The Functions And Mechanisms Of LUCAT1-miR-9-5p-CXCR4/CXCL12 Axis In Human Glioma

Background:Glioma is the most common malignant tumor in the central nervous system,which is characterized by great heterogeneity,high malignancy,poor prognosis,and high mortality.In recent years,the progress of treatment methods has greatly improved the prognosis of patients,but there is still no breakthrough in the overall survival of patients with glioma.Therefore,the screening of diagnostic markers for glioma and the study of the malignant evolution mechanism of glioma still need to be studied.Long noncoding RNA(lncRNA)is a gene with a length of more than 200bp that cannot encode proteins.More and more studies have shown that many lncRNA are abnormally expressed in liver cancer,leukemia,lung adenocarcinoma,prostate cancer,colorectal cancer,and other cancer tissues,which are related to the prognosis of patients,and participate in the formation and development of tumor cells to play important functionsAims:This study intends to select LUCAT1 as the key lncRNA in this study through bioinformatics analysis and further explore the potential mechanism of LUCATlin glioma and its evaluation value in glioma prognosis through bioinformatics mining and in vitro cell experiments,providing a new biomarker for the prognosis assessment of glioma.Methods:The present study can be divided into two parts:the bioinformatics mining part and in vitro cell experiment part.Bioinformatics mining part:lncRNA markers of glioma were explored using bioinformatics methods;GEPIA database was used to analyze the expression difference of LUCA T1 between tumor tissue and adjacent normal tissue;Diana Lncbase determine miR-9-5p as the key downstream target molecules of LUCAT1.Targetscan,microCDT and Multiple miRwalk databases determine CXCR4 as the key downstream target molecule of miR-9-5p;the STRING database was used to analyze the interaction between CXCR4 and CXCL12 proteins.Survival analysis and Cox analysis was used to evaluate overall survival rate of glioma patients;glioma single-cell data in TISCH database was studied to explore the role of CXCR4 and CXCL12 proteins in glioma tumor environment.In vitro cell experiment part:Firstly,the interaction between LUCAT1 and miR-9-5p and the interaction between miR-9-5p and CXCR4 were verified by dualluciferase reporter experiments;secondly,the relationship between LUCAT1 and its downstream molecules and the biological phenotype of glioma was studied.qRTPCR and Western-blot were used to detect the gene expression and protein expression of CXCR4 and CXCL12.Finally,the human glioma cell lines were divided into siLUCAT,siNC,siLUCATl+miR-NC,siLUCAT1+miR-9-5p inhibitor groups,respectively.CCK-8 assay was used to detect the proliferation of glioma cells after transfection;and the correlation of LUCAT1 and glioma cell division was analyzed by studying glioma cell cycle distribution and cell apoptosis.Results:Bioinformatics mining part:Expression of LUC AT1 in low-grade glioma and high-grade glioma tissues was significantly higher than that in normal brain tissue;Diana Lncbase confirmed that the key downstream target molecule of LUC AT1 is miR-9-5p;Targetscan,microCDT and miRwalk databases confirmed that the key downstream target molecule of miR-9-5p is CXCR4,and the STRING database determined the interaction between CXCR4 and CXCL12 proteins;Expression of LUCAT1,miR-9-5p,CXCR4 are related to the overall survival rate of glioma patients by survival analysis and Cox analysis;glioma single-cell data showed that CXCR4 and CXCL12 proteins participate in intercellular communication as ligands and receptors on cells in the glioma microenvironment.In vitro cell experiment part:Dual-luciferase reporter experiments proved that there is a binding site between LUCAT1 and miR-9-5p,as well as miR-9-5p and CXCR4;after silencing the expression of LUCAT1 in human glioma cells,CXCR4 and CXCL12 expression levels were significantly down-regulated,while miR-9-5p expression was increased;after promoting miR-9-5p in human glioma cells,CXCR4 and CXCL12 expression levels were significantly decreased.The silencing of LUCAT1 and the promotion of miR-9-5p in glioma cells will affect the expression of the downstream CXCL12-CXCR4 signaling pathway.The proliferation of glioma cells in the siLUCATl group was significantly lower than that in the siNC group,while the tumor cell cycle arrest and apoptosis rate were significantly increased,indicating that silencing LUC AT1 can promote apoptosis by inhibiting the malignant proliferation of glioma cells.The proliferation of glioma cells in the siLUCAT1+miR-9-5p inhibitor group was significantly higher than that in the siLUCATl+miR-NC group,the apoptosis rate of tumor cells decreased,while the tumor cells in the division phase(S phase)increased,indicating that LUC AT1 can participate in the regulation of glioma cell proliferation and apoptosis cell cycle by regulating miR-9-5p.Conclusions:1.LUC AT1 is related to the malignant proliferation of glioma,and the high expression of LUCAT1 is related to the poor overall survival rate of patients,and it is expected to become a molecular marker for glioma to predict the prognosis of glioma patients.2.LUCAT1 negatively regulates the targeting of miR-9-5p,and affects the proliferation and apoptosis of glioma cells through miR-9-5p.3.miR-9-5p has a targeted negative regulatory effect on CXCR4.LUCAT1 can increase the expression of oncogene CXCR4 by down-regulating the level of miR-95p,which is related to the regulation of tumor cell cycle and participates in the regulation of tumor cell cycle.