Antibodies against iASPP (cat. no. ab115605) and GAPDH (cat. no. KC-5G5) were purchased from Abcam (Cambridge, UK) and Kangcheng Bio (Beijing, China), respectively. Hoechst staining kit (cat. no. H1399) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). IgG-HRP antibody (cat. no. BA1054) was purchased from Wuhan Boster Biological Technology Ltd. (Wuhan, China).

CRC specimens were collected from 30 patients (from July 2015 to October 2016) in The Chinese People's Liberation Army General Hospital. The specimens were fixed and prepared in paraffin sections. The patients enrolled in the present study were diagnosed with primary CRC and had detailed clinicopathological and prognostic information. Screening, inspection and data collection were approved by the Ethics Committee of The Chinese People's Liberation Army General Hospital, and a written informed consent form was signed by all subjects. The procedures performed adhered to the Declaration of Helsinki.

Immunohistochemistry was used to detect the expression of iASPP protein in tissues, according to the operation guide of immunohistochemistry. Paraformaldehyde (4%) fixed tissues and paraffin embedded sections for antigen retrieval were used. Samples were then incubated overnight at 4°C with iASPP antibodies (1:400). After being washed with PBS, samples were incubated for 50 min at RT with IgG-HRP antibodies (1:500). Then the samples were observed and images were captured by an optical microscope (CX41-23C02; Olympus Corp., Tokyo, Japan).

Human CRC cell lines FHC, HCT116, HCT8, HT29, H1299 and SW480 were obtained from Shanghai Bioleaf Biotech, Co., Ltd., (Shanghai, China). The cells were cultured in minimum essential medium (MEM, M2279; Sigma-Aldrich, St. Louis, MO, USA) with 15% fetal bovine serum (FBS; 10099-141; Gibco, Carlsbad, CA, USA) and 1% (v/v) antibiotics mix and maintained in an atmosphere of 95% air and 5% CO₂ at 37°C. The expression level of miR-150 was determined using reverse transcription real-time PCR (qPCR) as described in the following sections. The cell line with the lowest expression level of miR-150 was selected for subsequent assays and, based on the results of qPCR, SW480 cell line had the lowest expression level of miR-150 (Fig. 1D) and was employed as an in vitro model for CRC.

Specific siRNA targeting iASPP (5′-AGTTCATGTCCAGAAAGTCCC-3′) and non-targeting siRNA (5′-ACGUGACACGUUCGGAGAATT-3′) were used to knockdown the expression of iASPP. Coding sequences were cloned through amplification reaction using primers (iASPP forward, 5′-GGGGTACCATGGACAGCGAGGCATTCC-3′ and iASPP reverse, 5′-CCGCTCGAGCTAGACTTTACTCCTTTGAGGCTTCAC-3′). Subsequently, the PCR product (2487 bp) was ligated to the pcDNA3.0 plasmid, and recombinant plasmid was confirmed by sequencing after digestion with KpnI/XhoI. SW480 cells were transfected with different vectors using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific). The coding sequence of iASPP was ligated into the pcDNA plasmid to form the pcDNA-iASPP vector for overexpression of the gene.

To detect the function of miR-150 in the oncogenesis of CRC, SW480 cells were divided into two groups: i) NC group, SW480 cells transfected with NC mimics; and ii) mimics group, SW480 cells transfected with miR-150 mimics. Each group was represented by at least five replicates.

To elucidate the key role of iASPP in the progression of CRC, SW480 cells were divided into three groups: i) Blank group, SW480 cells; ii) NC group, SW480 cells transfected with pcDNA-NC plasmid; and iii) siRNA group, SW480 cells transfected with pcDNA-siiASPP plasmid. Each group was represented by at least five replicates.

The interaction between miR-150 and iASPP was further assessed with four groups: i) blank group, SW480 cells; ii) NC group, SW480 cells transfected with NC mimics; iii) mimics group, SW480 cells transfected with miR-150 mimics; and iv) mimics+pcDNA group, miR-150 stably overexpressed in SW480 cells transfected with pcDNA-siiASPP plasmid.

The direct regulating function of miR-150 on the 3′UTR of iASPP was determined with a Dual-Luciferase assay. Luciferase activity was detected by Dual-Luciferase assay kit (E1960; Promega, Madison, WI, USA) after 24 h of transfection and co-transfection of Renilla luciferase plasmid, used as the internal control for transfection efficiency.

