Effects And Mechanism Of LncRNA-CCAT2 On Biological Function Of Human Glioma Cells

Background and Objective:Glioma is the most common malignant tumor in the central nervous system,,its proliferation and invasion increased with the degree of glioma malignancy,which resulted the current difficult histological resection of the tumor,and the post-surgery recurrence rate is also at high level.In recent years,genome-wide detection of glioma has made a major breakthrough.Gene chips have confirmed the differential expression of LncRNA in different types and grades of glioma,and closely related to the malignant degree and biological behavior of gliomas.This provides a new idea for revealing the intrinsic mechanism of glioma development and subsequent gene therapy.LncRNA-CCAT2 is involved in gene regulation of malignant tumors,such as colorectal cancer,which can promote the growth and metastasis of tumor by upregulating MYC,miR-17-5p and miR-20 a.Esophageal cancer,breast cancer,gastric cancer also found LncRNA-CCAT2 expression was significantly higher than normal tissue.However,whether LncRNA-CCAT2 expression level was changed in glioma,and whether it involved in the regulation of glioma biological characteristics,needs further explored.Wnt / ?-catenin signaling pathway has been proved to be involved in the regulation of glioma,promoted the translocation of ?-catenin can promote glioma cell proliferation and subsequent tumor formation.Whether LncRNA-CCAT2 affec-ted glioma development by regulating Wnt / ?-catenin signaling pathway is not clear.Therefore,the aim of this study was to analyze the correlation between LncRNACCAT2 and Wnt / ?-catenin signaling in gliomas by using large clinical data,and to verify whether LncRNA-CCAT2 glioma biological function by regulating Wnt/ ?-catenin signaling.Through this study,we want to determine the role of LncRNACCAT2 / Wnt / ?-catenin axis in the regulation of glioma,and to provide a new strategy for the treatment of glioma,as well as provide a new theoretical basis for the development of new drugs and glioma molecular target treatment.Methods:In this study,glioma tissues and its adjacent normal tissues from the Department of Neurosurgery,the Second Affiliated Hospital of Nanchang University;human glioblastoma cell lines U87-MG and U251 were purchased from Shanghai Cell Bank of Chinese Academy of Sciences.1.LncRNA-CCAT2 and Wnt / ?-catenin pathway proteins were differentially expressed in gliomas1.1 qRT-PCR was used to detect the expression of LncRNA-CCAT2 in glioma tissue and corresponding adjacent normal tissue.The relationship between LncRNA-CCAT2 and the malignancy of glioma was determined according to the WHO pathological grade.1.2 Western blot was used to detect the expression of Wnt / ?-catenin pathway related proteins ?-catenin,c-Myc,MMP-7 and CyclinD1 in glioma tissues and their corresponding normal tissues.The relationship between Wnt / ?-catenin pathway and the malignancy of glioma was determined according to the WHO pathological grade.2.LncRNA-CCAT2 affected glioma cells proliferation,migration and tumorige-nesis2.1 The lentivirus was transfected into U87-MG and U251 glioma cells to stably knocked down LncRNA-CCAT2.2.2 The proliferation of glioma cells was detected by CCK8 assay.The colony formation ability of glioma cells was detected by plate clone assay.The cell cycle distribution of glioma cells was detected by flow cytometry,and calculated the proliferation index.Transwell migration assay was used to detect the migration ability of glioma cells.2.3 Subcutaneous tumor formation test in nude mice was used to detect the in vivo tumorigenicity of glioma cells.3.LncRNA-CCAT2 regulate Wnt / ?-catenin signaling pathway in glioma3.1 Dual-luciferase reporter gene detection LncRNA-CCAT2 regulated LEF / TCF promoter in glioma cells.3.2 Western blot was used to detect the expression of Wnt / ?-catenin pathway related proteins ?-catenin,c-Myc,MMP-7 and CyclinD1 in glioma cells.Results: 1.LncRNA-CCAT2 and Wnt / ?-catenin pathway proteins were differentiallyexpressed in gliomas1.1 qRT-PCR results showed that the expression level of LncRNA-CCAT2 was closely related to glioma,and there was a significant up-regulation of LncRNACCAT2 in gliomas.Further analysis of the patient's clinical data(age,sex,tumor size)and the relationship between gliomas WHO level and LncRNA-CCAT2 expression found that LncRNA-CCAT2 and glioma grade are closely related.qRT-PCR also showed that the expression level of LncRNA-CCAT2 in high-grade glioma was significantly higher than that in low-grade gliomas and normal brain tissues,and the expression level of LncRNA-CCAT2 was also significantly higher in low-grade glioma than in normal brain tissue.1.2 Western blot results showed that there was no significant difference in total ?