A Novel LncRNA ARST Represses Glioma Progression By Inhibiting ALDOA-mediated Actin Cytoskeleton Integrity

As a representative of central nervous system malignant tumors,glioma has become the focus in recent years.The clinical features of gliomas are occult onset,rapid progress,poor prognosis and high mortality.The typical behavioral characteristics of gliomas are strong invasiveness in brain tissue and high recurrence rate after operation.Therefore,it is crucial to study the mechanism of glioma progress intracranially which can further consummate the treatment of glioma and improve the prognosis of glioma patients after neurosurgery.Cytoskeleton whose integrity is crucial to the proliferation,migration,invasion and apoptosis of glioma cells is an important factor in regulating the activities and dynamics of cellular proliferation.It is known that actin filament is the main component of cytoskeleton.Under normal conditions,the depolymerization of old actin filaments and the polymerization of new actin filaments maintain a dynamic balance,which is conducive to cellular migration and invasion.As a member of anaerobic metabolic enzyme family,aldolase A(ALDOA)has the capacity to combine the gamma-actin(G-actin),promoting its mutual polymerization process in order to form the fibrous actin(F-actin)besides the metabolic enzyme functions which are widely understood.If the interaction between ALDOA and G-actin is blocked,the polymerization of F-actin will be downregulated and reversed to form monomer G-actin.Therefore,the accumulation of the monomer G-actin in cells will be used to promote the assembly of new F-actin fibers.Moreover,cofilin,one kind of actin depolymerization protein(ADP),plays an important role in the depolymerization of actin filaments.In our previous high-throughput microarray data of glioma clinical specimens,it was found that a novel long non-coding RNA NONHSAT138818.2(lncRNA ARST)which was not registered in Ensemble and TCGA Database.In our study,we mainly focused on the specific mechanism of lncRNA ARST and the regulation of non-enzymatic functions of ALDOA affecting the stability of glioma cytoskeleton.OBJECTIVE:1.To study the expression and epidemiological characteristics of lncRNA ARST in glioma.2.To verify the biological effect of lncRNA ARST in vitro and vivo experiments.3.To clarify the effect of lncRNA ARST on the biological characteristics of aldolase A during the process of malignant behavior change of glioma cells.4.To study the detail molecular biological mechanism of lncRNA ARST affecting the invasion and migration of glioma cell lines by changing the biological characteristics of aldolase A.METHODS:Part ?:1.Microarray data were utilized to screen the differentially expressed lncRNA ARST in clinical glioma samples,and to compare the expression levels of lncRNA ARST in normal tissues and adjacent tissues.2.To verify the conservation of lncRNA ARST.3.qRT-PCR was performed to detect the expression levels of lncRNA ARST in different pathological grades of glioma tissues and different glioma cell lines.4.To analyse the epidemiological characteristics of lncRNA ARST.GEPIA database was applied for obtaining the effect of LINC00632(ARST maternal transcript)expression level on the survival analysis of patients with glioma.Part II:1.Fluorescence in situ hybridization(FISH)was performed to detect the subcellular localization of lncRNA ARST in glioma cell lines U87MG and U251.2.Establishment of the glioma cell model with lncRNA ARST overexpression.3.To study the effects of lncRNA ARST on the proliferation,migration,invasion and apoptosis of glioma cells.3.1 CCK-8 and EdU techniques were performed to detect the effect of lncRNA ARST on the proliferation of glioma cells after overexpression.3.2.Transwell assay and scratch test were performed to detect the effect of lncRNA ARST on the invasion and migration movements of glioma cells.3.3.Annexin V-FITC/PI staining and flow cytometry assay were utilized to detect the effect of lncRNA ARST overexpression on apoptosis of glioma cells.4.The lncRNA ARST plasmid containing luciferase marker was designed and transfected into gliomas cell line GL261.The effects of lncRNA ARST on the progression of gliomas were detected in vivo by using the luminescent imaging device of small animals after glioma cells intracranially implantation in C57 mice.Part ?:1.To identify the interaction protein and functional localization of lncRNA ARST.The total protein solution of U87MG was extracted for ARST RNA pull-down assay,and the protein