Abstract
BackgroundBrain metastasis (BM) is associated with poor prognosis in patients with non-small cell lung cancer (NSCLC). Recent studies demonstrated that microRNA-330-3p (miR-330-3p) was involved in NSCLC brain metastasis (BM). However, the exact parts played by miR-330-3p in BM of NSCLC remain unknown. Discovery and development of biomarkers and elucidation of the mechanism underlying BM in NSCLC is critical for effective prophylactic interventions. Here, we evaluated the expression and biological effects of miR-330-3p in NSCLC cells and explored the underlying mechanism of miR-330-3p in promoting cell migration and invasion in NSCLC.MethodsStable over-expression and knockdown of miR-330-3p in NSCLC cells was constructed with lentivirus. Expression levels of miR-330-3p in NSCLC cells were quantified by quantitive real-time PCR (qRT-PCR). The effects of miR-330-3p on NSCLC cells were investigated using assays of cell viability, migration, invasion, cell cycle, apoptosis, western blotting, immunohistochemical, and immunofluorescence staining. A xenograft nude mouse model and in situ brain metastasis model were used to observe tumor growth and brain metastasis. The potential target of miR-330-3p in NSCLC cells was explored using the luciferase reporter assay, qRT-PCR, and western blotting. The miR-330-3p targets were identified using bioinformatics analysis and verified by luciferase reporter assay. The correlation between GRIA3 and DNA methyltransferase (DNMT) 1 and DNMT3A was tested by RT-PCR, western blotting, and co-immunoprecipitation (IP).ResultsmiR-330-3p was significantly up-regulated in NSCLC cell lines. MTT assay, transwell migration, and invasion assays showed that miR-330-3p promoted the growth, migration, and invasion of NSCLC cells in vitro and induced tumor growth and metastasis in vivo. Luciferase reporter assays showed that GRIA3 was a target of miR-330-3p. qRT-PCR and western blotting exhibited that miR-330-3p promoted the growth, invasion, and migration of NSCLC cells by activating mitogen-activated protein kinase (MAPK)/extracellular-regulated protein kinases (ERK) signaling pathway. Furthermore, miR-330-3p up-regulated the total DNA methylation in NSCLC cells, and co-IP-demonstrated GRIA3 was directly related with DNMT1 and DNMT3A.ConclusionsmiR-330-3p promoted the progression of NSCLC and might be a potential target for the further research of NSCLC brain metastasis.
Highlights
Brain metastasis (BM) is associated with poor prognosis in patients with non-small cell lung cancer (NSCLC)
Up-regulation of miR-330-3p in NSCLC cell lines and BM+ patients Our result showed that miR-330-3p was expressed in the normal human bronchial epithelial cell line BEAS2B, and five NSCLC cell lines, including A549, H460, HCC827, H1975, and PC-9 cells. miR-330-3p expression in those NSCLC cell lines was significantly higher than in BEAS-2B (P < 0.001, Fig. 1a). miR-330-3p expression levels were evaluated in a series of 122 NSCLC primary lung tissue samples, using quantitive real-time PCR (qRT-PCR), and were found to be significantly up-regulated in subjects with BM compared to subjects without BM upon diagnosis (P = 0.006, Fig. 1b)
Since AMPAR signaling to KRAS and mitogen-activated protein kinase (MAPK) pathways, promoted migration and invasion of cells [51], and Glutamate receptor 3 (GRIA3) acted as an important mediator of survival, proliferation, and migration of tumor cell, which, in pancreatic cancer, are regulated by CUX1 downstream of Phosphoinositide 3-kinase (PI3K)/AKT [52], we investigated the MAPK/extracellular-regulated protein kinases (ERK) and PI3K/AKT signaling pathways in the migration and invasion of NSCLC cells
Summary
Brain metastasis (BM) is associated with poor prognosis in patients with non-small cell lung cancer (NSCLC). Recent studies demonstrated that microRNA-330-3p (miR-330-3p) was involved in NSCLC brain metastasis (BM). Discovery and development of biomarkers and elucidation of the mechanism underlying BM in NSCLC is critical for effective prophylactic interventions. Lung cancer is the first leading cause of cancer-related deaths [1, 2], and non-small cell lung cancer (NSCLC) accounted for about 88% of primary lung malignancies [3,4,5]. Brain metastasis (BM) developed in approximately 25% of these patients [6]. MicroRNAs (miRNAs) have been utilized for the characterization of tumors [9, 10]. MiRNAs are small non-coding RNAs of 18–25 nucleotides that might impact various stages of development and progression of cancer [11, 12]. It has been reported that miRNAs can function as oncogenes or tumor suppressors [15,16,17]
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