| Objective: Glioma is the most common primary intracranial malignant tumor,and its main treatment methods are mainly surgical treatment,combined with radiotherapy and chemotherapy and other comprehensive treatment after surgery,but after systematic treatment,the average median survival time of patients with glioma is only 12-18 months.Glioma tissues mainly maintain their own nutrition supply through angiogenesis,but the effect of antiangiogenic targeted drugs in the treatment of glioma is not satisfactory.In a1999 study of melanoma,a form of vasculogenic mimicry was found that does not rely on blood vessels but deforms the extracellular matrix to supply blood to the tumor.This provides a new target for the study of vasculogenic mimicry targeted therapy for gliomaRNA binding proteins(RBPs)accompany the formation and metabolism of RNA,regulate RNA stability,and participate in mediating RNA transport and translation.Poly(A)binding protein cytoplasmic 5(PABPC5),a member of the cytosolic poly(A)binding protein family,is involved in DNA and RNA metabolism in mitochondria.The PABPC5 gene consists with at least two exons and one intron and an uninterrupted ORF(open reading frame).PABPC5 is studied on ovarian disease.PABPC5 is located on the translocation breakpoint DX214 of genes associated with premature ovarian failure,and that the high expression of PABPC5 is closely related to the poor prognosis of ovarian cancer patients.There are no related reports has been reported in glioma research.LncRNAs are a large class of important regulatory molecules in the human transcriptome and exert their biological functions in a variety of ways,and play important regulatory roles in tumorigenesis and development,including epigenetic regulation,transcriptional regulation and post-transcriptional regulation.It has important and significance for the diagnosis and treatment of glioma.HCG15(HLA complex group15)is located on chromosome 6p21.At present,HCG15 has not been reported in glioma and vasculogenic mimicry.SMD(STAU1-mediated mRNA decay)constitutes an important post-transcriptional regulatory pathway.STAU1 mediated mRNA degradation pathway refers to the recognition and pairing of the Alu element of lncRNAs with the specific sequence of the Alu element of the target gene mRNA 3’-UTR to form a STAU1 binding site(SBS),recruit STAU1 to bind to it,and promote the mRNA of the target gene is combined with frameshift increasing protein 1(UPF1)to specifically accelerate the degradation of the target gene mRNA.ZNF331(zinc finger protein 331),which encodes a zinc finger protein containing the KRAB(Kruppel related box)domain found in transcriptional repressors.In gastric cancer cells,the ZNF331 promoter region is inactivated after methylation and loses the role of tumor suppressor genes,thereby changing the growth and invasion ability of gastric cancer cells.The low expression of ZNF331 indicates a poor prognosisin colorectal cancer tissues.At present,no research report on ZNF331 regulating vasculogenic mimicry of gliomas has been reported.LAMC2(laminin subunit gamma 2)is a family of extracellular matrix glycoproteins and is the main non-collagen component of the basement membrane.It is involved in regulating a variety of biological processes,including cell adhesion,differentiation,migration,signaling,neurite growth and metastasis.LAMC2 promotes the migration and invasion of lung cancer cells through the AKT signaling pathway.LAMC2 is highly expressed in glioma cells U87 and U251,and is related to the formation of vascular mimicry.LAMC2 plays a key role in the formation of tumor vascular mimicry through AKT and ERK signaling pathways.In gliomas,LAMC2 promote the formation of vasculogenic mimicry through the ERK or AKT signaling pathway and increase the malignancy of gliomas.ZNF331 has not been seen to regulate the transcription of LAMC2 and thus to regulate the vasculogenic mimicry of gliomas.This study first investigated the expression and function of PABPC5,HCG15 and ZNF331 in glioma tissues and glioma cell lines.Further study the role of PABPC5,HCG15 and ZNF331 in regulating glioma VM.The aim is to provide a new basis for the development of gliomas.Methods:1.Real-time PCR was used to detect the expression levels of the target PABPC5,HCG15 and ZNF331.2.Western blot was conducted to detect the