Yana Gao1,2, Hai Yu3,4,5, Yunhui Liu3,4,5, Xiaobai Liu3,4,5, Jian Zheng3,4,5, Jun Ma1,2, Wei Gong1,2, Jiajia Chen1,2, Lini Zhao1,2, Yu Tian3,4,5, Yixue Xue1,2. 1. Department of Neurobiology, College of Basic Medicine, China Medical University, Shenyang, China. 2. Key Laboratory of Cell Biology, Ministry of Public Health of China, and Key Laboratory of Medical Cell Biology, Ministry of Education of China, China Medical University, Shenyang, China. 3. Department of Neurosurgery, Shengjing Hospital of China Medical University, Shenyang, China. 4. Liaoning Clinical Medical Research Center in Nervous System Disease, Shenyang, China. 5. Key Laboratory of Neuro-oncology in Liaoning Province, Shenyang, China.
Abstract
BACKGROUND/AIMS: Vasculogenic mimicry (VM) has been reported to be a novel glioma neovascularization process. Anti-VM therapy provides new insight into glioma clinical management. In this study, we revealed the role of the long non-coding RNA HOXA cluster antisense RNA 2 (HOXA-AS2) in malignant glioma behaviors and VM formation. METHODS: Quantitative real-time PCR was performed to determine the expression levels of HOXA-AS2 in glioma samples and glioblastoma cell lines. CD34-periodic acid-Schiff dual-staining was performed to assess VM in glioma samples. CCK-8, transwell, and Matrigel tube formation assays were performed to measure the effects of HOXA-AS2 knockdown on cell viability, migration, invasion, and VM tube formation, respectively. RNA immunoprecipitation, dual-luciferase reporter and Western blot assays were performed to explore the molecular mechanisms underlying the functions of HOXS-AS2 in glioblastoma cells. A nude mouse xenograft model was used to investigate the role of HOXA-AS2 in xenograft glioma growth and VM density. Student's t-tests, one-way ANOVAs followed by Bonferroni posthoc tests, and chi-square tests were used for the statistical analyses. RESULTS: HOXA-AS2 was upregulated in glioma samples and cell lines and was positively correlated with VM. HOXA-AS2 knockdown attenuated cell viability, migration, invasion, and VM formation in glioma cells and inhibited the expression of vascular endothelial-cadherin (VE-cadherin), as well as the expression and activity of matrix metalloproteinase matrix metalloproteinase (MMP)-2 and MMP-9. miR-373 was downregulated in glioma samples and cell lines and suppressed malignancy in glioblastoma cells. HOXA-AS2 bound to miR-373 and negatively regulated its expression. Epidermal growth factor receptor (EGFR), a target of miR-373, increased the expression levels of VE-cadherin, as well as the expression and activity levels of MMP-2 and MMP-9, via activating phosphatidylinositol 3-kinase/serine/threonine kinase pathways. HOXA-AS2 knockdown combined with miR-373 overexpression yielded optimal tumor suppressive effects and the lowest VM density in vivo. CONCLUSION: HOXA-AS2 knockdown inhibited malignant glioma behaviors and VM formation via the miR-373/EGFR axis.
BACKGROUND/AIMS: Vasculogenic mimicry (VM) has been reported to be a novel glioma neovascularization process. Anti-VM therapy provides new insight into glioma clinical management. In this study, we revealed the role of the long non-coding RNA HOXA cluster antisense RNA 2 (HOXA-AS2) in malignant glioma behaviors and VM formation. METHODS: Quantitative real-time PCR was performed to determine the expression levels of HOXA-AS2 in glioma samples and glioblastoma cell lines. CD34-periodic acid-Schiff dual-staining was performed to assess VM in glioma samples. CCK-8, transwell, and Matrigel tube formation assays were performed to measure the effects of HOXA-AS2 knockdown on cell viability, migration, invasion, and VM tube formation, respectively. RNA immunoprecipitation, dual-luciferase reporter and Western blot assays were performed to explore the molecular mechanisms underlying the functions of HOXS-AS2 in glioblastoma cells. A nude mouse xenograft model was used to investigate the role of HOXA-AS2 in xenograft glioma growth and VM density. Student's t-tests, one-way ANOVAs followed by Bonferroni posthoc tests, and chi-square tests were used for the statistical analyses. RESULTS:HOXA-AS2 was upregulated in glioma samples and cell lines and was positively correlated with VM. HOXA-AS2 knockdown attenuated cell viability, migration, invasion, and VM formation in glioma cells and inhibited the expression of vascular endothelial-cadherin (VE-cadherin), as well as the expression and activity of matrix metalloproteinase matrix metalloproteinase (MMP)-2 and MMP-9. miR-373 was downregulated in glioma samples and cell lines and suppressed malignancy in glioblastoma cells. HOXA-AS2 bound to miR-373 and negatively regulated its expression. Epidermal growth factor receptor (EGFR), a target of miR-373, increased the expression levels of VE-cadherin, as well as the expression and activity levels of MMP-2 and MMP-9, via activating phosphatidylinositol 3-kinase/serine/threonine kinase pathways. HOXA-AS2 knockdown combined with miR-373 overexpression yielded optimal tumor suppressive effects and the lowest VM density in vivo. CONCLUSION:HOXA-AS2 knockdown inhibited malignant glioma behaviors and VM formation via the miR-373/EGFR axis.