Tumor tissues were collected from 134 patients who received surgery between 2003 and 2013 at Osaka University Hospital and its three related facilities: 96 primary CRC tumors with stage I (n=18), II (n=38), and III (n=40) disease without metastasis, 22 primary CRC tumors with simultaneous liver metastasis (stage IV), and 16 liver metastatic lesions of CRC. All samples were immediately frozen in RNAlater (Ambion, Austin, TX, USA) and stored at −80°C until RNA extraction. This study was approved by the institutional review board of each institution, and all subjects provided written informed consents before participation. The clinical parameters of the validating cohort are shown in Table I.

Human CRC cell lines DLD-1 (KRAS G13D), HCT116 (KRAS G13D), HT29 (BRAF V600E), and SW480 (KRAS G12V) were purchased from the American Type Culture Collection (Rockville, MD, USA) in 2001. Cell lines were maintained in Dulbecco's modified Eagle's medium (Sigma-Aldrich, St. Louis, MO, USA) with 10% fetal bovine serum in a humidified 5% CO₂ at 37°C.

miR-487b and negative control miRNA were purchased from Gene Design Inc. (Osaka, Japan). These were transfected into cells 24 h after seeding with Lipofectamine RNAiMAX reagent (Invitrogen, Carlsbad, CA, USA) at a final concentration of 30 nmol/l, according to the manufacturer's protocol.

Transfected cells were seeded at a density of 2.5–3.0×10⁴ per well in 24-well dishes. After cultured for 24, 48 and 72 h, cells were trypsinized and stained with trypan blue solution (Invitrogen). The total number of cells in each well was determined with Countess Automated Cell Counter (Invitrogen).

Transfected cells were seeded at a density of 500 per well in 6-well plates. After incubation at 37°C for 10 days, cells were washed with PBS, fixed with formalin and stained with Giemsa solution. The number of visible colonies was counted with ImageJ software (National Institutes of Health).

To measure cell invasion, transwell inserts with 8-µm pores (BD Biosciences, San Jose, CA, USA) were used according to the manufacturer's protocol. The invading cells were fixed and stained with Diff-Quik (Sysmex, Hyogo, Japan), and counted under a light microscope in three random fields (×40 magnification).

Total RNA was isolated using miRNeasy Mini kit (Qiagen, Hilden, Germany) for clinical tissues and TRIzol reagent (Invitrogen) for cell lines following the manufacturer's protocol. RNA concentration and purity were assessed with NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE, USA).

For miRNA quantification, TaqMan microRNA assays (Applied Biosystems, Foster City, CA, USA) were used: hsa-miR-487b ID 001285, and RNU6B ID 001093. Total RNA was reverse transcribed using TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems). qRT-PCR was performed with ABI PRISM 7900HT Sequence Detection system (Applied Biosystems) using TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems). Relative expression was quantified with the ΔΔCq method (20).

For mRNA quantification, total RNA was reverse transcribed using High Capacity RNA-to-cDNA kit (Applied Biosystems). To measure KRAS expression level, qRT-PCR was performed with LightCycler 480 Real-Time PCR system (Roche Diagnostics, Mannheim, Germany) using the specific primers and LightCycler-DNA Master SYBR Green I (Roche Diagnostics) (21). The specifically designed primers were as follows: ACTB forward, 5′-GATGAGATTGGCATGGCTTT-3′; reverse, 5′-CACCTTCACCGTTCCAGTTT-3′. KRAS forward, 5′-ATTCCTTTTATTGAAACATCAGCA-3′; reverse 5′-TCGGATCTCCCTCACCAAT-3′. For LRP6 expression analysis, qRT-PCR was performed with ABI PRISM 7900HT Sequence Detection system (Applied Biosystems) using TaqMan Gene Expression Assays (Applied Biosystems) and TaqMan Universal PCR Master Mix, No AmpErase UNG (Applied Biosystems). The product numbers of TaqMan Gene Expression assay were as follows: ACTB ID Hs99999903_m1, LRP6 ID Hs00999795_m1. Relative expression was quantified using the ΔΔCq method (20).

