LncRNA HDAR Promotes Glioma Proliferation And Metastasis By Binding To HnRNPQ-DHX9 Scaffold

BackgroundGliomas are the most aggressive brain tumors in the central nervous system.For most high-grade glioma patients,glioma cells proliferate and migrate rapidly.Due heterogeneity of glioma tumor,the overall survival of patients is about only 15 months.However,the current treatment methods,including surgery,chemotherapy and radiotherapy,are not ideal for the prognosis of glioma,and there is still no effective solution for postoperative recurrence.Although there are many studies on glioma all over the world,the pathogenesis is still not clear.Current researches have shown that the proliferation,invasion and migration of glioma cells are related to the regulation of oncogenes and transcription factors.Therefore,the method of molecular targeted therapy has obvious clinical advantages,which can cause tumor cell-specific death without affecting normal tissue cells around the tumor.With the in-depth study of molecular biology and gene expression of glioma,it provides a new therapeutic target for molecular therapy of glioma,in the hope of helping early treatment and prognosis of glioma.LncRNA(Long Noncoding RNA)is a type of non-coding RNA longer than 200 nt,which was previously thought to be transcriptional "noise",but as increased researches,it became more and more versatile.It has been shown that IncRNA can play a role in transcriptional regulation and post-transcriptional regulation,and plays an important role in regulating development of tumors and expression of cancer related gene.Numerous studies have shown that non-coding RNAs have key functions in the biological processes that regulate the development and progression of gliomas.Recently,new data have emerged suggesting that cross-regulation between long non-coding RNAs and small non-coding RNAs contributes to the phenotypic diversity of glioma subclasses.HnRNPQ(Heterogeneous Nuclear Ribonucleoprote Q),encoded by the SYNCRIP gene,is a highly conserved RNA-binding protein that plays an important role in the development of nerve and muscle in drosophila and mammals.Dysfunction or dysfunction of hnRNPQ can lead to severe cardiomyopathy and neurodegenerative diseases.In mammals,hnRNPQ controls the length and number of synapses in mouse embryonic cortical neurons and the growth of new axons.In drosophila embryos,hnRNPQ regulates cytoplasmic vesicle-based messenger RNA(mRNA)transport.At the molecular level,hnRNPQ regulates the editing,transport,translation and degradation of mRNA,but there is currently no research on it in gliomas.DHX9(DExH-box Helicase 9),also known as RHA(RNA Helicase A),is an ATP-dependent RNA helicase and a member of the helicase deah protein family.DHX9 contains 2 copies of the double-stranded RNA binding domain,the DEXH core domain and the RGG box.The RNA binding domain and the RGG cassette can affect and regulate RNA helicase activity.DHX9 is a multifunctional protein that can be involved in the regulation of DNA replication,transcription,translation,and processing and translocation of RNA,and plays an important role in dna damage repair and maintenance of gene stability.Because DHX9 can participate in multiple processes of cell activity,regulate gene expression at different stages,degrade abnormal polynucleotides,ensure the fidelity and efficiency of DNA replication,and maintain gene stability.Therefore,abnormal function of DHX9 protein will cause disease,and may even lead to tumor.DHX9 plays different roles in different tumors depending on the intracellular environment and which molecular chaperone interacts.VGF(Nerve Growth Factor Inducible)is a cDNA sequence rapidly induced and massively copied by Levi et al.After they treated pheochromocytoma cells(PC 12)with nerve growth factor(NGF)in 1985.The protein with 67KD encoded by this sequence is also called VGF.Previous studies have shown that this secreted neuropeptide is involved in energy homeostasis,neurogenesis,synaptogenesis and the development of mental illness.In recent years,some studies have suggested that VGF also plays an important role in the pathogenesis of tumors.In our current study,lncRNA expression profiling was performed on glioma tumor tissues and