| Objective: Glioma is the most common primary malignant tumor of the human CNS.Although the applications of surgery,chemotherapy,and radiotherapy have made significant progress in the treatment of glioma in recent years,the prognosis of patients with glioma remains poor,with the lowest 5-year survival rate among all cancers;in addition,the median survival time of patients with gliomas of high pathological grade is approximately 15 months.The aim of present study is to explore whether the RNA-binding protein DGCR8 and the long non-coding RNA ZFAT-AS1 play an important role in regulating the biological behavior of glioma cells,and to study its underlying molecular mechanism,that is,DGCR8 induces the cleavage of ZFAT-AS1 to attenuated the transcriptional repression of CDX2 induced by ZFAT-AS1 in a PRC2 dependent manner,up-regulates CDX2 expression,and promotes the biological behavior of glioma cells.Methods: Human glioma cells U87,U251 and astrocytes cells HA were cultured.The expression levels of DGCR8,ZFAT-AS1,CDX2 and RRM2 B in normal brain tissue adjacent glioma,glioma tissue,human astrocytes,and glioma cells were determined by quantitative Real-time PCR and Western blot.DGCR8 knockdown cell lines and ZFATAS1,CDX2,and RRM2 B knockdown and overexpression cell lines were constructed by cell stable transfection methods,respectively.To clarify their effect on the biological behavior of glioma cells,cell proliferation,migration and invasion,and apoptosis were measured using CCK-8,Transwell assays,and flow cytometry analysis,respectively.RIP assays were utilized to validate the binding effect of DGCR8 and ZFAT-AS1.RIP assays and RNA pull-down assays were used to verified the binding of ZFAT-AS1 to the subunit EZH2,Ch IRP assays was used to clarify the presence of ZFAT-AS1 in the CDX2 promoter region,and Ch IP assays was utilized to detect the H3K27me3 modification in the CDX2 promoter region.Ch IP assays and luciferase reporter system assays were used to analyze the direct binding of CDX2 to RRM2 B and DGCR8 promoter regions,respectively.Quantitative Real-time PCR and Western Blot methods were used to detect the m RNA and protein changes of DGCR8,CDX2,and RRM2 B,as well as ZFAT-AS1 expression changes.Nude mouse xenograft experiments were performed to test the tumor inhibition effect of DGCR8 and ZFAT-AS1 on tumor transplantation in nude mice and the survival time of nude mice with brain tumor in situ.Results: This study found that DGCR8,CDX2,and RRM2 B were upregulated in glioma tissues and cells,and ZFAT-AS1 was lowly expressed.Knockdown of DGCR8,CDX2 and RRM2 B and over-expression of ZFAT-AS1 could restrain the proliferation,migration and invasion of glioma cells,and facilitate apoptosis.DGCR8 promoted the biological behavior of glioma cells by promoting ZFAT-AS1 cleavage and down-regulating its expression.ZFAT-AS1 bound to the catalytic subunit EZH2 of the PRC2 and co-presented in the promoter region of CDX2.ZFAT-AS1 induced H3K27me3 modification in the CDX2 promoter region by recruiting and binding the PRC2,inhibiting the transcription and expression of CDX2 and inhibiting the biological behavior of glioma cells.CDX2 bound to the promoter region of RRM2 B,promoted its transcription and expression,and then promoted the malignant biological behavior of glioma cells.CDX2 also promoted its transcription and expression by binding to the DGCR8 promoter region,forming a positive feedback loop to regulate the biological behavior of sarcoma cells.Knockdown of DGCR8 combined with overexpression of ZFAT-AS1 inhibited the growth of xenografts in nude mice and prolonged the survival of nude mice with orthotopic tumors.Conclusions: 1.DGCR8,CDX2 and RRM2 B were highly expressed in glioma tissues and cells,and ZFAT-AS1 was downregulated.Knockdown of DGCR8,CDX2 and RRM2 B and overexpression of ZFAT-AS1 could supresses the proliferation,migration and invasion of glioma cells,and accelerate apoptosis.2.DGCR8 can reduce the stability of ZFAT-AS1.DGCR8 knockdown up-regulated its expression by enhancing the stability of ZFAT-AS1,inhibited the proliferation,migration and invasion of glioma cells and promote apoptosis.3.ZFAT-AS1 bound to the catalytic subunit EZH2 of the PRC2.ZFAT-AS1 enhanced the H3K27me3 modification induced by PRC2 in the CDX2 promoter region,and inceased the transcriptional inhibitory effect on CDX2 to reduce the expression of CDX2,which inhibited the proliferation,migration and invasion of glioma cells,and promoted apoptosis.4.CDX2 promoted its transcription by binding to the promoter region of RRM2 B,promoted the proliferation,migration and invasion of glioma cells,and inhibited apoptosis.CDX2 promoted its transcription by binding to the promoter region of DGCR8,forming a feedback loop DGCR8/ZFAT-AS1/CDX2,regulating the biological behavior of glioma cells. |
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