HCT8 (ileocecal colorectal adenocarcinoma), HCT116 (colorectal carcinoma) and SW480 (Dukes' type B, colorectal adenocarcinoma) cell lines were purchased from the Cell Resource Center, Institute of Basic Medical Sciences (IBMS), Chinese Academy of Medical Sciences and Peking Union Medical College (CAMS/PUMC) (Beijing, China). They were maintained in Gibco™ Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) (both from Thermo Fisher Scientific, Inc., Waltham, MA, USA) in a humidified 5% CO₂/95% air atmosphere at 37°C.

The synthetic miR-197 mimic, miR-197 inhibitor, mimic control and inhibitor control were purchased from GenePharma Inc. (Shanghai, China), and their sequences are listed in Table I. The siRNAs for IGFBP3 and siRNA control were synthesized (Invitrogen; Thermo Fisher Scientific, Inc.) and the sequences are listed in Table II.

Transwell chambers (8-µm pore size; Costar; Corning Costar, Cambridge, MA, USA) were used in the in vitro migration assay. Colorectal cancer cells were transfected with the miR-197 mimic or the mimic control. Following 48 h, cells were detached with trypsin, washed with PBS and resuspended in serum-free medium. Two hundred microliters of cell suspension (1×10⁵ cells/ml for miR-197 mimic and its control, 2×10⁵ cells/ml for miR-197 inhibitor and its control) was added to the upper chamber, and 500 ml of complete medium was added to the bottom well. The cells that had not migrated were removed from the upper surfaces of the filters using cotton swabs, and the cells that had migrated to the lower surfaces of the filters were fixed with 4% paraformaldehyde solution and stained with crystal violet. Images of 3 random fields (×10 magnification) were captured from each membrane, and the number of migratory cells was counted as previously described (14). Similar inserts coated with Matrigel were used to determine the invasive potential.

Total RNA was extracted using the Invitrogen™ TRIzol total RNA isolation reagent (Thermo Fisher Scientific, Inc.) and purified with the Column DNA Erasol kit (Tiangen Biotech Co., Ltd., Beijing, China) according to the manufacturer's instructions. mRNA levels were assessed with RT-qPCR using SYBR-Green I (Takara Biotechnology Co., Ltd., Dalian, China). The gene expression level was normalized to the endogenous reference gene GAPDH. The experiments were performed in triplicate. The primers for RT-qPCR are listed in Table III. The primers for miR-197 and U6 were purchased from Qiagen (Duesseldorf, Germany) (Table IV). Reverse transcription of miRNAs was performed with a miScript Reverse Transcription kit (Qiagen). The expression of mature miRNAs was determined using miRNA-specific quantitative RT-qPCR (Takara Biotechnology Co., Ltd.). The expression levels were normalized to the U6 endogenous control and assessed by the comparative Ct (ΔΔCq) method (15).

Since miRNAs commonly act on posttranscriptional regulation by targeting 3′UTRs of mRNAs of target genes, in order to ascertain the mechanism underlying the involvement of miR-197 in biological processes, we first searched for possible target genes of miR-197 in the database on the web server ‘Targetscan’ (http://www.targetscan.org/vert_71/). Subsequently, we determined the candidate genes for our study from this repertoire of target genes by carefully reviewing corresponding literature and selecting the most possible target genes which had been reported to participate in cell migration, invasion and tumor metastasis.

Fragments from 3 candidate genes including ADAMTS5, CBL and IGFBP3 whose 3′-UTRs contained the predicted binding site for hsa-miR-197, and flanking sequences on each side were synthesized with a short extension containing cleavage sites for XhoI (5′-end) and NotI (3′-end) (Table V); second fragments containing mutated binding site sequences were also synthesized. The two constructs were termed WT (Gene-wild type) and MT (Gene-mutant). The fragments were cloned into the psiCHECK™-2 vector (Promega Corporation, Madison, WI, USA). Ten nanograms of WT, MT or control vector and 200 nmol/l miR-197 mimic were transfected into 293T cells (293T cells were supplied by the Cell Center of Institue of Basic Medical Sciences, Chinese Academy of Medical Sciences) using Invitrogen™ Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The cells were harvested 24 h following transfection and assayed for Renilla and firefly luciferase activities using the Dual-Luciferase Reporter Assay System (Promega Corporation).