Western blot analysis was performed as described previously (22). Briefly, cells were collected and lysed in RIPA buffer, containing phosphatase inhibitor and protease inhibitor cocktail. The protein lysates (20 µg) from each sample were separated with sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE), and transferred electronically to a polyvinylidene difluoride membrane. The membrane was blocked with 5% skimmed milk and incubated with anti-human polyclonal antibodies against ERK, phosphorylated ERK, phosphorylated LRP6 (Cell Signaling Technology, Beverly, MA, USA), ACTB (Sigma-Aldrich), and anti-human monoclonal antibodies against AKT1, phosphorylated AKT, cleaved PARP, LRP6 (Cell Signaling Technology), KRAS (Sigma-Aldrich). The membrane was incubated with secondary antibodies, and visualized with the ECL Detection system (GE Healthcare, Little Chalfont, UK).

For serum response element (SRE) luciferase reporter assay, the vector (pGL4.33[luc2P/SRE/Hygro]) was obtained from Promega (Madison, WI, USA). For pmirGLO luciferase reporter assay, the vector was made as follows. The 3′-UTR of human LRP6 mRNA containing the putative miR-487b binding site was amplified by polymerase chain reaction (PCR). The primer sequences were; forward, 5′-GCTCGCTAGCCTCGAAGCAGGATGGGCGATAGA-3′; reverse 5′-ATGCCTGCAGGTCGATGGACAAGGGCTGACCAA-3′. The amplified DNA product was cloned into the restriction site downstream of the firefly luciferase gene in pmirGLO vector (Promega). The vector with mutant 3′-UTR sequence was constructed using the QuikChange Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer's protocol.

Cells were seeded in 96-well plates and transfected with both reporter vector and miRNA using Lipofectamine 2000 (Invitrogen), and collected 48 h after co-transfection. Luciferase activity was measured using Dual Luciferase Reporter assay system (Promega), as described previously (16,23). Briefly, the cell extracts were prepared by rinsing each plate twice with PBS and lysing the cells in Passive Lysis buffer (Promega). The transfection efficiency was evaluated with renilla luciferase activity, and firefly luciferase activity was normalized.

The data are expressed as the mean ± standard deviation. Statistical differences were analyzed by the Student's t-test for continuous variables and by the Chi-square test for the others. Survival curves were drawn by the Kaplan-Meier method, and compared using the log-rank test. The Cox proportional hazard regression model was used to estimate the hazard ratio (HR) and the 95% confidence interval (CI). All statistical analyses were conducted with JMP ver. 11.0.0 (SAS Institute, Inc., Cary, NC, USA). P-values of <0.05 were considered to be statistically significant.

As a testing cohort, we used microarray data that were previously registered in the NCBI Gene Expression Omnibus database available through GEO with accession number GSE72199, consisting of tumor tissues derived from 36 patients: 16 primary CRC tumors with stage II and III disease without metastasis, 12 primary CRC tumors with simultaneous liver metastasis (stage IV), and 8 liver metastatic lesions of CRC (16). Consequently, 38 miRNAs that showed significantly differential expression with fold change >2.0 in microarray analyses were identified by comparison between primary tumors without metastasis and liver metastatic lesions (Table II). Among them, we found that the expression of miR-487b, which is reportedly shown as an anti-oncomir in several human malignancies (17–19), significantly decreased in liver metastatic lesions as compared to primary tumors without metastasis (P=0.030, Fig. 1A). qRT-PCR validated the result in the same testing cohort (n=36, P=0.001, Fig. 1B). To confirm the statistical correlation between the results of microarray and qRT-PCR, Spearman's rank order correlation coefficient was calculated, which showed a significant correlation (ρ=0.487, P=0.003, Fig. 1C).

To confirm the differential expression of miR-487b between primary tumors without metastasis, and liver metastatic lesions, miR-487b expression was measured by qRT-PCR in the validating cohort of 134 patients: primary tumors without metastasis (n=96), primary tumors with liver metastasis (n=22), and liver metastatic lesions (n=16). It was confirmed that miR-487b expression in liver metastatic lesion was significantly lower than that in primary tumors without metastasis (P=0.041, Fig. 2A). In addition, we found that miR-487b expression significantly decreased in primary tumors with liver metastasis, as compared to that without metastasis (P=0.049, Fig. 2A). When we examined miR-487b expression in a pair of CRC tissue and its corresponding synchronous liver metastasis (n=7), metastatic lesions tended to express lower levels of miR-487b than the primary tumors (P=0.0996, Fig. 2B). miR-487b expression in tumor tissues was significantly higher than that in their pair-matched adjacent normal mucosal tissues (P=0.007, Fig. 2C).

