Long Noncoding RNA LINC00460 Targets MiR-539/MMP-9 To Promote Meningioma Progression And Metastasis

Meningioma is one of the most frequent primary tumors occuringin central nervous system,and accounts for about 30%~40% of intracranial tumors,whereas,the statistics was13%~26% in ten years ago.Meningioma is a type of common neoplasms originated from the meningeal coverings of the brain or meningeal spaces.According to the classification of World Health Organization(WHO),meningioma is classified into WHO grade I,grade II,and grade III.Although large portion of meningioma is classified as benign tumors and small portion of meningioma is classified as malignant onesin primary qualitative classification,clinical research still found that malignant meningiomas always occur accompanied by rapid growth,invasive and metastasis.In recent years,although significant progress have achieved on diagnosis and treatment for meningioma,the recurrence rate is still pessimistic due to the ambiguous pathogenesis.Among the transcriptome,the vast portion of products is the non-coding RNAs(ncRNAs),besides,these ncRNAs are divided into short non-coding RNAs and long non-coding RNAs(lncRNAs)according to their length.Increasing evidences have manifested the extensive biological role of lncRNAs on multiple tumor pathological process,for example proliferation,invasion,metastasis and recrudesce.Although the studies on meningiomas has a deep exploration,only few lncRNAs have been found to be associated with the occurrence and development of meningiomas,and more and more studies are needed to explore lncrnas involved in the regulation of the pathological process of meningiomas.LncRNA LINC00460 has been reported to act as oncogenic RNA in several human cancers,e.g.lung cancer,nasopharyngeal carcinoma,ovarian carcinoma,thyroid cancer,stomach carcinoma,colorectal carcinoma,sophageal carcinoma,laryngeal squamous cell carcinoma.However,the role of lncRNA LINC00460 in meningioma is still unclear.In consideration of the vital role of ncRNAs in the tumorigenesis of other types of tumors,we choose LINC00460 as the target and explore its role in meningioma cell phenotype.We performed series of validation experiments to verify the underlying role of LINC00460 in meningioma tumorigenesis.We found the aberrant high-expression of LINC00460 in meningioma tissue samples and cells.In addition,miR-539 has also been proved to be a tumor suppressor in a variety of carcinomas,and its expression in glioma cell lines and tissues decreased,while overexpression of miR-539 significantly inhibit the proliferation and invasion of glioma cells.By using bioinformatics,it was found the binding sequence between LINC00460 and miR-539.So it was aimed to explore the mechanism of LINC00460 by targeting miR-539/MMP-9 in meningioma.OBJECTIVE:1.In the present study,we firstly investigated the roles of lncRNA LINC00460 in meningioma tissuesand malignant meningioma cell lines,and uncover the correlation of LINC00460 expression with the malignant characteristics of meningioma.2.Then we study the effect of lncRNA LINC00460 on meningioma cell lines by using LINC00460 deletion assays.3.According to the theory of competitive endogenous RNA,the binding sequence of LINC00460 with its targetedmicroRNA,miR-539,was analyzed by bioinformatics analysis.And their correlation was explored in vitro in malignant meningioma cell lines by using luciferase assay and other assays.It was aimed to reveal the molecular mechanism of LINC00460 promoting malignant transformation of meningioma.METHODS:1.33 samples from meningioma and 18 samples from normal meningestissues were collected.21 of 33 were WHO I meningioma,9 of 33 were WHO II meningioma,and 3 of33 were WHO III meningioma.Then the relative expression levels of LINC00460 in the two kinds of tissues were detected by RT-qPCR.2.The relative expression levels of LINC00460 in benign meningioma cell line Ben-Men-1 andmalignant meningioma cell lines(IOMM-Lee and CH157-MN)were detected by RT-qPCR.3.Three LINC00460 siRNA duplexes were designed and transfected into malignant meningioma cell lines(IOMM-Lee and CH157-MN)using Lipofectamin 2000.The downregulated level of LINC00460 was detected by RT-qPCR after48 