Total RNA in the cells was extracted by RNA purification using RNA Extraction kit (9109; Takara Bio, Inc., Otsu, Japan) accordingly. β-actin and U6 were selected as the internal reference genes. cDNA templates were achieved by using Super MMLV Reverse Transcriptase (DBI-2342; DBI Bioscience, Shanghai, China), and the final RT-qPCR reaction mix contained 10 µl Bestar^® SYBR Green qPCR Master Mix, 0.5 µl of each primer (miR-150, forward 5′-ACACTCCAGCTGGGTCTCCCAACCCTTGTACC-3′ and reverse, 5′-CTCAACTGGTGTCGTGGA-3′; iASPP, forward, 5′-GAAAGCCTGGAACGAGTCTGA-3′ and reverse, 5′-GCGCTAGTGAGGTTGTCCT-3′; U6, forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; and GAPDH, forward, 5′-TGTTCGTCATGGGTGTGAAC-3′ and reverse, 5′-ATGGCATGGACTGTGGTCAT-3′), 1 µl cDNA template and 8 µl RNase-free H₂O. Amplification was performed as follows: a denaturation step at 94°C for 2 min, followed by 40 cycles of amplification at 94°C for 20 sec, 58°C for 20 sec and 72°C for 20 sec. The reaction was stopped at 25°C for 5 min. The relative expression levels were detected and analyzed by Exicycler™ 96 (Bioneer Corp., Daejeon, Korea) based on the formula of 2^−∆∆ct.

CCK-8 assay was performed to detect cell viability. Briefly, SW480 cells (1×10⁵ cells/ml) that underwent different treatments were seeded into one well of a 96-well plate and incubated for 72 h. Every 24 h, 10 µl of CCK-8 solution was added to every well and incubated at 37°C for a minimum of another 4 h. The OD values were detected at 450 nm and employed as the representative of cell viability.

Cell cycle distribution was determined using flow cytometry. Cells were stained with propidium iodide (PI) in the dark for 20 min at room temperature. The results were analyzed using a FACS flow cytometer (BD Accuri C6; BD Biosciences, San Jose, CA, USA).

DNA damage in cell nuclei was detected using a Hoechst staining method after cells were transfected for 24 h. Cells were stained with Hoechst 33258 (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's instructions.

To evaluate cell mobility, a scratch assay was performed on the transfected cells. Cells at a density of 2×10⁴ cells/well were incubated in one well of a 24-well plate. After marking reference points, cells were cultured to confluence at 37°C for 48 h. Then, a cell-free line was made (regarded to as a scratch), and debris at the edges was removed. Twenty-four hours after the scratch was made, gap distances between the midline were assessed using an optical microscope (Olympus Corp.) in reference to the reference points.

Transwell assays were performed to detect the mobility of SW480 cells. In brief, cells at a density of 2×10⁴ cells/well were incubated in the upper chamber (Corning Costar, Cambridge, MA, USA) after a 24-h serum-free incubation, and the chamber was pre-coated with 40 µl Matrigel (0.8 µg/µl) at 37°C for 2 h. Then, the system was placed at 37°C for 24 h and, subsequently, cells in the upper surface were completely removed. The lower surfaces of the chamber were stained for 5 min using 1% (w/v) crystal violet and the cell number was recorded using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

Total cellular protein was extracted using the protein lysate. Western blot analysis was conducted according to Lin et al (22). The membranes were incubated with primary antibodies against iASPP (1:4,000) and GAPDH (1:10,000) for 1 h at room temperature. Secondary HRP-conjugated IgG antibodies (1:20,000) were then added and incubated for 45 min at room temperature. The blots were developed and the results were recorded in the Gel Imaging System (ScanWizard Bio; Microtek International, Inc., Taipei, Taiwan). Then, data were analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Inc.).

Data were expressed as the mean ± standard deviation (SD). Student's t-test was performed with a significance level of 0.05. Statistical analyses and graphing were performed using GraphPad Prism 6.01 (GraphPad Software, Inc., Chicago, IL, USA).