Following washing twice with PBS, cells were lysed in ice-cold Radio Immunoprecipitation Assay (RIPA) lysis buffer (Beyotime Institute of Biotechnology, Nanjing, China) and manually scraped from the culture plates. Proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels, electroblotted onto a polyvinylidene difluoride (PVDF) membrane and incubated with anti-IGFBP3 antibody (1:1,000 dilution; cat. no. sc-135947; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-GAPDH antibody (1:1,000 dilution; cat. no. sc-47724; Santa Cruz Biotechnology, Inc.), followed by incubation with a secondary anti-rabbit or anti-mouse horseradish peroxidase-conjugated antibody (1:1,000; Zhongshan Golden Bridge Biotechnology, Beijing, China). Antibody-antigen complexes were detected using a chemiluminescent ECL reagent (EMD Millipore, Billerica, MA, USA).

Fresh frozen samples of colorectal adenocarcinomas were collected from 21 patients who underwent surgery at Peking Union Medical College Hospital (PUMCH) from May 2015 to July 2015. All patients had signed written informed consents prior to enrolling in the study before admission. The use of human subjects in this study was also approved by the Ethics Committee at Peking Union Medical College Hospital (Beijing, China). This cohort includes 11 males and 10 females patients with average age of 60.5 (46–72 years). Basic clinicopathological features of these 21 patients are listed in Tables VI and VII.

IGFBP3 expression was evaluated by IHC staining. Briefly, after 5-µm sections were deparaffinized, antigen retrieval was performed by use of heat-induced epitoperetrieval with 10 mM citrate buffer. Sections were incubated with a monoclonal anitbody against IGFBP3 (cat. no. sc-135947 Santa Cruz Biotechnology, Inc.) at 1:250 dilution. The IGFBP3 antibody was detected using the avidin-biotin-peroxidase technique (Dako LSAB kit; Dako, Carpinteria, CA, USA). The expression levels of IGFBP3 were determined by a pathologist. The classification of ‘- and +’ was defined by the percentage of IGFBP3 positive cells at the level of <5 and ≥5%, respectively.

Comparisons between different groups in migration and invasion assays were analyzed using t-tests (two-sided). ANOVA followed by Dunnette's test was applied to compare intergroup differences in luciferase reporter assays. Correlation between miR-197 and IGFBP3 expression was determined by correlate bivariate Spearman assay. Chi-square Fisher's exact test were used to calculate differences of miR-197 expression between different groups of clinicopathological factors. All data were calculated using SPSS 19.0 (IBM Corp., Armonk, NY, USA) and two-sided P-value <0.05 was considered to indicate a statistically significant difference.

We first evaluated the efficacy of miR-197 overexpression and inhibition using synthetic miR-197 mimic, miR-197 inhibitor and corresponding controls in CRC cell lines (Fig. 1). We then investigated the effect of miR-197 on the migration and invasion in colorectal cell lines. HCT8, HCT116 and SW480 cells were transfected with either miR-197 mimic, mimic control, miR-197 inhibitor or inhibitor control and then subjected to cell migration and invasion assays (Fig. 2). In the HCT8 cell line, significantly more tumor cells in the miR-197 mimic group had migrated to the lower surface of the filter compared with the miR-197 mimic control group (33.50±4.65 vs. 18.00±3.91, P<0.05) in the migration assay and similarly, significantly more cells invaded to the lower surface in the miR-197 mimic group than its control group in the invasion assay (24.75±2.21 vs. 18.00±3.91, P<0.05, Fig. 2A). On the contrary, significant less HCT8 cells in miR-197 inhibitor group were found on the lower surface of the filter compared with its control group in the migration assay (32.25±2.98 vs. 47.25±3.20, P<0.05) and also in the invasion assay (31.00±3.16 vs. 46.75±4.42, P<0.05, Fig. 2A). Furthermore, similar results were also achieved in the HCT116 and SW480 cells (Fig. 2B and C), indicating that miR-197 could regulate migration and invasion in these 3 CRC cell lines in vitro.

The mRNAs which may be targeted by miR-197 were predicted, and genes associated with tumor invasion and metastasis were selected. According to this principle, we screened out 3 candidate target genes including IGFBP3, ADAMTS5 and CBL. Predicted miR-197 binding sites within the 3′UTR of mRNAs of these 3 genes are listed in Figs. 3A, 4B and D, respectively.

