| Objective Glioma is the most common and fatal intracranial malignant tumor in adults.Diffuse lower grade gliomas(DLGG)refer to the WHO grade II and grade Ⅲ adult glioma located on the supratentorial area,accounting for about 15% of all gliomas.Complete surgical resection is difficult due to the migration of the tumor cells into multiple lobes.Therefore,understanding the mechanisms of the invasion and migration of the DLGG cells is the key to the treatment of DLGG.Previous research by our research team found that the central nervous system axonal growth inhibitor Nogo-66 and its receptor NgR are expressed in gliomas and have an effect on the invasion and metastasis of glioma.To further investigate the role of Nogo-66 and NgR in DLGG and related mechanisms,we conducted a series of experiments in the hope of providing new ideas for the diagnosis and treatment of glioma.Materials and Methods A total of 25 cases of DLGG specimens were collected from Wuhan Union Hospital from September 2016 to December 2017.1.The same tumor specimens can be divided into two parts,one part was examined by immunohistochemistry to analysis the expression of Nogo-66 and NgR in the glioma specimens.For the other part of the specimens,the primary cells were extracted,subsequently,the expression of NgR gene in primary cells was inhibited by SiRNA transfection.In order to detect the expression of NgR gene in primary cells of DLGG before and after SiRNA transfection,q RT-PCR was conducted 48 hours after transfection,and Western blot 72 hours after transfection.2.According to the difference of transfected SiRNA,and the presence or absence of Nogo-66,the primary DLGG cells cultured in vitro were divided into four groups,the first group: NC(SiRNA negative control)+Nogo-66,the second group: NC(SiRNA negative control)+PBS,the third group: SiRNA+Nogo-66,the fourth group: SiRNA+PBS.Then,Scratch wound migration assay,cell migration assay and Cell invasion assay were conducted to test the migration and invasion ability of the primary cells.Results 1.DLGG specimens were incubated with NgR and Nogo-66 antibodies,respectively,and DLGG tumor cells showed high NgR expression,while Nogo-66 was highly expressed in in the interstitium of tumor cells.2.The immunofluorescence results of DLGG specimens showed that DLGG cells had strong NgR expression.NgR and Nogo-66 were colocalization on the surface of the MBP-labeled white matter fiber,and NgR positive glioma cells were distributed along the MBP-labeled white matter fiber.3.q RT-PCR assay showed that the m RNA of NgR gene was significantly decreased 48 hours after SiRNA transfection,and the Western-blot indicated that the expression of NgR protein was significantly decreased 72 hours after SiRNA transfection.4.The scratch wound migration assay showed that the number of cell migration in the experimental group(SiRNA+Nogo-66 group)was lower than that in the control group(NC+Nogo-66 group),and the cell migration assay showed that the migration number of cell of the experimental group(SiRNA+Nogo-66 group)was significantly lower than that of the control group(NC +Nogo-66 group)(P<0.05).The cell invasion assay showed that the number of cells invaded by the experimental group(SiRNA+Nogo-66 group)was significantly lower than that of the control group(NC+Nogo-66 group)(P<0.05).Conclusion These results suggested that NgR is highly expressed in the DLGG cells.In addition,we confirmed that the distribution of DLGG cells along the white matter fiber in morphology.Nogo-66 can promote the migration and invasion of DLGG cells,and RNA interference can block this effect by silencing NgR gene. |
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