| Backgrounds: Glioblastoma is a common central nervous system malignancy that seriously affects patients and their families and brings a heavy psychological and economic burden on them.With advances in molecular diagnostics,more and more studies have found that long non-coding RNAs play an important role in cancer initiation and progression.We have found that long non-coding RNA-LINC00978 is upregulated in severe cancers and participates in and regulates cancer initiation and progression.However,the key biological functions and mechanisms of LINC00978 in the development and progression of glioblastoma have not been fully elucidated.Purposes : Identification of biological functions and possible mechanisms of long non-coding RNA-LINC00978 involved in glioblastoma initiation and progression.Provide a theoretical basis for molecular diagnosis and treatment of glioblastoma and further explore potential drug targets for glioblastoma.Part Ⅰ Expression of LINC00978 in Glioma Tissue and Cell Line and Its Clinical SignificanceMethods:(1)LINC00978 was selected as the gene to be studied by screening genes with differential expression based on RNA-seq transcriptome sequencing data from our previous study of glioma tissue samples.(2)LINC00978 expression levels were measured by RT-q PCR in glioma tissues at different levels.Kaplan-Meier method was used to analyze the prognostic relationship between LINC00978 and glioma patients.Bioinformatics was used to validate LINC00978 expression levels and patient prognostic relationships in the TCGA and CGGA databases,respectively.(3)LINC00978 expression levels in glioblastoma cell lines T98 G,LN229,and U251 were assessed by RT-q PCR.Identification of LINC00978 in glioma cell lines by FISH assay.Results:(1)Results from RT-q PCR assays showed that LINC00978 expression in gliomas increased with the progression of the World Health Organization Central Nervous System tumor hierarchy,and the higher the expression,the worse the prognosis.Similar results were obtained in the TCGA and CGGA data repositories.(2)LINC00978 was expressed in both glioblastoma cell lines and was highly expressed in U251 cell lines.The FISH assay revealed that LINC00978 is expressed in the nucleus and cytoplasm of glioblastoma cells.Part Ⅱ Effects of LINC00978 on Proliferation,Apoptosis,Migration,and Invasion of GlioblastomaMethods:(1)Instant lipofactamine3000 transfection of si RNA silenced LINC00978 and RT-q PCR was used to detect LINC00978 expression after transfection.(2)The ability of glioblastoma cells to proliferate and colony after silencing LINC00978 was measured by CCK-8 and panel clonogenicity assays.(3)Flow cytometry assessed the Effects of silencing of LINC00978 on cell apoptosis and cell cycle of glioblastoma.(4)Effects of silenced LINC00978 on glioblastoma cell line migration and invasion ability were measured by Transwell assay.(5)Effects of silenced LINC00978 on glioblastoma proliferation and survival in nude mice using nude mouse models of intracranial tumorigenesis.Results:(1)Results showed that transfection of si RNA-LINC00978 reduced the expression of LINC00978 in glioblastoma cells compared to the negative control group.(2)Cell proliferation was inhibited by silencing of LINC00978 by CCK-8 and panel clonogenicity assays.(3)Flow cytometry revealed that silencing of LINC00978 promoted apoptosis in glioblastoma cells,but had no significant effect on cell cycle.(4)Transwell results showed that silencing LINC00978 inhibited glioblastoma cell migration and invasion.(5)Intracranial orthotopic tumorigenesis in nude mice showed that silencing LINC00978 inhibited glioblastoma formation in vivo,and Kaplan-Meier analysis showed that silenced LINC00978 nude mice lived longer than negative controls,with statistically significant differences.Part Ⅲ.LINC00978 Promotes Glioblastoma Proliferation by Positive Regulation of AKR1B1 Expression.Methods:(1)We selected AKR1B1 for study using bioinformatics analysis of potential target genes and pathway enrichment downstream of LINC00978.(2)Bioinformatics and RT-q PCR were used to analyze AKR1B1 expression levels in gliomas at different WHO grades in TCGA,CGGA databases,and glioma tissues.To analyze the prognostic relationship between AKR1B1 and glioma patients.(3)The correlation between LINC00978 and AKR1B1 expression levels was analyzed in the TCGA and CGGA databases and glioma tissues,validated by immunofluorescence and fluorescence in situ hybridization.Silencing of LINC00978 affected AKR1B1 expression using RT-q PCR.(4)AKR1B1 overexpression plasmids were constructed and AKR1B1 expression levels were measured by RT-q PCR and Western Blot.(5)The effects of silenced AKR1B1 on glioma cell proliferation were detected by CCK-8 and the effects of silenced LINC00978 and rescued AKR1B1 on glioma cell proliferation and ATP synthesis by Ed U and ATP assay kit.Results:(1)Bioinformatics analysis and RT-q PCR assays result showed that AKR1B1 expression in glioma increased with the grade of WHO classification in TCGA,CGGA database,and glioma tissues.The expression of AKR1B1 was associated with poor prognosis in glioma patients.(2)Correlation analysis revealed a positive correlation between AKR1B1 and LINC00978 expression.RT-q PCR results showed that silencing of LINC00978 reduced AKR1B1 expression,whereas silencing of AKR1B1 did not affect LINC00978 expression in glioblastoma.(3)AKR1B1 overexpression plasmids were detected by RT-q PCR and Western Blot to increase AKR1B1 expression in glioma cells.(4)Silencing AKR1B1 inhibited glioblastoma proliferation by CCK-8 assay,Ed U,and ATP assay showed that silencing LINC00978 reduced cell proliferation and overexpress AKR1B1 rescued silencing LINC00978 from inhibiting cell proliferation and ATP synthesis.Part Ⅳ Involvement of LINC00978 in AKR1B1-mediated sensitivity to 2DG in glioblastoma.Methods:(1)The expression levels of AKR1B1 and LINC00978 in glioblastoma cells treated with 2DG were detected by RT-q PCR,along with chronological relationships.(2)AKR1B1 expression in glioblastoma cells treated with 2DG was detected by RT-q PCR after silencing of LINC00978.(3)Effect of knockdown of LINC00978 on sensitivity of glioblastoma to 2DG by CCK-8 assay.(4)Bioinformatics predicted and RT-q PCR validated transcriptional regulators upstream of LINC00978.Results:(1)CCK-8 results showed that glioblastoma was sensitive to 2DG toxicity,and cell activity decreased with increasing 2DG concentration.(2)RT-q PCR showed that 2DG induced upregulation of AKR1B1 and LINC00978,with LINC00978 expressing upregulation first.Silencing of LINC00978 inhibited 2DG upregulation of AKR1B1 expression.(3)Silencing of LINC00978 by CCK-8 assay reduced sensitivity of glioblastoma cells to 2DG.(4)NRF2 is an upstream transcription factor that regulates the expression of LINC00978.Conclusions:(1)LINC00978 is highly expressed in glioblastoma and its high expression is associated with poor prognosis in glioblastoma patients.(2)Knocking down LINC00978 significantly inhibits glioblastoma proliferation,clonogenicity,migration,invasion,and intracranial tumorigenesis,and promotes apoptosis.(3)LINC00978 positively regulates AKR1B1 expression in glioblastoma.High expression of LINC00978 promotes 2DG sensitivity in glioblastoma cells.There are 44 figures,10 tables,and 117 references... |
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