Abstract
BackgroundNon-small cell lung cancer (NSCLC) is one of the major types of lung cancer, which is a prevalent human disease all over the world. LncRNA LINC01503 is a super-enhancer-driven long non-coding RNA that is dysregulated in several types of human cancer. However, its role in NSCLC remains unknown.MethodsThirty NSCLC patients were recruited between April 2012 and April 2016. Luciferase reporter assay, qRT-PCR, Cell Counting Kit-8 (CCK-8), Transwell migration assay, RNA pull-down assay, western blotting, 5-ethynyl-29-deoxyuridine (EdU) assays, and flow cytometry were utilized to characterize the roles and relationships among LINC01503, miR-342-3p, and LASP1 in NSCLC. The transplanted mouse model was built to examine their biological functions in vivo.ResultsWe demonstrated that the expression of lncRNA LINC01503 and LIM and SH3 domain protein 1 (LASP1) were upregulated and miR-342-3p was downregulated in NSCLC samples and cell lines. Functional experiments revealed that inhibiting the expression of LINC01503 or over-expression of miR-342-3p inhibited NSCLC growth and metastasis both in vitro and in vivo. In addition, LINC01503 could bind to miR-342-3p and affect the expression of LASP1.ConclusionThese results provide a comprehensive analysis of the roles of LINC01503 as a competing endogenous RNA (ceRNA) in NSCLC progression.
Highlights
Non-small cell lung cancer (NSCLC) is one of the major types of lung cancer, which is a prevalent human disease all over the world
The expression of LINC01503 was upregulated in NSCLC patients and NSCLC cells To identify the roles of LINC01503 in NSCLC progression, qRT-PCR analysis was used to investigate the expression of LINC01503 in 30 pairs of NSCLC samples compared to that in adjacent normal samples
The expression of miR-342-3p was negatively related to the expression of LINC01503 in NSCLC patients (Fig. 1c)
Summary
Patients and samples A total of 30 NSCLC patients were recruited in the First Hospital of China Medical University, P.R. LASP1WT (wild-type) or LSAP1-MUT (mutant) had amplification using PCR, and transfection to A549 cells with Lipofectamine 2000 (Invitrogen, USA). Luciferase reporter assays LINC01503 and LASP1 wild-type or mutant binding miR-342-3p were cloned to pMIR Basic vector (OBiO Biology, Beijing) to generate pMIR-REPOR-LINC01503wt or pMIR-REPOR-LINC01503-mt, respectively. After being cultivated in 24-well plates for 24 h, A549 cells were transfected with miR-342-3p mimics or control (GenePharma, China) and co-transfected with pMIRREPOR-NC or constructed recombinant luciferase vectors by Lipofectamine 3000 (Invitrogen, USA). The membranes were incubated by anti-LASP1 (1:1000; Abcam, UK) and anti-GAPDH (1:1000; Cell Signal Technology, USA) at 4 °C overnight. Flow cytometry Cell apoptosis was measured by Annexin V (Biosea, China) at 2 d post-transfection and Cell Quest (BD, USA).
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