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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
Targeting TRIM9 by miR-218-5p Restricts Cell Proliferation and Epithelial-Mesenchymal Transition in Non-Small Cell
Zunqiao Wang1,2,3, Bin Wang1,2,3, Keping Wang4,2,3
1Department of Thoracic Surgery, Nanjing Chest Hospital, Nanjing, Jiangsu, China.
Objective:
Tripartite motif-containing protein 9 (TRIM9) has been studied in several human tumors. MicroRNA-218-5p (miR-218-5p) was predicted to target TRIM9. We aimed to investigate the roles of miR-218-5p/TRIM9 axis in non-small cell lung cancer (NSCLC).
Methods:
TRIM9 and miR-218-5p expression levels in NSCLC tissues and cell lines (95D and H1299) were determined by reverse transcription quantitative PCR. UALCAN and Kaplan-Meier (KM) plotter were employed to analyze the expression level of TRIM9 in lung cancer. The interaction between TRIM9 and miR-218-5p was explored by luciferase reporter assay and Spearman correlation test. Immunohistochemistry assay was used to confirm the protein expression of TRIM9 in NSCLC tissues. Regulatory effects of TRIM9 or miR-218-5p on cell proliferation, migration and invasion, and epithelial-mesenchymal transition (EMT) process of NSCLC cells were assessed by CCK-8 assay, transwell assay and western blot analysis.
Results:
MiR-218-5p was predicted to specifically target TRIM9 and confirmed to negatively regulate TRIM9 expression in NSCLC cells. Online bioinformatics analysis showed TRIM9 overexpression in lung cancer and predicted poor prognosis. The data from collected clinical specimens showed that miR-218-5p was downregulated, while TRIM9 was upregulated in NSCLC tissues, whose expression levels were negatively correlated. The in vitro experiments demonstrated that TRIM9 knockdown imitated the suppressive effects miR-218-5p overexpression on cell proliferation, migration, invasion and EMT process. In addition, overexpression of TRIM9 reversed the effects of miR-218-5p in NSCLC cells.
Conclusions:
Our results suggest that TRIM9 functions as an oncogene in NSCLC in vitro and is regulated by miR-218-5p.
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