combined lncRNA ARST was obtained and listed by mass spectrometry,clarifying the interaction between lncRNA ARST and aldolase A(ALDOA).2.The binding region between lncRNA ARST and ALDOA was verified.Based on the stem-loop structures,ARST was segmented in order to explore the binding region of ALDOA on lncRNA.3.To explore the effect of lncRNA ARST overexpression on ALDOA expression in U87MG.4.To analyse the epidemiological characteristics of ALDOA.Part ?:1.To clarify the mechanism of lncRNA ARST on aldolase A.1.1.To explore the related effects of ARST combined with aldolase A on cell life activities utilizing the core module analysis method in Metascape database.To analyse the metabolic level of U87MG after ARST overexpressed.1.2.To analyse the differences in the cytoskeleton structure of U87MG glioma cells in different treatment groups by confocal laser imaging technique.1.3.To analyse the differences in aldolase A localization and distribution of U87MG glioma cells in different groups by immunofluorescence and cytoskeleton co-staining assay after lncRNA ARST overexpression.1.4.To analyse the interaction capacity changes between aldolase A and F-actin after lncRNA ARST overexpression by immunoprecipitation assay.2.To analyse the changes of cofilin after lncRNA ARST overexpression.2.1.To analyse the interaction changes between cofilin and F-actin byimmunoprecipitation assay after lncRNA ARST overexpression.2.2.To analyse the differences in protein localization and distribution of cofilin and Factin in different U87MG groups by immunofluorescence and cytoskeleton costaining.2.3.To analyse cofilin epidemiological features utilizing TCGA database.3.To explore the interaction mechanism of F-actin,aldolase A,cofilin and ARST.3.1.To predict the binding domain of aldolase A and lncRNA ARST by catRAPID database.3.2.To detect the binding capacity changes of aldolase A and ARST with aldolase A mutant plasmids by RNA pulldown assay.3.3.To analyse the interaction status changes between F-actin and aldolase A(or cofilin)after different aldolase A mutant plasmids transfection.4.Rescue experiments.RESULTSPart ?:1.Microarray data were applied for screening the differential expression of lncRNA ARST in clinical glioma samples.The expression levels of ARST in normal tissues and tumor adjacent tissues were both lower than that in glioma tissues.2.According to the relevant information of NCBI and UCSC database,it was found that ARST,which encoded no polypeptides with biological significance,was consistent with chain RNA.3.qRT-PCR was performed to detect the expression of lncRNA ARST in different pathological grades of glioma tissues and different glioma cell lines.The results demonstrated that the ARST expression level decreased with the raise of glioma clinical grade.4.GEPIA database was used to obtain the effect of LINC00632 gene expression level on the survival time of patients.The results showed that the higher malignant degree of glioma,the lower expression level of LINC00632;The expression level of LINC00632 is positively correlated with the prognosis of glioma patients.Part ?:1.Fluorescence in situ hybridization was performed to observe the distribution of lncRNA ARST which was mainly in the cytoplasm of glioma cell lines U87MG and U251.2.To establish a glioma cell model with lncRNA ARST overexpressing.The lncRNA ARST overexpression plasmid was constructed.Glioma cell lines U87MG and U251 were selected for transient transfection.The transfection efficiency of transfected glioma cells was determined and labeled as oeARST.3.To study the effects of lncRNA ARST on the proliferation,migration,invasion and apoptosis process of glioma cells.3.1 CCK-8 and EdU assays showed that lncRNA ARST overexpression could effectively inhibit the proliferation of glioma cells.3.2 Transwell assay and scratch test demonstrated that invasion and migration of glioma cells was inhibited with lncRNA ARST overexpression.3.3 Annexin V-FITC/PI staining and flow cytometry were performed to indicate that glioma cell apoptosis process was promoted after lncRNA ARST overexpression.3.4.Small animal luminescent imaging system was utilized for showing that overexpression of lncRNA ARST could effectively inhibit the progression of glioma in C57 mice.Part ?1.To identify the interaction protein and functional localization of lncRNA ARST.The total protein solution of U87MG was extracted for RNA pull-down experiments.The interacting proteins of lncRNA ARST were obtained by mass spectrometry and sequenced according to the