Abstract
BackgroundLung adenocarcinoma (LAD) is considered to be a highly aggressive disease with heterogeneous prognosis and the molecular mechanisms underlying tumor progression remain elusive. Growing evidence demonstrates that the dysregulation of microRNAs (miRNAs) plays an important role in various tumor processes. The aim of this study is to discover prognostic miRNA and investigate its role involved in progression of LAD.MethodsPrognosis related miRNA was detected by miRNA microarray using formalin-fixed paraffin-embedded (FFPE) specimens from 87 patients with IIIA-N2 LAD. The cell proliferation was evaluated by Cell Titer 96 AQueous One Solution Cell Proliferation Assay (MTS), and the migration/invasion was evaluated by transwell assay. The bioinformatics methods and luciferase reporter assay were applied to detect the relationship between miRNA and its target. The mRNA and protein levels of miRNA target were determined by quantitative real time polymerase chain reaction (qRT-PCR) analysis, western blot and enzyme-linked immunosorbent assay (ELISA). Changes of angiogenesis induced by miRNA was evaluated by human umbilical vein endothelial cell (HUVEC) tube formation assay. Immunohistochemistry (IHC) analysis was performed in FFPE specimens of patients to evaluate the correlation between miR-29c with microvessel density (MVD) and vascular endothelial growth factor A (VEGFA) expression.ResultsMiR-29c expression downregulation was significantly associated with unfavorable prognosis in IIIA-N2 LAD. MiR-29c inhibited cell proliferation, migration and invasion in cell lines. Integrated analysis revealed that VEGFA was a direct target of miR-29c. MiR-29c reduced the capability of tumor cells to promote HUVEC tube formation. The compromised cell proliferation, migration/invasion and angiogenesis induced by miR-29c mimic transfection were reversed by transfection of VEGFA expression plasmid. Furthermore, the correlation of miR-29c with MVD and VEGFA was confirmed in patients’ samples.ConclusionsMiR-29c acts as a tumor suppressor by targeting VEGFA and may represent a promising prognostic biomarker as well as a potential therapeutic target for LAD.
Highlights
Lung adenocarcinoma (LAD) is considered to be a highly aggressive disease with heterogeneous prognosis and the molecular mechanisms underlying tumor progression remain elusive
We evaluated whether miR-29c could regulate vascular endothelial growth factor A (VEGFA) at both mRNA and protein levels (Fig. 3c, d and e). quantitative real time polymerase chain reaction (qRT-PCR) analysis indicated that overexpression of miR-29c in A549 cells resulted in down-regulation of VEGFA mRNA compared with control
The compromised cell proliferation, migration, invasion and angiogenesis induced by miR-29c mimic transfection were almost completely reversed by transfection of VEGFA expression plasmid (Fig. 5a-e). These results suggest that miR-29c involves in regulation of cell proliferation, migration, invasion and angiogenesis at least partially depending on regulation of VEGFA
Summary
Lung adenocarcinoma (LAD) is considered to be a highly aggressive disease with heterogeneous prognosis and the molecular mechanisms underlying tumor progression remain elusive. The aim of this study is to discover prognostic miRNA and investigate its role involved in progression of LAD. With high invasiveness and early metastasis, LAD often presents with locally advanced or metastatic disease at prognosis [3]. Stage IIIA-N2 LAD presents substantial heterogeneity ranging from resectable microscopic lymph node disease to unresectable, bulky nodal metastases [4], with 5-year overall survival (OS) ranging from 13 to 58.3%[5,6,7].
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