Effect Of LncRNA ASB16-AS1 On Biological Behaviors Of Glioma Cells

Objective: Learn R language and bioinformatics,as well as LncRNAs were filtered out by bioinformatics.qPCR and RTCA were used to determine whether differential LncRNAs are differentially expressed in the samples and whether the expression level of LncRNA are correlated with WHO(World Health Organization)grades.Moreover,cell function experiments were conducted on lncRNA showing differences in tissues to verify whether knocking-down the lncRNA would affect the proliferation,invasion and migration function of glioma cells.Plus,novel molecular markers and therapeutic targets were identified for the diagnosis and treatment of gliomas.Methods: The TCGA database was used to filter out differential genes and R code was written to perform the ROC analysis(Receiver operating characteristic analysis)on every differential gene.The genes with the area under the curve(AUC value)> 0.85 were screened out,along with the construction of the ceRNA network of these selected genes based on data from starbase v2.0.Additionally,the ceRNA network was analyzed using Cytoscope 3.6 software,along with the identification of the hub genes with the top 10% internal connectivity.In the hub genes,4 lncRNAs were randomly selected within rarely reported lncRNAs to perform qPCR in the sample tissues to determine whether there was differential expression and whether there was correlation between the expression level and the WHO grades.Finally,ASB16-AS1 was selected for functional experiments,and the siRNA technique was used to knock down ASB16-AS1.Furthermore,U87 and U251 were incubated with growth factor to propagate U87 MG and U251 glioblastoma stem-like cells(U87GS,U251GS).Each cell line was divided into three groups,namely,NC(Negative control,transfected with NC),siRNA-1 group(silence,transfected with ASB16-AS1 siRNA-1),siRNA-2 group(silence,transfected with ASB16-AS1 siRNA-2).RTCA(Real-time cellular analysis)technology was used by functional experiment to detect changes in cell proliferation,invasion,and migration,along with application of flow cytometry to detect difference of cell cycle.Results: A total of 240 differential genes which are not all lncRNAs with AUC > 0.85 were identified through differential analysis and ROC analysis.A total of 1650 predicted ceRNA relationships,based on the official symbol of these genes and the ceRNA prediction network data provided by starbase v2.0,were identified.Predictive relationships can't be found among all differential genes in starbase v2.0.Only a total of 72 lncRNAs were included in the output network.The ceRNA network of differential genes has a total of 417 nodes,along with a total of 42 hub genes with the top 10% connectivity.Genes which is not lncRNA was removed,and reported frequently.We selected HOXA-AS2,HOTAIRM1,ASB16-AS1,and LINC00339 for qPCR validation of clinical samples,with the results suggesting that only ASB16-AS1 was significantly different in clinical samples from our hospital and was positively correlated with glioma grades(normal group 1.01±0.84,low grade glioma 4.34±2.69,high grade glioma 11.29± 6.33),however,with factor analysis of variance(P < 0.01).The expression level of ASB16-AS1 was positively correlated with the diagnosis of glioma,with ROC analysis showing that the area under the curve reached 0.94.In the TCGA database,ASB16-AS1 was significantly up-regulated in tumor tissues compared with that in normal tissues(normal group 1.36±0.70,tumor group 4.03±1.67,t-test P < 0.05)..There was no significant difference in ASB6-AS1 expression in recurrence group of the tumor group and primary group(recurrence group 3.59±1.92,primary group 4.07±1.65,t test P>0.05).The results of qPCR detection showed that siRNA-1 and siRNA-2 can effectively knock down ASB16-AS1,with the inhibition efficiency as about 50%.The results of RTCA proliferation,invasion and migration assays show that the proliferation,invasion and migration ability of glioma cells significantly decreased by ASB16-AS1 knockdown(P<0.001)while the percentage of cells in G2/M phase increased significantly in the ASB16-AS1 siRNA group(U87GS NC 19.3±4.2,siRNA 30.9±5.5;U251GS NC 18.3±3.2,siRNA 27.9±4.6).Conclusion: The expression level of ASB16-AS1 is significantly up-regulated in glioma tissues compared with that in normal tissues,with its expression increasing with the increase of glioma grade.ASB16-AS1 is involved in glioma progression,and the knockdown of ASB16-AS1 in glioma U87 and U251 cell line can exert a significant effect on the proliferation,invasion and migration of glioma.ASB16-AS1 serves as a potential biomarker and drug target.