correlation score.The binding relationship of ARST to aldolase A was discovered.2.The binding domains between lncRNA ARST and aldolase A were verified.Based on the stem-loop structure,lncRNA ARST was segmented into 5 segmentations in order to be explored the binding region of ALDOA.3.To demonstrate the effects of lncRNA ARST overexpression on ALDOA expression in U87MG.Western blot and qRT-PCR results showed that lncRNA ARST could not affect the expression of ALDOA.4.To obtain the epidemiological characteristics of ALDOA.The expression level of ALDOA in different grades of gliomas and the effects of ALDOA expression level on the survival time of glioma patients were obtained by GEPIA database.However,with higher expression level of ALDOA,the glioma patients would obtain the worse prognosis.Part ?1.To explore the mechanism of lncRNA ARST on ALDOA.The interaction proteins obtained by mass spectrometry were classified into core modules,and the cell dynamics function mediated by the cytoskeleton was determined to be related to the effect of lncRNA ARST on aldolase A.1.1.Lactate level and metabolic enzymes did not change significantly after ARST overexpression.1.2.After cytoskeleton staining experiments,the laser confocal imaging system was utilized to detect the results.It was observed that the function of lncRNA ARST was destroying the cytoskeleton structures and interfering with the process of new cytoskeleton formation.1.3.The co-localization status of aldolase A and F-actin was destroyed after ARST overexpression.1.4.The results of co-immunoprecipitation demonstrated that lncRNA ARST could effectively inhibit the binding ability of ALDOA to F-actin.2.To explore the changes of cofilin after lncRNA ARST overexpression.2.1.The results of co-immunoprecipitation assay showed that the binding capacity of cofilin and F-actin was inhibited after lncRNA ARST overexpression.2.2.The results of cytoskeleton and immunofluorescence co-staining demonstrated that after overexpression of lncRNA ARST,the binding of cofilin and F-actin was weakened,the fibrous structure of F-actin was destroyed,new F-actin could not be formed,and cofilin could not bind to the remaining skeleton structure and showed irregular free The phenomenon of distribution.2.3.Results in TCGA database demonstrated that cofilin was highly expressed in glioma cells,while the expression level was negatively correlated with prognosis.3.The interaction mechanism of F-actin,aldolase A,cofilin and ARST.3.1.It was predicted that 289-340 amino acid fragment of aldolase A was the binding domain on lncRNA ARST according to the catRAPID database.However,this specific domain overlapped with the structure of aldolase A binding to F-actin in the former researches.3.2.The results of RNA pulldown showed that the mutant plasmid aldolase A(ALDOA 78-364AA)could interact with ARST,which demonstrated that the binding regions of F-actin or lncRNA ARST on aldolase A both included the 289-364AA fragment.3.3.In glioma cells,F-actin fibers are depolymerized by cofilin into G-actin monomer.However,overexpression of ARST inhibits the combination of aldolase A and F-actin,which makes it difficult to form F-actin fibers.Therefore,the binding capacity of cofilin/aldolase A to F-actin is weakened with monomer G-actin accumulating.4.The results of rescue experiments are consistent.CONCLUSION1.In glioma samples,the expression of lncRNA ARST was significantly decreased,and the higher grade of glioma obtained the lower ARST expression level.The expression of lncRNA ARST was positively correlated with the prognosis.2.Overexpression of lncRNA ARST could significantly inhibit the proliferation,invasion and migration of U87MG,and promoted apoptosis process of glioma cells.3.Overexpression of lncRNA ARST could not affect the expression level of ALDOA.However,ARST could effectively occupy the binding region of ALDOA and cytoskeletal protein F-actin.Moreover,cofilin,a cytoskeleton depolymerizing protein with overlapping localization of ALDOA on F-actin,could bind to F-actin more efficiently instead,playing the role as a depolymerizing factor to destroy the skeleton structure after ARST overexpression.4.LncRNA ARST,which occupies the F-actin binding region of ALDOA,could inhibit the cytoskeleton stabilizing function of ALDOA,resulting in cofilin continuously destroying the old cytoskeleton without new cytoskeleton structures forming after depolymerization process.Such effect could inhibit malignant activities of glioma cells,especially the invasion and migration abilities.