The expression status of miR-150 was determined with qPCR validation in clinical CRC samples and corresponding para-carcinoma tissues from patients (Table I). As displayed in Fig. 1, the level of miR-150 in CRC samples was lower than that in para-carcinoma samples and the difference was statistically significant (Fig. 1B; P<0.05). On the contrary, the expression of iASPP at the mRNA level was induced in CRC samples (Fig. 1C). Based on the detection in clinical samples, it was inferred that miR-150 had an anti-CRC effect during the progression of the tumor. Furthermore, miR-150 expression was assessed, and lower expression was detected in SW480 and HCT116 cells, and relatively higher expression presented in SW620 and HT29 cells compared with the FHC cell line (Fig. 1D). The expression of iASPP was also investigated using immunohistochemistry in clinical tissues from CRC patients. As displayed in Fig. 1A, the expression of iASPP was relatively higher in tumor tissue than para-carcinoma tissue.

The effect of miR-150 overexpression on normal cell features was determined to validate the antitumor effect of miR. Being induced by transfection of specific mimics, the upregulated expression of miR-150 significantly decreased the OD450 value in the mimics group at 72 h (Fig. 2A), representing impaired viability of SW480 cells. Furthermore, the overexpression of miR-150 induced G1 cell cycle arrest and cell apoptosis in the mimics group, as a larger proportion of cells were distributed in the G1 phase (Fig. 2B) and more Hoechst-positive cells (Fig. 2C) were detected in cells transfected with miR-150. Detection focusing on the proliferation potential of SW480 cells confirmed the conclusion that miR-150 is an anti-CRC molecule that influences CRC cells both by decreasing cell proliferation and inducing cell apoptosis.

The effect of miR-150 overexpression was further assessed by detecting its effect on the metastasis potential of SW480 cells. For cells subjected to the scratch assay, induced expression of miR-150 resulted in a delayed closure rate of the gap (wider gap width) (Fig. 3A). Furthermore, impaired invasion ability was also detected in SW480 cells with overexpression of miR-150 (Fig. 3B), as less cells penetrating the membranes were recorded in the mimics group. The results of the scratch and Transwell assays together indicated the inhibitory effect of miR-150 on the metastasis potential of CRC cells.

Based on bioinformatic analysis, iASPP was a potential target of miR-150. In the present study, a possible interaction between the two indicators was determined using a dual-luciferase assay. The results indicated that only cells transfected with miR-150 mimics and wild-type iASPP 3′UTR demonstrated a decreased relative luciferase activity (Fig. 4), which indicated a direct and specific modulating effect of miR-150 on the iASPP gene.

To confirm the results that iASPP promoted the progression of CRC, the effects of iASPP knockdown on cell viability, cell apoptosis, cell cycle distribution and cell migration and invasion were also assessed. The expression of iASPP was confirmed by western blot analysis after transfection with siRNA (Fig. 5A). The results indicated that iASPP knockdown exhibited a similar effect on SW480 cells to that of miR-150 overexpression: in the siRNA group, cell viability was decreased (Fig. 5B), G1 cell cycle arrest and apoptosis were induced (Fig. 5C and D) and cell migration and invasion were impaired (Fig. 6).

Given the direct regulating function of miR-150 in the transcription of iASPP, it was hypothesized that the antitumor effect of miR-150 on CRC was dependent on the suppressed function of iASPP. Therefore, the expression of iASPP was induced in miR-150 overexpressed SW480 and HCT116 cells (Figs. 7A and 9A, respectively). Subsequently, the growth and metastasis potential of SW480 and HCT116 cells in different groups were assessed. Based on the results, induced expression of iASPP counteracted the effect of miR-150 overexpression on SW480 and HCT116 cells. Concerning growth potential, induced iASPP expression increased cell viability, relieved cells from G1 cell cycle arrest and inhibited cell apoptosis in the miRNA+pcDNA group when compared with the miRNA group (Figs. 7B-D and 9B-D). Additionally, upregulated iASPP levels also improved the metastatic potential of SW480 and HCT116 cells, as a faster closure rate and more cells penetrating the membrane were detected in the miRNA+pcDNA group as demonstrated by scratch and Transwell assays (Figs. 8 and 9E-G). It was hypothesized that, without the low level of iASPP, the suppressing function of miR-150 on CRC cells was confounded, which indicated that the anti-CRC function of miR-150 was exerted via the inhibition of iASPP.