Abstract
BackgroundLung cancer is the major cause of cancer-related death worldwide, and 80% patients of lung cancer are non-small-cell lung cancer (NSCLC) cases. MicroRNAs are important gene regulators with critical roles in diverse biological processes, including tumorigenesis. Studies indicate that sphingosine kinase 2 (SphK2) promotes tumor progression in NSCLC, but how this occurs is unclear. Thus, we explored the effect of miR-338-3p targeting SphK2 on proliferation and apoptosis of NSCLC cells.MethodsExpression of miR-338-3p and SphK2 in NSCLC A549 and H1299 cell lines was measured using qRT-PCR and Western blot. CCK-8 and colony formation assays were used to assess the effect of miR-338-3p on NSCLC cell line proliferation. Flow cytometry was used to study the effect of miR-338-3p on NSCLC apoptosis. Luciferase reporter assay and Western blot were used to confirm targeting of SphK2 by miR-338-3p. Finally, in vivo tumorigenesis studies were used to demonstrate subcutaneous tumor growth.ResultsmiR-338-3p expression in 34 NSCLC clinical samples was downregulated and this was correlated with TNM stage. miR-338-3p significantly suppressed proliferation and induced apoptosis of NSCLC A549 and H1299 cells in vitro. SphK2 was a direct target of miR-338-3p. Overexpression of miR-338-3p significantly inhibited SphK2 expression and reduced luciferase reporter activity containing the SphK2 3′-untranslated region (3′-UTR) through the first binding site. SphK2 lacking 3′-UTR restored the effects of miR-338-3p on cell proliferation inhibition. miR-338-3p significantly inhibited tumorigenicity of NSCLC A549 and H1299 cells in a nude mouse xenograft model.ConclusionsCollectively, miR-338-3p inhibited cell proliferation and induced apoptosis of NSCLC cells by targeting and down-regulating SphK2, and miR-338-3p could inhibit NSCLC cells A549 and H1299 growth in vivo, suggesting a potential mechanism of NSCLC progression. Therapeutically, miR-338-3p may serve as a potential target in the treatment of human lung cancer.
Highlights
Lung cancer is the major cause of cancer-related death worldwide, and 80% patients of lung cancer are non-small-cell lung cancer (NSCLC) cases
Results miR‐338‐3p expression is significantly reduced and sphingosine kinase 2 (SphK2) expression is significantly increased in NSCLC tissues Analysis of patient data indicated that SphK2 expression was significantly correlated with NSCLC TNM stage (p = 0.017), and expression of miR-338-3p was significantly correlated with NSCLC TNM stage (p = 0.023)
The results of half quantitative analysis for protein by Western blot indicated that the relative expression of SphK2 in NSCLC tumor tissues was much higher than in normal tissues (p < 0.001; Fig. 1b), and the relative expression of SphK2 in NSCLC I + II stage tumor tissues was lower than in NSCLC III stage tumor tissues (p = 0.017; Fig. 1c). qRT-PCR results showed that the relative expression of miR-338-3p in NSCLC tumor tissues was much lower than in normal tissues (p < 0.001; Fig. 1d), and the relative expression of miR-338-3p in I + II stage tumor tissues was higher than in III stage tumor tissues of NSCLC (p = 0.023; Fig. 1e)
Summary
Lung cancer is the major cause of cancer-related death worldwide, and 80% patients of lung cancer are non-small-cell lung cancer (NSCLC) cases. MicroRNAs are important gene regulators with critical roles in diverse biological processes, including tumorigenesis. We explored the effect of miR-338-3p targeting SphK2 on proliferation and apoptosis of NSCLC cells. MicroRNAs (miRNAs) are small, endogenous, noncoding RNAs of approximately 22 nt that regulate the expression of target mRNA by binding to 3′-untranslated regions (3′-UTRs), resulting in target mRNA degradation or silencing [7, 8]. Recent studies indicate that microRNAs (miRNAs) are important subtypes of noncoding RNAs in the regulation of diverse biological processes, especially those involved in critical pathways linked to cancer cell proliferation, apoptosis, and metastasis [9,10,11]. Little is known about the role of miR-338-3p in NSCLC proliferation and apoptosis so we investigated NSCLC progression and development by identifying miRNA targets
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