| Neurogliocytoma,which also called glioma,originates from human glial cell’s malignant tumor.As one of the high grade malignancy cancer,it occupies 80%of the central neurological tumors.According to the data showed by National Cancer Center in 2017,about 95,900 people were newly diagnosed with neurological tumors every year.Besides,about 55,100 people died from neurological tumors per year.Because of its high proliferation and invasiveness,the current standard treatment(craniotomy combined with radiotherapy and chemotherapy)is still confirmed of low curability.Thus,revealing glioma’s pathogenesis ulteriorly and seeking the effective targets for the treatment of glioma is a problem that needs to be solved presently.As we all known,the occurrence and development of glioma is regulated by multiple genes and factors.Abnormal expression of related factors can lead to changes in the function of glioma cells,and thus it can affect the whole process of the occurrence and development of glioma.The purpose of this study is to find the proteins or genes that affects the occurrence and development of glioma,and to further explore its regulation of glioma cell function and the specific molecular mechanism.The purpose of this study is to explore the expression quantity of LncRNA-LYPLAL1-2(Long non coding RNA lysophospholipase-like 1-2,LncRNA-LYPLAL1-2β LYPLAL 1-2)in brain glioma and the role of LYPLAL1-2 in the occurrence and development of glioma,which is identified to provide new evidence for the effective treatment of brain glioma.Firstly,combined with relevant literature,this study did bioinformatics analysis by means of the GEO database(which is established and maintained by NCBI).Dependent on the analysis,there exists some relationship between LYPLAL 1-2,miR-217-5p and CCR7.That is,LYPLAL 1-2 regulates the expression of CCR7 through miR-217-5pβ thereby inhibiting the occurrence and development of malignant glioma.In the mean time,RNA was extracted from 55 patientsβ glioma tumor tissues that associated with adjacent pathological cancer.And qRT-PCR results showed that the expression level of LYPLAL 1-2 in glioma tissues was significantly decreased.Meanwhile in the vitro experiment,we observed that LYPLAL 1-2 overexpression inhibited the migration and invasion ability of glioma cells signally.Ulteriorly in the situ experiment,we used tail vein injection of OEC(Lentiviral vector constructed U87 blank control group)or OE-LYPLAL1-2(Lyplall-2 group was overexpressed by U87 constructed with lentiviral vector)in nude mice,and tumor tissue was taken for size detection,we found that LYPLAL1-2 overexpression inhibited the growth of glioma.Finally,DLRTM,Western blot and bioinformatics analysis showed that LYPLAL1-2 negatively regulated mir-127-5p,then decreased the expression of CCR7 in glioma cells,thereby inhibiting the occurrence and development of malignant glioma.In addition,this study also confirmed the promotion effect of TGF-β1(transforming growth factor-β)on glioma cell invasion,and the specific molecular mechanism of CCR7 in TGF-β1-mediated glioma cell function.Malignant glioma cells(U251 and U87)were cultured in basal culture medium and culture medium contained TGF-β1 respectively for 6 h,12 h and 24 h.Then Western blot was performed to evaluate the expression level of the E-cadherin,N-cadherin and Vimentin.All of these three markers could be used to measure epithelial-mesenchymal transition(EMT).We observed the upregulation of N-cadherin and Vimentin and the dowm*egulation of E-cadherin,which indicating that TGF-β1 could decrease the E-cadherin of the U251 and U87,while elevate the N-cadherin and Vimentin,thereby promoting the EMT in glioma cells.The results of transwell invasion and migration assay also revealed that the cell invasion and migration ability,including U251 and U87,was accelerated with TGF-β1.U251 and U87 cells were divided into four groups:Control(Con),TGF-β1,siCCCR7+TGF-β1 and Ab CCR7+TGF-(31.TGF-β1 significantly elevated the expression of CCR7 in U251 and U87 cells.Western blot exposed that the expression level of N-cadherin and Vimentin was obviously attenuated in siCCR7 cells treated with TGF-β1,while E-cadherin going opposite.The U251 and U87 cell proliferation,migration,and invasion ability induced by TGF-β1 was retarded when CCR7 was of low-expression.And TGF-β1 reduced cell cycle arrest in glioma cell lines,which was reversed by CCR 7 siRNA or CCR7 neutralizing antibody.These results indicated that CCR7 mediated the glioma cell cycle progression induced by TGF-β1.Besides,Western blot results showed that TGF-β1 increased the expression of MMP2/9(matrix metalloproteinase 2/9)in glioma cells,while interference with CCR7 reduced the promoting effect of TGF-β1 on MMP2/9 expression.The change of mmp2/9 activity was consistent with the change of MMP2/9 expression level,that is,the activity of MMP2/9 was enhanced after TGF-1 treatment,and the enhanced effect of TGF-β1 on MMP2/9 activity was weakened when CCR7 expression was low.It has been proved that TGF-β1 enhances the activity of MMP2/9 and promotes the proliferation,migration and invasion of glioma cells by up-regulating the expression of CCR7.In addition,the enzyme-linked immunosorbent assay results in this study showed that the expression of NF-κB p65 was significantly increased after TGF-β1 treatment,and the effect was decreased after interference with CCR7.Further experiments using qRT-PCR and double luciferase reporter gene assay confirmed that the transcription of NF-κB p65 was regulated by MMP2/9(cis-acting element).Based on the above data,it can be concluded that TGF-β1 activates MMP2/9 by regulating CCR7,and MMP2/9,as a cis-acting element,regulates NF-κB p65 to promote the proliferation,migration and invasion of glioma cells.In summary,the project concluded that LYPLAL 1-2 inhibits malignant glioma by regulating miR-217-5p/CCR7,and CCR7 can regulate malignant glioma biological function induced by TGF-β1 via MMP2/9 and NF-κB pathway. |
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