corresponding tumor surrounding and normal brain tissues.We analyzed the chip results,screened and identified the up-regulated lncRNA NONHSAT069243,which we named HDAR(hnRNPQ-DHX9 Associated RNA)as the research goal.Subsequent experiments in vivo and in vitro demonstrated that low expression of lncRNA HDAR can inhibit the proliferation of glioma and inhibit the invasion and metastasis of gliomas.In addition,our study shows that lncRNA HDAR can directly interact with RNA helicase A(DHX9).heteronuclear nuclear ribonucleoprotein Q(hnRNPQ),and promote the migration and invasion of gliomas by promoting the stability of mRNA VGF.Research methods1.Defining the expression status and cellular localization of lncRNA HDAR in glioma tumor tissues and glioma cell lines.We collected different grades of glioma tumor tissues,used Q-PCR to detect the expression of lncRNA.and analyzed the effect of the expression level on the prognosis and survival cycle of glioma patients.The expression position of lncRNA HDAR in the glioma cell line U87,U251 was detected by in Fluorescent In Situ Hybridization(FISH).2.Study the effect of lncRNA HDAR on the biological function of glioma cells(1)The effect of lncRNA HDAR on the proliferation of glioma.Cell-value-adding ability was measured using the previously constructed stable cell line using cck8 colorimetric assay and edu incorporation assay,respectively.(2)The effect of lncRNA HDAR on the invasion and migration of glioma.The effect of lncRNA HDAR knockout on invasion and migration of glioma cells was examined using a previously established stable cell line using transwell migration assay and matrigel invasion assay.3.Animal experimentTwenty nude mice were randomly divided into two groups,and U87NC cells and U87siHDAR cells were collected,respectively.U87NC cells and U87siHDAR cells were respectively collected for intracranial injection in nude mice for in situ tumor formation experiment.The survival conditions of 10 nude mice in each group were observed,and brain tissue samples were taken from the mice after execution,and then dehydrated and fixed for HE staining.1.Explore the mechanism of lncRNA HDAR on the development of glioma(1)Using the RNA pull down method,the captured proteins were identified by mass spectrometry.(2)Western Blot was performed to detect the pull down results of RNA using protein-corresponding antibodies.(3)The above experimental results were verified by RNA pull down experiment and RIP experiment.(4)LncRNA HDAR fragment sequences were constructed,and the RNA pull down experiment was repeated to determine the binding sites of IncRNA HDAR and downstream proteins.(5)To explore the downstream targets of lncRNAHDAR,hnRNPQ and DHX9 protein complex regulation.(6)Q-PCR and other methods were used to verify the downstream targets.Research result1.Q-PCR showed that the expression of lncRNA HDAR was significantly increased in glioblastoma tumor tissues,and FISH experiment showed that IncRNA HDAR was expressed in the nucleus and cytoplasm of glioma cells.2.(1)Compared with the control group,the cck8 value of cells in the IncRNA HDAR knockout group was significantly reduced at 24h,48h and 72h,indicating that IncRNA HDAR knockout could inhibit the proliferation of glioma cells.The EDU results also showed that the proportion of positive cells in the lncRNA knockout group decreased.(2)Transwell migration assay and Matrigel invasion assay showed that the number of cells passing through the chamber in the lncRNA HDAR knockout group was significantly reduced,indicating that the ability of cell migration and invasion was reduced.3.In vivo experiments also proved that lncRNA HDAR knockout can inhibit the proliferation of glioma,thus slowing down the growth of subcutaneous tumors.4.RNA pull down experiment proved that hnRNPQ and DHX9 bind to IncRNA HDAR chain at different positions.5.LncRNA HDAR binds hnRNPQ-DHX9 complex to regulate the stability of VGF mRNA.Conclusion1.The expression of IncRNA HDAR in glioma showed a significant upward trend.2.LncRNA HDAR promotes the proliferation,invasion and migration of glioma cells.3.Physical binding of lncRNA HDAR with hnRNPQ-DHX9.4.LncRNA HDAR knockout can improve the stability of VGF mRNA and slow down the degradation of VGF.