| Background and Objective: Gliomas is the most common primary malignancy of the central nervous system,the 5 year survival rate of patients with gliomas is less than 5%.However,the molecular mechanisms underlying gliomas formation still remain elusive.Plenty of research indicates that angiogenesis is a necessary prerequisite for tumor progression because it fulfills the nutrient and oxygen requirements of the tumors,and removes the metabolic wastes and promotes the distal metastasis of tumor cells.It is said that lncRNA plays an important role in tumor angiogenesis.XIST is the lncRNA which essential for transcriptional silencing of one X-chromosome during mammalian female development.Using an online software program starbase v2.0,we noticed that there is a complementary sequence between miR-429 and XIST;XIST might be a lncRNA target of miR-429.Therefore,in this study we performed a series of molecular biology and cell function experiments to identify the expression of XIST and miR-429 in gliomas and the relationship between XIST and miR-429,to explore the molecular mechanism of XIST involvement in tumorigenicity and angiogenesis of gliomas,and provide theoretical support for the anti-angiogenic therapy of gliomas.Method: First,we explored the expression of XIST in different grades of gliomas by analyzing the gene expression profiles in GEO and CGGA public datasets,and then compared the expression of XIST in U251 and A172 glioma cells with the normal glial cells(HA1800)by Real-time PCR,to confirm the role of XIST in gliomas.Second,after constructing lentiviral vector and silencing XIST in U251 and A172 cells,we performed a series of cellular functional assays(MTT assay,clone formation,flow cytometry,transwell,and tube formation assays),as well as molecular biology experiments to detect the effect of XIST on glioma cells induced HBMVEC proliferation,migration and tube formation.The effect of XIST on the tumorigenicity of gliomas was verified by subcutaneous xenografts in nude mice.Immunohistochemical analysis of tumors was performed to further demonstrate that down-regulation of XIST expression could inhibit the angiogenesis of gliomas.Third,we synthesized miR-429 mimic,inhibitor,mimic negative control and inhibitor negative control,and then transfected into glioma cells to detect the effect of miR-429 on XIST expression.The dual-luciferase reporter system was used to evaluate the interaction between miR-429 and XIST.Finally,miR-429 inhibitor was transfected into a stable XIST silencing glioma cell line.MTT analysis,clone formation,flow cytometry,transwell,and tube formation assays were performed to detect whether the effect of XIST on glioma cells induced HBMVEC proliferation,migration and tube formation is mediated by miR-429.Results: The results of analyzing dataset GSE2223 and GSE4290 showed that the expression of XIST in glioma tissue was significantly higher than that in normal brain tissue,and this aberrant expression is mainly formed in high-grade gliomas.The difference between low-grade glioma tissues and normal brain tissue is not statistically significant,suggesting that there is no significant correlation between XIST expression and the grades of gliomas.The CGGA dataset analysis revealed that XIST expression was significantly different in primary and recurrent glioma tissues,and XIST was overexpressed in recurrent glioma tissues.After establishing XIST silencing glioma cell lines,we performed MTT assay,clone formation assay,flow cytometry and found that XIST promoted the proliferation and colony formation of glioma cells.Then we performed Transwell,tube formation assays and found that XIST promoted HBMVEC proliferation,migration and tube formation inducing by glioma cells.The above experiment results strongly suggest the effect of XIST on glioma tumorigenicity and angiogenesis in vitro.Next,the nude mouse xenograft tumor model showed the effect of XIST on glioma tumorigenicity in vivo,and immunohistochemical analysis of tumors showed the effect of XIST on glioma angiogenesis in vivo.Real-time PCR results revealed that the expression of miR-429 was down-regulated in A172 and U251 cells compared with normal glial cells.We detected the expression of miR-429 in XIST silencing glioma cell line and found that silencing XIST induced the expression of miR-429.In addition,miR-429 mimic could significantly reduce the expression of XIST,while miR-429 inhibitor could significantly increase the expression of XIST,demonstrating that XIST is negatively correlated with the expression of miR-429 in gliomas.Using bioinformatics software,we predicted the target sequence between XIST and miR-429,and then constructed pmir GLO-WT-Luc vector containing the target sequence and pmir GLO-Mut-Luc vector containing mutated target sequence.Next,to determine whether XIST target miR-429 directly,we used dual-luciferase reporter system.The results revealed that co-transfection of miR-429 mimic and pmir GLO-WT could significantly reduce the intracellular luciferase activity,but there was no significant change in the luciferase activity in control group,demonstrating that lncRNA XIST regulates miR-429 through targeting 73045083-7345062 sequences directly.We transfected miR-429 inhibitor into XIST silencing glioma cell lines A172 sh-XIST and U251 sh-XIST,and found that miR-429 inhibitor restored the expression of XIST in A172 sh-XIST and U251 sh-XIST.In addition,miR-429 inhibitor partly restored the proliferation and colony formation of these two XIST silencing glioma cell lines.Conclusion: lncRNA XIST is upregulated in glioma cell lines and tissues,especially in recurrent glioma tissues,suggesting that lncRNA XIST based treatment strategies may be more effective for recurrent gliomas.Silencing XIST inhibit the tumorigenicity and angiogenesis of glioma,indicating that XIST plays an important role in the development of gliomas.XIST inhibit the level of miR-429 in glioma cells by acting as a molecular sponge.Overexpression of XIST in gliomas suppresses the level of miR-429,and then to induce the proliferation,migration and angiogenesis of HBMVEC. |
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