MicroRNA-130b functions as an oncomiRNA in non-small cell lung cancer by targeting tissue inhibitor of metalloproteinase-2

Non-small cell lung cancer (NSCLC) is the most frequent cause of cancer-related death worldwide. Although many molecular-targeted drugs for NSCLC have been developed in recent years, the 5-year survival rate of patients with NSCLC remains low. Therefore, an improved understanding of the molecular mechanisms underlying the biology of NSCLC is essential for developing novel therapeutic strategies for the treatment of NSCLC. In this study, we examined the role of miR-130b in NSCLC. Our results showed that high expression of miR-130b in clinical specimens was significantly associated with poor overall survival in patients with NSCLC. Moreover, miR-130b expression was significantly increased in NSCLC clinical specimens from patients with vascular and lymphatic invasion. Consistent with this, overexpression of miR-130b promoted invasion and matrix metalloproteinase-2 (MMP-2) activity in A549 cells. Argonaute2 immunoprecipitation and gene array analysis identified tissue inhibitor of metalloproteinase-2 (TIMP-2) as a target of miR-130b. Invasion activity promoted by miR-130b was attenuated by TIMP-2 overexpression in A549 cells. Furthermore, TIMP-2 concentrations in serum were inversely correlated with relative miR-130b expression in tumor tissues from the same patients with NSCLC. Overall, miR-130b was found to act as an oncomiR, promoting metastasis by downregulating TIMP-2 and invasion activities in NSCLC cells.

in 96-well plates (500 cells/well) for anchorage-dependent assays or Nunclon Sphera 96F-well plates (1000 cells/well; Nunc) for anchorage-independent assays. Cells were thenincubated for the indicated times, and WST-8 reagent (Dojindo, Japan) was then added. Cells were incubated for 2 h at 37°C in an atmosphere containing 5% CO 2 , and the optical density was determined at a wavelength of 450/630 nm (measurement/reference) using an iMark microplate reader (Bio-Rad, Hercules, CA, USA).

Wound healing assay
A549 cells stably overexpressing miR-130b were seeded in 24-well plates (1 × 10 5 cells/well) and incubated for 48 h. A wound was created in the cell monolayer at ~90% confluency using a sterile 1-ml pipette tip. Images were acquired at 0 and 48 h after wound creation using an OLYMPUS IX71 fluorescence microscope (Olympus, Japan).

RNA extraction
miRNAs were purified from NSCLC cell line 24h after transfection using an miRNeasy mini kit (Qiagen). Total RNA was isolated 24h after transfection using TRIzol reagent (Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions.

Cell invasion assay
NCI-H520 cells were reseeded into inserts in 96-well plates (1.25 × 10 4 cells/well) in serum-free conditions 24 h after transfection. NCI-H520 cells were incubated for 24 at 37°C in an atmosphere containing 5% CO 2 .

Ago2-IP and gene array analysis
Ago2-IP was performed using an IP kit (RNA-binding protein IP-assay kit for microRNA; MBL, Japan) following the manufacturer's instructions. Briefly, RNAs immunoprecipitated with anti-ago2 antibodies were eluted from beads with hemagglutinin peptide (Wako), and QIAzol reagent (Qiagen) was added to extract RNAs. Ago2-bound RNAs were cleaned using a miRNeasy column and then subjected to microarray analysis. After confirming the purity of total RNAs by Experion (Bio-Rad), cDNA was synthesized using a WT expression kit (Applied Biosystems, Foster City, CA, USA), biotinylated using a GeneChip WT terminal labeling kit (Affymetrix, USA), and then injected into a GeneChip Human 1.0 ST array (Affymetrix). After hybridization, the GeneChip was washed and stained using a GeneChip hybridization wash and stain kit (Affymetrix) with a GeneChip fluidics station (Affymetrix). The stained GeneChip was then scanned using a GeneChip scanner 3000 (Affymetrix). Partek Genomics Suite 6.6 (Molsis, Japan) was used for data analysis (GEO accession number: GSE118274).

Sample mock 130b
Cell number ( zinc and ring finger 3

Supplementary Figure 2. miR-130b expression in NSCLC cell lines.
Expression of miR-130b in NSCLC cell lines (A) and in a clone isolated from A549 cells stably expressing miR-130b (B) was examined by real-time qPCR. Relative expression of miR-130b normalized to U6 snRNA is shown from duplicate experiments.