h transfection,and we selected the best one with the highest knockdown efficiency.4.LINC00460 siRNAwas transfected into IOMM-Lee cells using Lipofectamin 2000,and the cells transfected with scramble siRNA was used as the control group.CCK-8 was used to determine the cell proliferation from 1 day to 5 day with detection every 24 h.Annexin V-FITC/propidium iodide(PI)and flow cytometry was used to detect the apoptotic cells.The percentage of EdU positive cells after 48 h transfection was counted with EdU staining to evaluate the DNA replication.5.LINC00460 siRNAwas transfected into IOMM-Lee cells using Lipofectamin 2000,and the cells transfected with scramble siRNA was used as the control group.After 24 h transfection,the cells were seeded into the upper chamber of the Transwell system coated with Martrigel.Serum-free DMEM and DMEM containing 20% serum were added into the upper and lower chamber,respectively.After 24 h culture,crystal violet staining was used to visulize the invasive cells.RT-qPCR and western blot were used to detect the mRNA and protein expression levels of invasion-related molecules(MMP-2,MMP-9,ZEB1),respectively.6.The nude mice bearing tumor of IOMM-Lee cells were treated with scramble siRNAor LINC00460 siRNAsubcutaneously for 4 weeks with 2 ?g of siRNA per injection once every other day.The tumor growth curve and the expression level of Ki-67 in tumor were detected by tumor volume measurement and immunohistochemistry.7.Bioinformatics software RNA22 version 2.0 was used to predict the potential binding sites between LINC00460 and miR-539,as well as miR-539 andMMP-9 3'-UTR.The luciferase reporter gene pGL3 vectors were constructed with mutant or wildLINC00460,or mutant or wild MMP-9 3'-UTR.Then the pGL3 vectors were co-transfected with miR-539 mimic or miR NC.Luciferase activity was detected by using luciferase substrate.8.LINC00460 siRNA was transfected into IOMM-Lee cells using Lipofectamin2000,then miR-539 andMMP-9 mRNA were detected by RT-qPCR after 48 h transfection.miR-539 inhibitor was transfected into IOMM-Lee cells using Lipofectamin2000,and LINC00460 andMMP-9 mRNA were detected by RT-qPCR after 48 h transfection.Furthermore,LINC00460 siRNA was co-transfected with miR-539 inhibitor,and the protein expression of MMP-9 was detected by western blot to observe the reversal effect of LINC00460 siRNA on miR-539 inhibitor.RESULTS:1.Compared with normal meninges tissues,the expression level of LINC00460 in human meningioma tissues was significantly higher.It indicates that LINC00460 is correlated with the occurrence of meningioma.2.In order to investigate the correlation between LINC00460 and the tumorigenesisof meningioma in vitro,and to confirm a appropriate cell model for exploring the in vitro effect and mechanism study,we detected the expression levels of LINC00460 in one benign meningioma cell line and two malignant meningioma cell lines.Compared withbenign meningioma cell line,the expression levels of LINC00460 in two malignant meningioma cell lines were significantly higher.It suggests that LINC00460 hasthe correlation with the malignant occurrence of meningioma cells.IOMM-Lee andCH157-MN could be used in cellular assays.3.Since LINC00460 expression is associated with the occurrence of meningioma,we intend to delete LINC00460 to observe the effect of LINC00460 on malignant meningioma cell lines in vitro.Firstly,the effects on cell proliferation and the related mechanism were observed.When LINC00460 level was decreased by siRNA,the proliferation of IOMM-Lee and CH157-MN cells were inhibited.On the fifth day,the cell proliferation abilities were the most obvious between the LINC00460 knockdown group and the non-transfection cell(p <0.05).When LINC00460 expression was inhibited for 48 h,apoptotic cells in bothIOMM-Lee andCH157-MN cell lines were significantly increased(p < 0.05).The apoptotic rate of IOMM-Lee cell line increased from(9.8%±0.8%)to(22.5%±2.7%),and the apoptotic rate of CH157-MN cell line increased from(8.8%±0.7%)to(18.2%±2.1%),suggesting the statistically significant difference.In addition,it was also found thatLINC00460 knockdown reduced the DNA replication levels of the two malignant meningioma cell lines.The proliferative