| Objective: Glioma is the most common primary brain tumor in adults.Though surgery,radiotherapy,and chemotherapy treatment are improving,the prognosis of glioma patients is still very poor.Current studies show that due to the fact that genes encoding only genome sequence at the length of 2% and below,encoding gene is not merely sufficient to elucidate the molecular mechanism of glioma formation and malignant disorders.The dysregulation of non-coding RNA,which accounts for the vast majority of genomic sequences,also affects the development of tumors.Long non-coding RNAs,lnc RNAs,and mi RNAs are all typical and classical non-coding RNAs.Lnc RNAs cannot encode proteins,but their interactions with other molecules produce complex gene regulatory functions at multiple regulatory levels.In the study of glioma,Small Nucleolar RNA Host Gene 1,or SNHG1 can be used as a risk factor for the diagnosis and prognosis of the glioblastoma multiforme(GBM)disease.Some scholars have found that the expression of SNHG1 can reduce the proliferation and invasion of glioma cells,increase cell apoptosis.The increase in regulation of the SNHG1 expression in gliomas is associated with poor prognosis,but the molecular mechanisms underlying the biological effects of SNHG1 have not been investigated.Mi RNAs,the most representative of short-chain non-coding RNAs,can be combined completely or incompletely with the 3'UTR of the target messenger RNA(m RNA).This process can degrade or inhibit the targeted protein translation.Mi RNAs participate in the gene expression regulation and play an important role in cell biological behaviors such as development,cell proliferation,apoptosis and differentiation.Bio-informatics software predicts that two mi RNAs are associated with SNHG1.One is mi R-154-5p,and recent studies have shown that it can act as a tumor suppressor,by inhibiting the proliferation and metastasis of glioblastoma cells by binding PIWIL1,and predicting the prognosis of glioma patients.The second is mi R-376b-3p.Studies have shown that mi R-376b-3p is involved in the development of malignant tumors such as liver cancer and other tissues,but its potential involvement in glioma genesis remain unknown.Our team found that the two mi RNAs were downregulated in glioma tissues.We found that the common target of the above mi RNAs action is the fork-head box protein P2(FOXP2)gene,FOXP2 gene belongs to the fork-head box transcription factor family.It is expressed in many tissues,especially playing an important role in the process of brain development and maturation.The relationship between FOXP2 and glioma has not been reported yet.FOXP2 acts as a transcription factor on histone lysine specific demethylase 5B(KDM5B)promoter region.Many studies have confirmed that KDM5 B,as a tumor promoting factor,is involved in the tumorigenesis of multiple tissues.Overexpression of KDM5 B can enhance the tumorigenicity and drug resistance of neuroblastoma in the nervous system diseases.In this study,we would identify the endogenous expression of SNHG1,mi R-154-5p,mi R-376b-3p,FOXP2 and KDM5 B in gliomas,and the biological behavior of glioma cells,to further investigate whether SNHG1 can regulate expression of mi R-154-5p and mi R-376b-3p,and the regulation of the expression of FOXP2 and KDM5B;The objective is to study the biological behavior and mechanism of glioma cells in order to provide the basis for targeted therapy of human glioma.Methods: 1.Cell culture of U87 and U251.2.Real-time PCR was used to detect the endogenous expression of SNHG1,mi R-154-5p,mi R-376b-3p and FOXP2,KDM5 B in glioma tissues and cells.3.Plasmids were constructed to silence SNHG1,over-express or silence mi R-154-5p,mi R-376b-3p and FOXP2.4.Luciferase reporter assay was used to detect the binding site between SNHG1 and mi R-154-5p,or between SNHG1 and mi R-376b-3p.5.RNA immunoprecipitation assay was conducted to verify the specific binding.6.Cell proliferation assay.7.Cell migration and invasion assay.8.Quantization of apoptosis by Flow Cytometry.9.Western Blot was used to detect the expressions of FOXP2 and KDM5 B.10.Luciferase reporter assay was used to detect the promoter activity of FOXP2 and KDM5 B.11.Chromatin immunoprecipitation assay was conducted to verify the specific binding between FOXP2 and KDM5 B 12.RNA immunoprecipitation and pull-down assay was conducted to verify the specific binding between KDM5 B and SNHG1.13.Tumor xenografts in nude mouse.Result: 1.Comparing the TCGA database analysis with normal brain tissue,the expression of SNHG1 was increased significantly in glioma samples(P<0.01).SNHG1 is highly expressed in glioma tissues and cells,and increases with the pathological grade(P<0.01).The silencing of SNHG1 inhibits proliferation,migration,and invasion of malignant glioma cells,and promotes apoptosis(P<0.01).2.In comparing the TCGA database analysis with the normal brain tissue,the expression of mi R-154-5p and mi R-376b-3p was significantly decreased(P<0.01).The expression of mi R-154-5p and mi R-376b-3p is low in glioma and malignant glioma cells,and decreases with the increase of the pathological grade(P<0.01).The overexpression of mi R-154-5p or mi R-376b-3p inhibits proliferation,migration,and invasion of malignant glioma cells,and promotes apoptosis(P<0.01).3.The TCGA database,via the Pearson Correlation Analysis showed that the expression of mi R-154-5p and mi R-376b-3p were significantly inverse in correlation with SNHG1(P<0.01).The silencing of SNHG1 increased the expression of mi R-154-5p and mi R-376b-3p(P<0.01),and there was a targeted binding between SNHG1 and mi R-154-5p,SNHG1 and mi R-376b-3p,and played the role of mi RNAs sponge.4.The overexpression of mi R-154-5p or mi R-376b-3p can reduce the expression level of FOXP2 by directly binding to the 3 'UTR region of FOXP2 m RNA,in turn inhibits proliferation,migration,and invasion of malignant glioma cells,and promotes apoptosis(P<0.01).5.FOXP2 was highly expressed in glioma tissues and cells,and increased with the pathological grade(P<0.01).The expression of silent transcription factor FOXP2 can reduce the transcription of its downstream KDM5 B,inhibit the proliferation,migration,and invasion of malignant glioma cells,and promote apoptosis(P<0.01).6.KDM5 B was highly expressed in glioma tissues and cells,and increased with the pathological grade(P<0.01).Silencing the expression of KDM5 B in malignant glioma cells inhibits the proliferation,migration,and invasion of malignant glioma cells by inhibiting the PI3K/Akt signaling pathway and promotes cell apoptosis(P<0.01).7.Silencing KDM5 B can also reduce the expression level of SNHG1 and form a positive feedback regulatory loop.8.SNHG1 knockdown combine with mi R-154-5p and mi R-376b-3p overexpression suppress tumor growth in a xenograft mouse model.Conclusion: 1.Knockdown of SNHG1 inhibited malignant behaviors of glioma cells.2.Mi R-154-5p and mi R-376b-3p acted as tumor suppressors in glioma cell lines.3.SNHG1 bound to and attenuated the expression of mi R-154-5p and mi R-376b-3p.4.FOXP2 acted as an oncogene in glioma cells lines,mi R-154-5p and mi R-376b-3p inhibited FOXP2 expression by targeting its 3'UTR.5.KDM5 B acted as an oncogene in glioma cells lines,FOXP2 bound to oncogene KDM5 B promotors and facilitated its expression.6.SNHG1 regulates the expression of FOXP2 by inhibiting mi R-154-5p and mi R-376b-3p,further regulating the expression level of KDM5 B.KDM5B bound to SNHG1 and formed a positive feedback loop. |
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