| IntroductionGlioma,the most frequent and most malignant primary brain tumor in adults,is characterized by high capacity for invasion,proliferation,and recurrence.Despite multiple therapeutic approaches comprising surgery,chemotherapy,and radiotherapy have been applied to prolong the survival time of patients with glioblastoma(GBM),the median survival was only 15 months.In addition to the traditional treatment methods,many studies focusing on the molecular mechanisms of malignant gliomas have been performed in last several years.Abundant evidence confirms that glioma cells undergo heterogeneous genetic molecular aberrations.Hence,numerous genes have been certified as regulator involving in glioma cells proliferation,invasion and apoptosis,which have non-coding RNA molecule such as MicroRNA-184,miR-218-5p,MicroRNA-16 and lncRNA-MALAT1,and coding RNA molecule such as AKT,PTEN and NFKBIA.Non-coding RNAs(nc RNAs)dysregulation are involved in various tumors progression.ncRNAs mainly comprise of two parts: long non-coding RNA molecule(lncRNAs)with an approximate length of >200 nucleotides and small non-coding RNA molecules(microRNAs)with an approximate length of 19-25 nucleotides.Compared to the exploration of microRNAs,lncRNAs have not been sufficiently studied,but partial evidence informs us that lncRNAs exert regulatory functions in different types of tumors,which includes stimulating tumor growth or suppressing tumor development.Similarly,as per some theory,lncRNAs play key roles in glioma progression[1].Therefore,lncRNAs research helps to explore potential therapeutic targets in different tumors,especially for glioma.Through the results of the study of early gene chip,we found a long non-coding RNA molecule,a member of lncRNAs family,named LncRNA-ZNF281 with 353 bp in LNCipedia.org,locates in chromosome 1q32.1 within nucleotides 200443207-20043559.ZNF281 maps to chromosome 1q32.1 However,the role of LncRNA-ZNF281 in human tumors,as well as in glioma cells,has not been studied thoroughly.In this study,we will explore structural feature and biological function of LncRNA-ZNF281 in glioma cells,and molecular mechanisms to influence glioblastoma process.MethodsHuman brain glioma tissue specimens were obtained from January 2010 through December 2013 at the Department of Neurosurgery of the First Affiliated Hospital of Soochow University.These samples came from 80 glioma patients including 28 cases of the low-grade tumor(WHO(grades II),52 cases of the high-grade tumor(WHO grades III and IV),and 5 patients that had presented with craniocerebral injuries whose brain tissues were partially resected to reduce intracranial pressure according to decompression treatment guidelines.In these glioma patients,there were 48 male patients(mean age at the time of surgery was 47.98 years,grade II,16 cases;grade III,17 cases;and grade IV,15 cases)and 32 women(mean age at the time of surgery was 48.91 years,grade II,12 cases;grade III,10 cases;and grade IV,10 cases).All tissue samples were immediately collected and stored in liquid nitrogen after resection from patients and then used for analysis.In addition,this study was authorized by the local ethics committee of our hospital.Real-time quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to analyze the expression of LncRNA-ZNF281 in 80 tissue specimens and 5 non-tumor brain tissues.qRT-PCR were used to detect the expression level of LncRNA-ZNF281,VEGF and MMP2 in lentiviral-lnc ZNF281-SHG139 and lentiviral-lnc ZNF281-U87,empty vector cells and untreated cells.And Infection efficiency was detected by fluorescence microscope and flow cytometer..In this study,we will probe the role of LncRNA-ZNF281 participating in glioma cells proliferation?invasion and apoptosis by Cell Counting Kit-8(CCK-8),transwell assay,migration assay and flow cytometry.Then,the protein levels of VEGF,MMP2,Caspase-3,BCL-2 and CyclinD1 were detected by Western blot analysis in increasing LncRNA-ZNF281 groups,negative groups and blank groups.In vivo,hematoxylin-eosin staining was used to prove existence of intracranial xenograft tumors in nude mice injected with SHG139 transfected with the lentiviral vector with LncRNA-ZNF281 simulative sequence or negative control oligonucleotide.Then Immunohistochemical(IHC)was used to analysed the protein expression levels of Ki-67,MMP 2,Caspase3 and VEGF.Results1.The expression level of LncRNA-ZNF281 in Microarray,glioma tissues and glioma cell linesThe result of Microarray analyzed comparing SHG139 cell samples with SHG139 S cell samples,showed that LncRNA-ZNF281 was differentially expressed gene between SHG139 and SHG139 S.The expression level of LncRNA-ZNF281 was measured by qRT-PCR.,The result revealed that LncRNA-ZNF281 expression significantly decreased in glioma tissues as compared with non-cancerous brain tissues.As expected,the expression levels of LncRNA-ZNF281 decreased gradually with the degree of tumor malignancy.Similarly,compared