cell number in IOMM-Lee cell line was reduced by51.5%,and the proliferative cells in CH157-MN cell line was reduced by 67.3%(p < 0.05).According to these results,the expression of LINC00460 was related to the proliferation of malignant meningioma cells,and the inhibition to DNA replication and upregulation to cell apoptosis might play an anti-proliferation role,and LINC00460 knockdown can reduce the malignant proliferation of meningioma cell lines.4.Then the relationship between LINC00460 and invasion of malignant meningioma cell lines was observed.By using the knockdown assays,when LINC00460 expression was reduced,the invasion abilities of IOMM-Lee and CH157-MN cells were inhibited.The number of cells attached to the inferior wall was significantly reduced in Transwell chamber by LINC00460 depletion.The invasion number of IOMM-Lee cell decreased from(56.4±6.2)to(22.1±2.4),and The invasion number ofCH157-MN cell decreased from(46.9±5.3)to(17.8±3.2).At the same time,the invasion related gene and protein expression levels were significantly decreased.These results suggested that LINC00460 expression is related to the invasion of malignant meningioma cells,and it is speculated that the extracellular matrix and basement membrane could be degraded by increasing MMP-2 and MMP-9,and the expression of ZEB1 transcription factor.It is speculated thatepithelial-mesenchymal transformation might be correlated with LINC00460 expression.5.In addition,we intend to verify the in vivo effect of LINC00460 on meningioma.Due to the relatively high malignancy of IOMM-Lee cells,the tumor of nude mice bearing IOMM-Lee cells were established and LINC00460 siRNA was used to inhibit the expression of LINC00460 in tumor.The results showed that LINC00460 inhibition in the tumor of nude mice could reduce the tumor growth ability and the expressionlevel of Ki-67 within 28 days.It indicated that LINC00460 siRNA could inhibit the growth of meningiomas in vivo,and confirmed that LINC00460 is correlated with the growth and cell proliferation of meningiomas in vivo.6.The binding sequences between LINC00460 and miR-539,and miR-539 and MMP-93'-UTR were detected by bioinformatics assays.After gene mutation at the binding sequences,the luciferase reporter gene vector was constructed.After co-transfection with a miR-539 mimic or miRNA NC,it was found that only wild-type LINC00460 could bind to miR-539,and only wild-type MMP-93'-UTR could directly bind to miR-539.It was confirmed that miR-539 was a miRNA for connecting LINC00460 and MMP-9.7.After confirming the direct binding between LINC00460 and miR-539,and miR-539 and MMP-93'-UTR,the interaction between LINC00460 and miR-539 and their influence on MMP-9 were further studied to reveal the existence of LINC00460/ miR-539/ MMP-9 axis in meningiomas.In meningioma cells,when LINC00460 level was inhibited,miR-539 was up-regulated,while MMP-9 was decreased.When miR-539 level was inhibited,theexpressions of both LINC00460 and MMP-9 were up-regulated.It indicated that there was interaction between LINC00460 and miR-539,and MMP-9 was the downstream gene of LINC00460/miR-539.Furthermore,miR-539 inhibitor was used to reduce miR-539 expression,then LINC00460 siRNA was used to reduce the effect of miR-539 inhibitor.It showed that miR-539 inhibitor downregulated the expression of miR-539 and upregulated the expression of MMP-9 protein,however,LINC00460 siRNA reversed the effect of miR-539 inhibitor.It indicated that LINC00460 was a competitive endogenous RNA of miR-539,blocked the regulation of miR-539 inhibitor to MMP-9 mRNA.This resultconfirmed the existence of LINC00460/ miR-539 / MMP-9 axis in meningioma cells.CONCLUSION:1.LINC00460 was significantly up-regulated in meningioma tissues and malignant meningioma cell lines,indicating that LINC00460 was associated with the malignant characteristics of meningioma.2.LINC00460 down-regulation can inhibit the proliferation and invasion of meningioma cells in vitro,and reduce the expression of MMP-2,MMP-9 andZEB1.3.LINC00460 down-regulation can inhibit the growth ability of meningioma tumors in vivo.4.LINC00460 promotes the proliferation and metastasis of meningiomas by targeting miR-539 and up-regulating MMP-9 mRNA.