Knockdown Of Long Non-coding RNA ANRIL In The Growth Inhibiton Of Glioma And Related Mechanisms

Background: Gliomas are the most common types of primary central nervous system malignancy found in adults.Gliomas are classified as astrocytomas,oligodendrogliomas,ependymomas,and mixed tumors according to the histological subtype,and are assigned malignancy grades I-IV.Gliomas is very aggressive and invasive,which grow rapidly.Furthermore multi-drug resistance is always occurred in chemotherapy.At present,the clinical application of radiotherapy and chemotherapy are very common after surgery,but the effect is still not obvious.Now it is more and more urgent to diagnose and treat glioma in molecular aspects.LncRNAs are endogenous non-coding transcripts of more than 200 nucleotides.Although thousands of these molecules have been identified,few have been assigned a biological function.Those that have been characterized have been found to be involved in a broad spectrum of processes such as apoptosis,invasion,histone protein modification,etc.Antisense noncoding RNA in the INK4 locus(ANRIL)is a 3.8 kb lncRNA antisense transcription of the INK4B-ARF-INK4 A genecluster located in the chromosome 9p21 region.It is mainly located in the nucleus.Variants in this region have been associated with several cancers,including glioma,leukemia,melanoma,basal cell carcinoma,nasopharyngeal carcinoma,breast cancer,etc.MiRNA is a class of non-coding small RNA molecules with a long range of 19 to 24 ribonucleotides.It is closely related to tumor growth,invasion,new angiogenesis and apoptosis.MiR-34 a is one of them and has the function of tumor suppressor genes.MiR-34 a is available as repressors of gene expression by binding to specific sites in the 3′-untranslated region(3′UTR)of mRNA molecules.Sirt1 is a member of the mammalian sirtuin family that are implicated in themechanisms underlying regulation of transcriptional silencing and cell survival.Aims: However,the potential clinical significance of ANRIL in glioma is unclear at present,which are not many related studies on glioma.Therefore,the purpose of this study was to explore the role of ANRIL,mir-34 a and Sirt1 in gliomas and the potential associations,as well as the mechanisms for the proliferation,invasion,migration and apoptosis of glioma cells.Methods: In this study,we use glioma cell lines,glioma tissue,normal brain tissue and xenograft models as the experimental subjects.We use CCK-8,colony formation,flow cytometry,transwell plate,westernblot,siRNA gene silencing,Real time-RT-PCR,transfection and generation of stably transfected cell lines,reporter vector constructs and luciferase reporter assay to investigate the biological effect of ANRIL on glioma as well as the roles of ANRIL,miR-34 a,and Sirt1 in glioma and their potential interaction in the regulation.Firstly,expression of ANRIL in normal gliacells and five glioma cell lines was measured.Then,effects of ANRIL suppression on cell proliferation,apoptosis,migration and invasion ofU251 cells as well as expression of miR-34 a were assessed.Meanwhile,effects of miR-34 a on U251 cells silencing ANRIL were tested.Whether Sirt1 is a target of miR-34 a was verified,followed by estimating the role of Sirt1 overexpression in U251 cells overexpressing miR-34 a.Finally,the involved signaling pathways were assessed.The main results are as follows:(1)ANRIL was upregulated in glioma tissue and glioma cells,its suppression inhibited cell proliferation,migration and invasion but promoted cell apoptosis.Whats more in vivo experiments,knockdown ANRIL significantly reduces tumor volume in nude mice.(2)ANRIL acted as a sponge of miR-34 a,mir-34 a is significantly upregulated after inhibiting ANRIL.The results of our study provide evidence that the inhibition of U251 cell proliferation,migration,and invasion observed following ANRIL suppression occurs via amechanism that involves upregulation of miR-34.(3)Sirt1 is a target of miR-34 a.MiR-34 a overexpression inhibited proliferation and decreased Sirt1 protein expression via post-translational regulation,which inhibited cell proliferation,migration and invasion but promoted cell apoptosis.(4)These observations indicate that Sirt1 may affect glioma cell lines via activation of the PI3K/AKT and mTOR signaling pathways.Thus,our findings indicate that the inhibition of glioma cell proliferation,migration,and invasion as well as the increase of glioma cell apoptosis observed following ANRIL suppression occurs via a mechanism that involves miR-34 a mediated downregulation of Sirt1 expression and concomitant inhibition of the PI3K/AKT and mTOR pathways.Conclusion:In summary,the results of our study provide evidence that the inhibition of glioma cell proliferation,migration,and invasion observed following ANRIL suppression occurs via a mechanism that involves upregulation of miR-34 a.Furthermore,we showed that the tumor suppressive function of mi R-34 a is likely to be mediated,at least partially,by downregulation of Sirt1 expression and concomitant inhibition of the PI3K/AKT and mTOR pathways.Thus,findings of the present study implicate mechanisms of ANRIL suppression and miR-34 a upregulation as therapeutic strategies for glioma.