expression levels of PABPC5,ZNF331,and LAMC.3.Glioma cells(U87,U251)stably transfected with knockdown PABPC5 plasmid,knockdown HCG15 plasmid,knockdown and overexpression ZNF331 plasmid,knockdown STAU1 and UPF1 were contructed.4.Migration and invasion ability were detected by Transwell.5.CCK-8 test was used todetect proliferation ability.6.The vascular mimicry of glioma cells was detected by in vitro tube formation experiment.7.The dual-luciferase reporter gene system verifiedthat HCG15 and ZNF331 mRNAs had targeted binding through Alu sequences.8.RNA-IP(RNA immunoprecipitation)verified that PABPC5 and HCG15,HCG15 and STAU1,and ZNF331 mRNA and STAU1 had binding effects.9.In order to verify the binding effect of ZNF331 with the promoter regions of LAMC2 and PABPC5,we applied chromatin immunoprecipitation experiment.10.RNA nascent experiments was used to detect stability of HCG15 and ZNF331 mRNA.11.Half-life experiments were used to detect HCG15 and ZNF331 mRNA RNA half-lives.12.Immunohistochemical staining of CD34-PAS was used to verify the tissue vasculogenic mimicry.13.Nude mouse xenograft experiments conducted to test tumor growth and VM ability.Results:1.PABPC5 is highly expressed in glioma tissues and cells.PABPC5 knockdown significantly inhibits the expression of LAMC2,inhibits glioma cell proliferation,migration and invasion,and VM of glioma cell.2.HCG15 is highly expressed in glioma tissues and cells.PABPC5 knockdown significantly reduces the expression of HCG15.HCG15 knockdown significantly inhibits the expression of LAMC2,inhibits the proliferation,migration and invasion of glioma cells,and the ability of VM.Overexpression of HCG15 significantly enhances LAMC2 expression,enhances glioma cell proliferation,migration and invasion,and the ability of VM.3.RNA-IP verifies the binding effect of PABPC5 and HCG15.PABPC5 knockdown lessens the half-life of HCG15.Knockdown PABPC5 and HCG15 significantly inhibit the expression of LAMC2,inhibit the proliferation,migration and invasion of glioma cells and the ability of VM.4.ZNF331 is lowly expressed in glioma tissues and cells.Knockdown PABPC5 or HCG15 significantly increase the expression of ZNF331.Konckdown STAU1 or knockdown UPF1 enhance ZNF331 expression.Overexpression of ZNF331 significantly reduces the expression of LAMC2,inhibites the proliferation,migration,invasion of glioma cells and the ability of VM.Knockdown ZNF331 significantly enhances the expression of LAMC2,enhances the proliferation,migration,invasion of glioma cells and VM.5.Dual-luciferase experiments verifies the targeted binding of HCG15 and ZNF331 mRNA via Alu sequence.RNA-IP experiments verifies that HCG15 and ZNF331 mRNAs bind to STAU1.HCG15 knockdown significantly prolongs the ZNF331 mRNA half-life.Knockdown STAU1 or UPF1 significantly prolong ZNF331 mRNA half-life.HCG15 knockdown and overexpression of ZNF331 significantly inhibit the expression of LAMC2,inhibit the proliferation,migration and invasion of glioma cells,and the ability to form VM.HCG15 knockdown reverses LAMC2 expression in ZNF331 knockdown cell and reversed glioma cell proliferation,migration and invasion,and VM.6.ZNF331 binds to LAMC2 and PABPC5 promoter regions and inhibits their transcription.7.Knockdown PABPC5,knockdown HCG15,overexpression of ZNF331 and simultaneous knockdown PABPC5 and HCG15 overexpression of ZNF331 significantly inhibit the growth of nude mice,prolong their survival,and reduce the formation of glioma VM.Conclusions:1.PABPC5 and HCG15 are highly expressed in glioma tissue and U87 and U251glioma cells,and ZNF331 is low.Knockdown PABPC5,HCG15 or overexpression of ZNF331 inhibit the vasculogenic mimicry of glioma cells.2.PABPC5 and HCG15 have specific binding effect.PABPC5 increases the stability of HCG15.HCG15 promotes the degradation of ZNF331 mRNA through the SMD pathway.3.ZNF331 binds to the promoter regions of LAMC2 and PABPC5 and inhibits its transcription,thereby inhibiting the formation of vasculogenic mimicry of glioma cells.4.Knockdown PABPC5 reduce the stability of HCG15 and reduce its expression;further weaken the HCG15 mRNA degradation of ZNF331 and increase the expression of ZNF331;and then inhibit LAMC2 and PABPC5 transcription,thereby inhibiting the formation of vasculogenic mimicry of glioma cells. |
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