-catenin expression between high-grade gliomas,low-grade gliomas and normal brain tissues,while the expression of ?-catenin in high grade gliomas was significa-ntly higher than that in low grade gliomas Levels of glioma and normal brain tissue,low-grade glioma nucleus ?-catenin expression is also higher than normal brain tissue.The expression of c-Myc in high-grade gliomas was significantly higher than in low-grade gliomas and normal brain tissues.The expression of c-Myc in low grade gliomas was also higher than that in normal brain.The expression of MMP-7 protein in high-grade glioma and low-grade glioma no significant difference,but in highgrade glioma and low-grade glioma were significantly higher than normal brain tissue.The expression of CyclinD1 in high grade gliomas was significantly higher than that in low grade gliomas and normal brain tissues.The expression of CyclinD1 in low grade gliomas was also higher than that in normal brain tissues.2.LncRNA-CCAT2 affected glioma cells proliferation,migration and tumorige-nesis2.1 Three groups of pGV248-CCAT2 shRNA(shRNA1,shRNA2,shRNA3)and scramble shRNA lentivirus were successfully infected on U87-MG and U251 cells.The qRT-PCR results showed that the interference efficiency of pGV248-CCAT2 shRNA3 was the highest in U87-MG and U251 cells,Therefore,U87-MG and U251 cells interfered with pGV248-CCAT2 shRNA3 were used for subsequent experiments.2.2 CCK8 results showed knockdown of LncRNA-CCAT2 expression in glioma cells,U87-MG and U251 glioma cell proliferation was significantly reduced.The results of plate cloning experiments showed that the ability of glioma cells to clone in vitro was significantly weakened after knockdown of LncRNA-CCAT2 expression in glioma cells.Flow cytometry cell cycle results showed knockdown of LncRNACCAT2 glioma cells showed significant G0 / G1 phase arrest.The results of Transwell migration showed that the migration ability of glioma cells was significantly reduced after knockdown of Lnc RNA-CCAT2 expression in glioma cells.2.3 The results of subcutaneous tumor formation in nude mice showed that the expression of LncRNA-CCAT2 in glioma cells was significantly decreased after subcutaneous tumor formation in nude mice,indicating that LncRNA-CCAT2 can significantly inhibit the proliferation of glioma cells in vivo.3.LncRNA-CCAT2 regulate Wnt / ?-catenin signaling pathway in glioma3.1 Double luciferase reporter gene showed low luciferase activity in the Negative control group and the LEF / TCF reporter group;addition of LiCl to the LEF / TCF reporter group activated the Wnt / ?-catenin signaling pathway and led to the LEF / TCF promoter The activation of luciferase was significantly higher than that of the negative control group and the LEF / TCF reporter group.In LncRNACCAT2-knockdown glioma cells,the luciferase activity was significantly lower than that of the Negative control group And LEF / TCF reporter group,but significantly lower than the LEF / TCF reporter + LiCl group.The results showed that knockdown of LncRNA-CCAT2 expression in glioma cells inhibited the transcrip-tional activity of Wnt / ?-catenin signaling pathway.3.2 Western blot results showed that the expression of total ?-catenin protein in LncRNA-CCAT2 knockdown glioma cells and control cells had no significant difference,while the expression of nuclear ?-catenin protein in knockdown LncRNACCAT2 tumor cells significantly less than the control group cells.The expression of cytoplasmic ?-catenin protein in knockdown of LncRNA-CCAT2 glioma cells was significantly more than control cells.The expression of c-Myc,MMP-7 and CyclinD1 in glioma cells knocked down by LncRNA-CCAT2 were significantly lower than those in control group.Conclution:1.LncRNA-CCAT2 was highly expressed in glioma,and its expression level increased with the increase of glioma WHO grade.The intranuclear transfer of ?-catenin and the expression of Wnt / ?-catenin signaling proteins c-Myc,MMP-7 and CyclinD1 were positively correlated with glioma grade.2.Knockdown of glioma cells LncRNA-CCAT2 expression can inhibit glioma cell proliferation,invasion,cell cycle,in vivo tumorigenesis and so on.This in turn proves that LncRNA-CCAT2 promotes glioma tumorigenesis.3.Knockdown of LncRNA-CCAT2 inhibits the intracellular transfer of ?-catenin in glioma cells and inhibits the expression of target proteins c-Myc,MMP-7 and CyclinD1 in Wnt / ?-catenin signaling.From the above studies,we found that LncRNA-CCAT2,which is overexpressed in gliomas,can promote its proliferation,invasion,progression,by activating Wnt / ?-catenin signaling pathway.Regulation of LncRNA-CCAT2 and Wnt / ?-catenin signaling pathways may become a new target for gene therapy of glioma.