to non-cancerous brain tissues,LncRNA-ZNF281 expression was lower in glioma cell lines A172,U251,U87,HG44,SU2 and SHG139.2.Lentivirus infection efficiencyTo investigate the function of LncRNA-ZNF281 in glioma process,the lentivirus LncRNA-ZNF281 and Lnc-control were transfected into glioma cell lines U87 and SHG139.Fluorescence microscopy,qRT-PCR and flow cytometer were used to confirm that the expression level of LncRNA-ZNF281,which increased in lentiviral-LncRNA ZNF281-SHG139 and lentiviral-LncRNA ZNF281-U87 as compared to empty vector cells and untreated cells,and the lentivirus infection efficiency is more than 80%.3.Up-regulation of LncRNA-ZNF281 suppressed cell proliferation,invasion and migrationAfter being infected with the lentivirus-LncRNA-ZNF281,the growth curve of glioma cells transfected lentivirus-LncRNA-ZNF281 was significantly inhibited compared to empty vector cells and untreated cells.Flow cytometer was used to further study the cell cycle of different groups,The result showed that up-regulation of LncRNA-ZNF281 induced more cells to rest on G1/G2 phase and fewer cells in the S-phase than the negative control groups and blank groups without treatment.Enhancing expression level of LncZNF281 resulted in weakening the invasive ability of U87 and SHG-139 compared to negative control groups and blank groups.4.Over-expression of LncRNA-ZNF281 promoted glioma cells apoptosisFlow cytometer was employed to investigate the function of LncRNA-ZNF281 in glioma cells U87 and SHG-139.The data from flow cytometer revealed that the glioma cells transfected lentivirus-LncRNA-ZNF281 had higher apoptosis rates compared to empty vector or untreated cells.5.LncRNA-ZNF281 regulate VEGF and MMP2 levels in glioma cell linesVEGF was the key gene in tumor development,which can promote angiogenesis and provided nutrient to feed tumors.RNA and protein expression of VEGF was also examined by qRT-PCR and Western blots in glioma cells that were transfected with the lentiviral vector with LncRNA-ZNF281 simulative sequence,the negative control oligonucleotides and untreated cells.We found that over-expression of LncRNA-ZNF281 in glioma cells significantly reduced VEGF levels in mRNA and protein.Meanwhile,MMP2 was differentially expressed gene between SHG139 and SHG139 S according to the result of Microarray.And MMP2 participated actively in glioma growth process with LncRNA-ZNF281 qRT-PCR and Western blots were used to examine the RNA and protein expression of MMP2.We found that over-expression of LncRNA-ZNF281 in glioma cells significantly reduced MMP2 levels in mRNA and protein.4.LncRNA-ZNF281 regulated Caspase3 ?BCL-2 and Cyclin D1 levels in glioma cell linesBased on above-mentioned experimental result,we have reasons to believe that strengthening expression level of LncRNA-ZNF281 can suppressed cell proliferation and invasion,simultaneously promoted cells apoptosis.In order to satisfy our curiosity on molecular mechanisms how LncRNA-ZNF281 influenced glioma cell growth processes,we tried to explored the relationships between LncRNA-ZNF281 and relative representative genes Caspase3 ? BCL-2 and CyclinD1 in glioma cells process.By qRT-PCR and Western blot,the results showed that over-expression of LncRNA-ZNF281 reduced mRNA and protein expression level of BCL-2 and CyclinD1 as compared to empty vector or untreated cells.On the contrary,up-regulated LncRNA-ZNF281 significantly increased mRNA and protein expression of Caspase3 than empty vector or untreated cells.5.LncRNA-ZNF281 regulated VEGF?MMP2?Ki-67 and Caspase3 expressoin levels in vivoTo further explored whether LncRNA-ZNF281 similarly regulated glioma progression in vivo,we achieve xenograft experiments.We obtained tumors from the armpit subcutaneous area of the left forelimb of nude mice where SHG139 cell lines transfected with lentivirus-LncRNA-ZNF281 were injected.The tumor mass from mice injected with SHG139 cells that were transfected with lentivirus-LncRNA-ZNF281 was significantly smaller and lighter than in those mice that had been injected with SHG-139 cells transfected with lentivirus LncRNA-control vetor.Simultaneously,hematoxylin-eosin staining proved existence of intracranial xenograft in nude mice.The outcome of immunohistochemical(IHC)showed that Ki-67,MMP2 andVEGF was lower expression as compared the negative control groups,and Caspase3 was higher expression as compared the negative control groups.ConclusionLncRNA-ZNF281 expression was significantly decreased in glioma tissues and glioma cell lines as compared with non-cancerous brain tissues.And,the expression levels of LncRNA-ZNF281 decreased gradually with the degree of tumor malignancy.In vivo and invitro,LncRNA-ZNF281 suppressed glioma cells proliferation,invasion,angiogenesis and promoted glioma cells apoptosis by regulating the expressoin levels of VEGF?Ki-67?CyclinD1 ?MMP2?BCL-2 and Caspase3. |
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