and U87MG, adherent cultured cells (serum-containing medium) were digested into single cells, and conduct cell counting, and planted in the ultra-low attachment dish with serum-free Neurobasal medium containing 10μM PHEN. After eight days suspension culture, the number and size of the spheres were detected. We use soft agar colony formation assay to detect the impact of PHEN on clones forming of U251/U87MG cells. Then, we performed in vivo experiments. By using 4-week-old nude mice, the left and right sides were subcutaneous injected with U87 cells. And the left and right sides were subcutaneous injected with DMSO-control and DMSO-PHEN every two days. Tumor size was measured every two days until 26 days feeding. The tumor was gained by surgical resection after the mice were killed. The tumor tissue to be fixed, and then conducted Ki67 immunofluorescence staining and HE staining.3、Result:We found that PHEN could inhibit glioma stem cell potential and the ability of clone formation in vitro. It was also demonstrated that PHEN could inhibit tumor formation compared to the control group in vivo. Ki67 expression levels were significantly decreased in the experimental groups. HE staining reflected looser for the organization and less angiogenesis in experimental group.4、Conclution:PHEN can produce anti-tumor formation effect on Glioma cells.PartⅦ、Change of related signal pathway after LINGO-1 activated by PHEN1、Purpose:According to the foregoing experimental results, we found that, LINGO-1 can play a significant anti-tumor effect after expression increased or activated. Then we explored the mechanism of LINGO-l’s inhibition on GBM cell lines at this stage experiments.2、Methods:We used immunofluorescence staining for the studies. After LINGO-1 treated with PHEN, associated membrane receptor protein and intracellular signaling molecules were dectect. On the other hand, we use the Western blot assay to verify the expression levels of LINGO-1 associated molecule pathway after dealt with PHEN.3、Result:Experimental results show that LINGO-1 can inhibit the phosphorylation levels of TrkB, and inhibit the downstream signal expression of Akt/p-Akt.4、Conclution:The antitumor effect of LINGO-1 on malignant glioma cells may be achieved by changing the TrkB/p-TrkB-Akt p-Akt signaling pathway.