Compared to the control group, miR-197 mimic significantly decreased the luciferase activity when co-transfected with IGFBP3-WT reporter plasmid (Fig. 3B, P<0.05). However, this inhibitory effect on luciferase activity mediated by miR-197 mimic was abolished by mutant reporter plasmid (Fig. 3B, P<0.05). In addition, miR-197 overexpression had no effect on the luciferase activities of ADAMTS5 (P=0.974, Fig. 4A) and CBL (P=0.098, Fig. 4C). We decided to choose IGFBP3 as research target of our study due to its tight connection with CRC according to multiple studies.

To demonstrate whether miR-197 could exert a similar influence on IGFBP3 expression in CRC, we transfected HCT8 cell line again with the miR-197 mimic, miR-197 inhibitor or their corresponding controls, and then determined IGFBP3 expression at the RNA and protein levels, respectively. The protein level of IGFBP3 in HCT8 cells was obviously decreased following transfection with miR-197 mimic and increased following transfection with miR-197 inhibitor (Fig. 3C), while the alterations of IGFBP3 mRNA did not reach statistical significance upon overexpression and inhibition of miR-197 (P>0.05, Fig. 3D). These findings revealed that miR-197 suppressed the protein output of IGFBP3 rather than degrading its mRNA, indicating that IGFBP3 was regulated by miR-197 in CRC cells at the post-transcriptional level.

We then investigated the role of IGFBP3 in CRC cell migration and metastasis. Small interfering RNA (siRNA) designed to target IGFBP3 was employed. The transfection of siRNA resulted in a significant reduction in IGFBP3 mRNA and protein level in HCT8 and HCT-116 cells compared to the siRNA-control (Fig. 5). Both cell lines were transfected with either IGFBP3 siRNA (si-IGFBP3) or siRNA-control, and then subjected to cell migration and cell invasion assay. In the migration assay, the number of migratory HCT8 cells in the si-IGFBP3 group was significantly greater than the migratory HCT8 cells in the si-control group (54.75±4.57 vs. 32.75±7.18, P<0.05, Fig. 6A and B). In addition, significantly more HCT8 cells in the si-IGFBP3 group had invaded to the lower surface of filters compared with those in si-control group (12.75±3.50 vs. 2.75±1.70, P<0.05) in the invasion assay (Fig. 6A and B). Similar results were also achieved in HCT-116 cells (Fig. 6C and D). Cell motility of both assays was significantly enhanced following IGFBP3 downregulation compared with the control in HCT-116 cells, with similar results in HCT8 cells, which indicated that IGFBP3 was a suppressor protein in the process of migration and invasion in CRC cells.

We assessed the miR-197 quantity of cancer tissues using RT-PCR in fresh frozen samples and IGFBP3 expression using IHC in corresponding paraffin samples in order to analyze the correlation between miR-197 and IGFBP3 expression in CRC patients. In this cohort of 21 stage III CRC patients, there were one stage IIIA cancer, 2 stage IIIC cancers, and 18 patients with stage IIIB cancer; 13 patients had colon cancer and 8 patients had rectum cancer. The median ΔΔCq value of miR-197 was 7.64 (2.2–8.77). We dichotomized IGFBP3 protein expression using ‘positive’ or ‘negative’ as displayed in Fig. 7. There were 8 IGFBP3-positive and 13 IGFBP3-negative tumors. We revealed that the miR-197 ΔΔCq level was significantly associated with the expression of IGFBP3 with statistical significance (P=0.026), so that miR-197 expression was negatively related to IGFBP3 protein expression in the CRC tumor tissues.

Furthermore, we divided miR-197 expression level into two groups using the median ΔΔCq value (≤7.64 vs. >7.64), and explored whether there was any difference in miR-197 expression between the different groups of clinicopathological factors. Although we discovered a trend that there were more miR-197 high expression tumors in the T4 group compared to the T2-3 group, it was not statistically significant (P=0.09), and thus were other factors such as lymph node stage, histology, lymphovascular involvement and tumor site (Table VI). In addition we did not detect any differences in the expression of IGFBP3 between different groups of pathological features (Table VI). Finally, we aimed to clarify the association between prognosis and differential expression of miR-197 plus IGFBP3, but we failed due to the limited events of recurrence and death. Basic clinical information of this cohort of CRC patients is listed in Table VII.