To reveal the impact of miR-487b expression on patient prognosis, 72 CRC patients were divided into two groups according to the median value of the miR-487b expression. Kaplan-Meier survival curve showed that the patients with high expression of miR-487b (n=36) demonstrated better prognosis for overall survival (OS) than those with low miR-487b expression (n=36, P=0.036, median follow-up 56.4 months, Fig. 2D). Univariate analysis showed that tumor depth (P<0.0001), tumor differentiation (P=0.040), lymph node metastasis (P=0.010), lymphatic invasion (P=0.002), clinical stage (P<0.0001), and miR-487b expression (P<0.032) were significant prognostic parameters (Table III). Multivariate analysis revealed that miR-487b expression was an independent prognostic factor for 5-year OS, (RR of 4.164, 95% CI of 0.035), in addition to clinical stage (Table III). Clinical and pathological survey showed that miR-487b expression was significantly associated with tumor differentiation (P=0.040, Table IV).

We then performed in vitro experiments using human CRC cell lines. First, qRT-PCR revealed that the miR-487b levels of representative four CRC cell lines were low compared to clinical normal tissue samples (Fig. 3A). The ectopic expression of miR-487b resulted in reduction of cell viability at 48 and 72 h after transfection in HCT116, and at 72 h in DLD-1 (Fig. 3B). Increased expression of miR-487b was validated at 48 h by qRT-PCR in both cell lines (Fig. 3C). In colony formation assay, the numbers of colonies significantly decreased in miR-487b transfected cells as compared to negative control cultures in both cell lines (Fig. 3D). We then assessed cell invasion, since it is thought to play a crucial role at an initial step of metastasis. miR-487b transduced cells showed a marked reduction in the invasion ability in both cell lines (Fig. 3E). These results indicate that miR-487b suppresses cell proliferation, and more evidently inhibits invasion ability in CRC.

To reveal the underlying mechanisms of how miR-487b would suppress proliferation and invasion ability, we explored KRAS signaling pathway because there is evidence that miR-487b targets 3′-UTRs of KRAS mRNA in lung cancer cell lines (17) (Fig. 4A). At 48 h after transfection, KRAS mRNA levels in miR-487b transfected cells significantly decreased compared with negative control cultures of HCT116 and DLD-1 cells (Fig. 4B), which was clearly demonstrated with western blot analysis at a protein level (Fig. 4C). Additional studies were undertaken to examine the impact of miR-487b on downstream molecules of the KRAS signaling pathway. Phosphorylation of ERK and AKT in the miR-487b transfected cells decreased when compared to negative control cultures (Fig. 4D). SRE reporter assay revealed that miR-487b significantly inhibited SRE luciferase activities, indicating that the MAPK/ERK signaling pathway was partially blocked by miR-487b (Fig. 4E). Since miR-487b could also regulate AKT signaling pathways, we next investigated whether miR-487b would induce apoptosis. We found that introduction of miR-487b increased cleaved PARP expression in HCT116 cells (Fig. 4F), but not in DLD-1 cells (data not shown).

By using the target prediction tool (TargetScan 7.0, and microRNA.org.), we found that miR-487b might bind to the 3′-UTR of LRP6, a Frizzled (FZD) co-receptor for WNT signaling pathway (Figs. 5A and 6). After miR-487b was co-transfected with a reporter plasmid harboring LRP6 3′-UTR sequence, luciferase activities of the reporter were significantly reduced in HCT116 and DLD-1 cell lines (Fig. 5B), whereas, such decrease was not observed with mutant LRP6 3′-UTR sequence, indicating that LRP6 could be a direct target of miR-487b. At 48 h after transfection, LRP6 levels significantly decreased in both cell lines at mRNA and protein levels by treatment of miR-487b (Fig. 5C and D). Moreover, transfection of miR-487b reduced the phosphorylated LRP6 protein (Fig. 5D), which was thought to be crucial for signal transduction in the WNT pathway.