The Expression And Functional Role Of Long Non-coding RNA AFAP1-AS1 In Glioma

Glioma is one of the most prevalent and malignant tumors of the brain.Surgery and postoperative chemotherapy and radiotherapy remain the major options for glioma.However,the prognosis of glioma is still poor in spite of the advance in chemotherapy.The five-year survival of patients with glioblastoma is the shortest among all types of glioma,and the median survival is less than 14 months,since glioblastoma is the most aggressive of all gliomas with WHO grade IV.Now the research interest is directed to hunt for molecular markers and the target of glioma in order to improve the treatment efficiency.Long non-coding RNAs(IncRNAs)are non-protein coding transcripts longer than 200 nucleotides.As byproducts of transcription,IncRNAs cannot code for any proteins in the presence of RNA polymerase ?.It is believed that IncRNAs are involved in various biological processes,including epigenetics,transcriptional regulation and posttranscriptional translation.IncRNAs are particularly noted for their roles in the occurrence and development of cancers.AFAP1-AS1,a newly discovered InRNA,is upregulated in many cancers,including esophageal cancer,nasopharyngeal cancer,lung cancer,pancreatic cancer and colorectal cancer.As a predictor of prognosis,upregulation of AFAP1-AS1 can promote invasion and migration of tumor cells.However,the role of AFAP1-AS1 in the biological behaviors and prognosis of glioma is not fully understood.The purpose was to clarify the significance of lncRNA ASAP1-AS1 in the occurrence,development and prognosis of glioma and to lay the foundation for targeted therapy of glioma.This study was divided into three parts and main methods and results are as follows.Part ? The expression of AFAP1-AS1 in gliomaObjective:To investigate the expression level of AFAP1-AS1 in glioma and normal brain tissues.To analysis the correlation of AFAP1-AS1 expression with clinicopathological features and the prognostic value of AFAP1-AS1 in patients.Methods:The expression pattern of AFAP1-AS1 in 5 normal brain tissues and 52 glioma tissues was deteched by RT-PCR.The correlation of AFAP1-AS 1 expression with clinicopathological features and the prognostic value of AFAP1-AS1 in patients were analyzed through systemic analysis of patient specimen and clinical data.Results:The results showed that AFAP1-AS1 expression was significantly higher in the high-grade glioma tissues compared with that in the normal brain tissues and low-grade glioma tissues,whereas there was no significant difference in AFAP1-AS1 expression between the low-grade glioma and normal brain tissues.Clinicopathological evaluation suggested that AFAP1-AS1 was significantly associated with WHO grade(?-? vs.?-?,P=0.003)and KPS scores.Kaplan-Meier survival analysis revealed that glioma patients with high AFAP1-AS1 expression had significantly shorter overall survival(OS)than patients with low AFAP1-AS1 expression.Conclusion:AFAP1-AS1 is overexpressed in high-grade gliomas.There is a significantly negative correlation between expression levels of AFAP1-AS1 and OS.AFAP1-AS1 may be a potential oncogenic lnc RNA in glioma.Part ? The Effects of AFAP1-AS1 knockdown in glioma cells in vitroObjective:To identify the functional role of AFAP1-AS1 on glioma cell proliferation and invasion.Methods:The expression pattern of AFAP1-AS1 in normal human astrocyte cells and glioma cells was deteched by RT-PCR.SiRNA was synthesized to knockdown the expression of AFAP1-AS1.The AFAP1-AS1 siRNA was transfected into U87 and U251 cell lines with Lipofectamine 2000.The silencing effect was evaluated by RT-PCR.And then the biological behaviours of glioma cells were tested at 24 h after transfection.Results:The results showed that AFAP1-AS1 expression was signiticantly higher in the glioma cells compared with that in the astrocyte cells.The AFAP1-AS1 expression level was down-regulated in AFAP1-AS1 siRNA group compared with NC siRNA group.AFAP1-AS1 siRNA notably impaired cell proliferation of U87 and U251 cells compared to NC siRNA cells.Slience of AFAP1-AS1 in U87 or U251 cells resulted in an increase of cells in the S phase,dramatically reduced the cell proliferation and invasion ability compared with cells transfected with NC siRNA.Conclusion:Slience of AFAP1-AS1 may inhibit cell proliferation and invasion.Part ? The expression of AFAP1 in gliomasObjective:To find out the possible target gene of AFAP1-AS1 and to investigate the expression of AFAP1 in glioma tissues and cells.To explore the possible molecular mechanism of AFAP1-AS1 in glioma.Methods:To identify the target gene of AFAP1-AS1 through bioinformatics analysis.The expression pattern of AFAP1 in glioma tissues and cells was detected by employing RT-PCR and Western blot.The protein level of AFAP1 in U87 and U251 cells were exaned after AFAP1-AS1 knockdown.Results:The expression level of AFAP1 was also significantly down-regulated in the high-grade glioma tissues compared with that in the normal brain tissues and low-grade glioma tissues(both P<0.05).AFAP1 expression was significantly lower in the glioma cells compared with that in the astrocyte cells.The protein level of AFAP1 was increased concomitantly with the AF AP1-AS1 downregulation in cultured glioma cell lines U8 7 and U251.Conclusion:AFAP1 is down-regulated in high-grade gliomas.AFAP1-AS1 may fulfill its oncogenic function partly by modulating the AFAP1 expression.