NCBP3/SNHG6 Inhibits GBX2 Transcription In A Histone Modification Manner To Facilitate The Malignant Biological Behavior Of Glioma Cells

Objective: Malignant gliomas are the most common intracranial tumors.The conventional treatment is still surgical resection followed by chemoradiotherapy.However,treatment effect of patients remain poor,recurrence rates are high,and the five-year survival rate is less than 10% due to the glioma characteristics of strong invasiveness and aggressive growth.Therefore,the use of molecular biological methods to explore biological behaviors,such as proliferation,migration,invasion,and apoptosis,is expected to reveal new mechanisms of tumor occurrence and development,which is of profound significance for molecular targeted therapy.RNA-binding proteins(RBPs)interacting with RNA and play a decisive role in posttranscriptional expression regulation,such as RNA splicing,transport,and regulating the RNA stability and degradation.Nuclear cap-binding protein 3(NCBP3)is located on chromosome 17 and can bind to NCBP1 to form the cap-bingding complex(CBC).NCBP3 binds to the 5’ end of RNA,promoting RNA splicing and export.Long non-coding RNAs(lncRNAs)are a class of non-coding RNAs with transcripts more than 200 nt,which play a crucial regulatory role in the occurrence and development of many tumors.Small nucleolar RNA host gene 6(SNHG6)is located on human chromosome 8q13.1 and plays a decisive role in transcriptional regulation and epigenetics.Knockdown of SNHG6 significantly inhibited gastric cancer cell proliferation and epithelial-mesenchymal transition.Moreover,SNHG6 is highly expressed in gliomas,and knockdown of SNHG6 significantly inhibited cell proliferation and promoted cell apoptosis.A variety of lncRNAs can regulate gene transcription by recruiting polycomb repressive complex 2(PRC2).Enhancer of Zeste homolog 2(EZH2),as the key catalytic subunit of PRC2,can be catalyzed histone H3 at lysine 27 trimethylation(H3K27me3),thereby inhibiting gene transcription.In breast cancer,lncRNA-MEG3 binds to the PRC2 complex and forms an RNA-DNA triplex structure with target gene chromatin through the chromatin interaction sequence on MEG3,and finally generates H3K27me3 in the distal regulatory region to regulate transcription.Gastrulation brain homeobox 2(GBX2)is a transcription factor localized to human chromosome 2q37.2.Studies have found that GBX2 acts as a tumor suppressor in head and neck cancer.PRC2 is often located in Cp G-rich chromatin regions in mammals.In addition,there are Cp G islands in the GBX2 promoter region.Moreover,TSPYL2 and EZH2 can bind to the GBX2 promoter region,increasing the level of H3K27me3 enrichment and significantly reduce the expression of GBX2.Flotillin protein family 1(FLOT1)is located on chromosome 6q21.33 and plays a crucial role in vesicle transport and maintenance of cell morphology.FLOT1 can initiate receptor kinase signaling,and its expression is significantly up-regulated in malignant tumors including esophageal squamous cell carcinoma and liver cancer.Moreover,knockdown of FLOT1 can inhibit the proliferation and tumorigenesis of breast cancer,lung adenocarcinoma,and renal cell carcinoma.The purpose of the study was to clarify the expression level of NCBP3,SNHG6,GBX2,and FLOT1 in glioma tissues and cell lines and to study their regulatory effects on the malignant biological behavior of glioma cells.In addition,GBX2 binding to NCBP3 and SNHG6 promotor regions to form a feedback loop to regulate the malignant progression of glioma cells.Methods: 1.The RNA expression levels of NCBP3、SNHG6、GBX2、FLOT1 were detected by Real-time PCR experiment;2.Western blot experiments were used to determine the protein expression levels of NCBP3、GBX2、FLOT1;3.U87 and U251 cells were stably transfected with NCBP3 knockdown,SNHG6 knockdown,SNHG6 overexpression,NCBP3 and SNHG6 co-transfection,GBX2 knockdown,GBX2 overexpression,SNHG6 and GBX2 co-transfection,FLOT1 knockdown plasmids;4.CCK8 assay to detect cell proliferation;5.Transwell assay to determine cell migration and invasion;6.Flow cytometry to test cell apoptosis;7.Chromatin immunoprecipitation assay to examine the binding of GBX2 to the FLOT1、NCBP1、SNHG6 promoter regions;8.Dual-luciferase reporter assay to verify the binding of GBX2 to FLOT1、NCBP1、SNHG6;9.RNA-binding protein immunoprecipitation assay to determine the binding of NCBP3 to SNHG6,and the binding of EZH2 to SNHG6;10.RNA regeneration experiment to detect the nascent RNA;11.Half-life experiment to examine the stability of SNHG6;12.Nude mouse xenograft experiments.Results: 1.NCBP3 was highly expressed in glioma tissues and cells.Knockdown of NCBP3 inhibited the SNHG6、GBX2 and FLOT1 expression.Meanwhile knockdown of NCBP3 inhibited the biological behavior of glioma cells.2.SNHG6 was highly expressed in glioma tissues and cells.Inhibition of SNHG6 decreased the GBX2 and FLOT1 expression.Knockdown of SNHG6 induced the biological behavior of glioma cells.3.Knockdown of SNHG6 enhanced the inhibitory effect of NCBP3 knockdown on the biological behavior of glioma cells,but the overexpression of SNHG6 reversed the inhibitory effects.Moreover,NCBP3 knockdown inhibited the SNHG6 expression,prolonged SNHG6 half-life.RIP experiment proved NCBP3 bound to SNHG6.4.NCBP3 was down-regulated in glioma tissues and cells.GBX2 inhibition decreased the FLOT1 expression.Knockdown of GBX2 induced the cell proliferation,migration,invasion and promoted cell apoptosis.5.Knockdown of SNHG6 and GBX2 rescued the reduction of proliferation,migration,invasion and the increase of apoptosis induced by cotransfection of SNHG6 knockdown with GBX2 overexpression.Overexpression of SNHG6 and GBX2 rescued the increase of proliferation,migration,invasion,and the decrease of apoptosis induced by co-transfection of SNHG6 overexpression with GBX2 knockdown.RIP experiment confirmed that NCBP3 bound to EZH2.Ch IP test verified that H3K27me3 was enriched in the 500～1000bp upstream of GBX2 TSS.6.FLOT1 was up-regulated in glioma tissues and cells.Knockdown of FLOT1 inhibited the cell proliferation,migration,invasion and promoted cell apoptosis.Ch IP test confirmed that GBX2 bound to FLOT1、NCBP3、FLOT1 promotor regions.Dual-luciferase experiment proved that GBX2 bound to FLOT1、NCBP3、FLOT1.8.Konckdown of NCBP3 and SNHG6 combined with overexpression of GBX2 led to the most suppressive tumor effect and the longest survival time.Conclusion: 1.NCBP3、SNHG6、FLOT1 was up-regulated in glioma tissues and cells.GBX2 was down-regulated in glioma tissues and cells.Konckdown of NCBP3,SNHG6,FLOT1,or overexpression of GBX2 inhibited the cell proliferation,migration,invasion and promoted cell apoptosis.2.NCBP3 bound to SNHG6,and stabilized SNHG6,upregulated its expression.3.SNHG6 mediated the H3K27me3 modification induced by PRC2 in the GBX2 promoter region to inhibit its transcription.4.GBX2 transcriptionally inhibited the expression of FLOT1,by directly binding to FLOT1 promoter regions.5.GBX2 transcriptionally inhibited the expression of NCBP3 and SNHG6 by directly binding to their promoter regions.,formed a positive loop to regulate the malignant progression of glioma cells.