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An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells
Published on: May 21, 2019
LncRNA GAS5-AS1 inhibits glioma proliferation, migration, and invasion via miR-106b-5p/TUSC2 axis
1Department of Neurosurgery, Nanjing Medical University Affiliated Changzhou No. 2 People's Hospital, Changzhou, 213003, Jiangsu, China.
Abstract:
Glioma is one of the most common malignant tumors and shows a high metastasis rate and poor prognosis. Abnormal expression of long non-coding RNAs (lncRNAs) contributes to various human tumors including gliomas. This study aimed to investigate the regulatory role of the antisense RNA of growth arrest special 5 (GAS5-AS1), a novel lncRNA, in gliomas. Expression of GAS5-AS1 and microRNA-106b-5p (miR-106b-5p) in glioma tissues and cells was detected by quantitative reverse transcription PCR, northern blotting, or fluorescent in situ hybridization. Cell proliferation, migration, and invasion were analyzed by CCK-8 and Transwell assays. BALB/c nude mice were used to establish a glioma xenograft animal model by subcutaneous injection of U251 cells transfected with small interfering RNA targeting GAS5-AS1. A dual-luciferase reporter assay was conducted to confirm the targeting relationship between GAS5-AS1 and miR-106b-5p. GAS5-AS1 expression was downregulated in glioma tissues and cells, and upregulation of GAS5-AS1 expression inhibited glioma cell proliferation, migration, and invasion. GAS5-AS1 expression was correlated with the glioma tumor grade. In nude mice, upregulation of GAS5-AS1 markedly suppressed glioma tumor growth. GAS5-AS1 overexpression significantly increased the miR-106b-5p level in glioma cells, and GAS5-AS1 expression was negatively related to miR-106b-5p expression in glioma tissues. Overexpression of miR-106b-5p reversed the inhibitory effects of GAS5-AS1 upregulation on glioma cell proliferation and metastasis, while restoration of TUSC2 rescued the proliferation and invasion of glioma cells transfected with miR-106b-5p mimics. In summary, lncRNA GAS5-AS1 inhibited glioma proliferation, migration, and invasion by sponging miR-106b-5p and regulating the expression of TUSC2. Our results suggest GAS5-AS1 as a novel target for the treatment and prognosis prediction of gliomas.
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