The Expression And Functional Role Of Long Non-Coding RNA FOXD3-AS1 In Glioma

Glioma is the most common primary malignant tumor in the central nervous system(CNS),which accounts for more than 40 % of all brain tumors.Despite major advances made in the conventional treatments of glioma,including surgery,radiotherapy,and chemotherapy in the last decades,the overall survival rate in patients with glioma remains poor.The development of glioma is a complex process with multigene interaction and multimolecular regulation.Although alterations in oncogenes and tumor suppressor genes have been reported in glioma,the precise molecular mechanisms remain largely unknown.Therefore,it is necessary to explore the specific molecular mechanisms underlying glioma for selecting suitable predictive biomarkers and seeking new therapeutic strategies for the treatments of glioma.Long non-coding RNAs(lnc RNAs),which were initially argued to be spurious transcriptional noise,are now recognized as a class of RNAs with transcripts longer than 200 nucleotides with no function of encoding proteins.Studies have found that lnc RNAs play critical regulatory roles in many human diseases,including cancer.So we predict that lnc RNA could be a new therapeutic target in cancer gene therapy.Unlike their shorter counterparts including mi RNAs and other smaller noncoding RNAs,lnc RNAs can control downstream target genes on the level of transcription,post transcription and epigenetics.Recent studies have reported that a growing number of lnc RNAs cooperate with neighbor genes and form ‘‘lnc RNA-m RNA’’ pairs that affect their function.We previously performed microarrays with the glioma specimens and found that FOXD3-AS1 was aberrantly expressed in glioma.In this study,we first investigated the expression of FOXD3-AS1 in glioma tissues and normal brain tissues.And we analyzed the correlation of FOXD3-AS1 expression with clinicopathological features and the prognostic value of FOXD3-AS1 in patients.To understand the tumorigenic mechanism of FOXD3-AS1,the expression pattern and functional role of FOXD3-AS1 in glioma were investigated using real-time PCR and Smart Silencer-mediated knockdown studies.With an attempt to find the relationship between FOXD3-AS1 and FOXD3,we then investigated the expression of FOXD3 in glioma tissues and normal brain tissues.Finally,we detected the protein level of FOXD3 after FOXD3-AS1 knockdown in glioma cells.This study is consisted of four main parts: the first part is to investigate the expression of FOXD3-AS1 in glioma.The second part is to investigate the biological effects of FOXD3-AS1 konckdown in glioma cells.The third part is to identify the target gene of FOXD3-AS1.The last part is investigate the relationship between FOXD3-AS1 and FOXD3 in glioma.Part I The expression of FOXD3-AS1 in gliomaObjective: To investigate the expression of FOXD3-AS1 in glioma tissues and normal brain tissues.To analysis the correlation of FOXD3-AS1 expression with clinicopathological features and the prognostic value of FOXD3-AS1 in patients.Methods: The expression pattern of FOXD3-AS1 in 44 glioma tissues and 6 normal brain tissues was investigated using real-time PCR.The correlation of FOXD3-AS1 expression with clinicopathological features and the prognostic value of FOXD3-AS1 in patients were analyzed through systemic analysis of patient specimen and clinical data.Results: The results showed that FOXD3-AS1 expression was significantly higher in the high-grade glioma tissues compared with that in the normal brain tissues and low-grade glioma tissues(both P<0.01),whereas there was no significant difference in FOXD3-AS1 expression between the low-grade glioma and normal brain tissues.Clinicopathological evaluation suggested that FOXD3-AS1 was significantly associated with WHO grade(I– II vs.III–IV,P=0.003).Kaplan–Meier survival analysis revealed that glioma patients with high FOXD3-AS1 expression had significantly shorter overall survival(OS)than patients with low FOXD3-AS1 expression.Our stratified analysis showed that low FOXD3-AS1 expression was significantly with better OS in patients with high-grade gliomas.Multivariate analysis of the prognosis factors confirmed that high FOXD3-AS1 expression was a poor independent predictor of poor survival in glioma.Conclusion: FOXD3-AS1 is overexpressed in high-grade gliomas.There is a significantly negative correlation between expression levels of FOXD3-AS1 and OS.FOXD3-AS1 may be a potential oncogenic lnc RNA in glioma.Part II The biological effects of FOXD3-AS1 knockdown in glioma cellsObjective: To identify the functional role of FOXD3-AS1 in glioma cells.Methods: Lnc RNA Smart Silencer,synthesized from Ribo Bio(Guangzhou,China),was used to knockdown the expression of FOXD3-AS1.The FOXD3-AS1 Smart silencer was transfected into U251 and A172 cell lines with Lipofectamine 2000.The silencing effect of the Smart Silencer was evaluated by real-time PCR.And then we test the effects on biological behaviours of glioma cells in vitro 24 h after transfection.Results: The FOXD3-AS1 expression level was down-regulated in FOXD3-AS1 Smart Silencer group compared with NC Smart Silencer group.FOXD3-AS1 Smart Silencer notably impaired cell proliferation of U251 and A172 cells compared to NC Smart Silencer cells.Depletion of FOXD3-AS1 in U251 or A172 cells resulted in an increase of cells in the S phase and a corresponding decrease of cells in the G1 and G2/M phases,which suggested that silencing of FOXD3-AS1 prevented glioma cells from S phase entering the G2/M phase.Depletion of FOXD3-AS1 dramatically reduced the cell migration and invasion ability compared with cells transfected with NC Smart Silencer.Conclusion: FOXD3-AS1 may promote cell proliferation,migration and invasion in glioma development and progression.Part III The expression of FOXD3 in gliomaObjective: To find out the possible target gene of FOXD3-AS1 and to investigate the expression of FOXD3 in glioma tissues and normal brain tissues.To analysis the correlation of FOXD3-AS1 expression with clinicopathological features and the prognostic value of FOXD3 in patients.Methods: Through bioinformatics analysis to identify the target gene of FOXD3-AS1.The expression pattern of FOXD3 in the aforementioned specimen was detected by employing real-time PCR,immunohistochemistry(IHC),Western blot(WB).The correlation of FOXD3 expression with clinicopathological features and the prognostic value of FOXD3 in patients were also analyzed through systemic analysis of patient specimen and clinical data.Results: The expression level of FOXD3 was also significantly up-regulated in the high-grade glioma tissues compared with that in the normal brain tissues and low-grade glioma tissues(both P<0.05).Clinicopathological evaluation suggested that FOXD3 was significantly associated with WHO grade(I– II vs.III–IV,P=0.026).Kaplan-Meier analysis shows glioma patients with high FOXD3 expression had significantly shorter OS(P<0.01)than patients with low FOXD3 expression.Conclusion: FOXD3 is overexpressed in high-grade gliomas.There is a significantly negative correlation between expression levels of FOXD3 and OS.FOXD3 may be a potential oncogenic molecular in glioma.Prat IV The relationship between FOXD3-AS1 and FOXD3 in gliomaObjective: To preliminarily clarify the relationship between FOXD3-AS1 and FOXD3 in glioma.To explore the possible molecular mechanism of FOXD3-AS1 in glioma.Methods: We assessed the correlation of FOXD3-AS1 and FOXD3 expression levels in glioma using Pearson’s correlation.We examed the protein level of FOXD3 after FOXD3-AS1 knockdown in U251 and A172 cells.Results: The expression of FOXD3-AS1 was positively correlated with FOXD3 m RNA.The protein level of FOXD3 was decreased concomitantly with the FOXD3-AS1 downregulation in cultured glioma cell lines U251 and A172.Conclusion: FOXD3-AS1 may fulfill its oncogenic function partly by modulating the FOXD3 expression.