Human CRC tissues and the matched adjacent non-tumor tissues were obtained from eight patients, including four males and four females, between 38 and 76 years old, with CRC and histopathologically diagnosed at the Department of General Surgery, Huizhou First Hospital (Huizhou, China) between 1 June 2015 and 31 December 2015. The present study was approved by the Ethics Committee of the Department of General Surgery, Huizhou First Hospital. Written informed consent was obtained from all patients. Tissue samples were collected during surgery, immediately frozen in liquid nitrogen and stored until total RNA or proteins were extracted.

Human CRC cell lines SW620, COLO320DM, SW403, SW480, HT-29 and COLO205 and normal colonic cell line FHC were purchased from the National Rodent Laboratory Animal Resources (Shanghai, China). All CRC cell lines were grown in Dulbecco's modified Eagle medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and normal colon FHC cells were grown in DMEM/F-12 medium with 10% FBS, 10 ng/ml cholera toxin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), 5 µg/ml transferrin, 5 µg/ml insulin, 100 ng/ml hydrocortisone and 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). Cell lines were cultured in a humidified 37°C incubator with 5% CO₂.

SW480 cells (5×10⁵) were transfected with 2.5 µg each of scrambled miRs as negative controls (NCs), miR-598 mimic and miR-598-in (miR-598-inhibitor; GeneCopoeia, Inc., Rockville, MD, USA), Mutant miR-598 was constructed by GeneCopoeia, Inc. INPP5E siRNAs and NCs (stQ0004501-1; http://www.ribobio.com/sitecn/product_info.aspx?id=338920) were purchased from Guangzhou RiboBio Co., Ltd. (Guangzhou, China). Transfection of plasmids and siRNAs was performed using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol.

Total RNA was extracted from culture cells and patient samples using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, then cDNA was synthesized with using reverse transcription reagent kit (Takara Biotechnology Co., Ltd., Dalian, China) at 37°C for 15 min, followed at 85°C for 5 sec, and then cooling to 4°C. RT-qPCR was carried out using an ABI 7900HT Fast Real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the SYBR-Green PCR kit (Takara Biotechnology Co., Ltd.). The primers selected by GeneCopoeia, Inc., were miR-598 (cat. no. HmiRQP0702), U6 (cat. no. HmiRQP9001), cyclin D1 (cat. no. HQP016204), cyclin-dependent kinase inhibitor 1B (p27; cat. no. MQP028863) and GAPDH (cat. no. HQP006940). Thermocycling conditions were 95°C for 30 sec, followed by 40 cycles of amplification at 95°C for 5 sec, 59°C for 30 sec and then 72°C for 30 sec.

Relative miR-598 or cyclin D1 and p27 mRNA expression were normalized to U6 or GAPDH, respectively. Relative quantification was calculated as 2^−∆∆Cq (14) and was used to calculate fold-changes.

Protein lysates were lysed with radioimmunoprecipitation buffer (Beyotime Institute of Biotechnology, Haimen, China), equal quantities (50 µg) of protein were separated on a 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes. Membranes were incubated with primary antibodies as follows: Rabbit anti-INPP5E monoclonal antibody (ab191520; 1:1,000); rabbit anti-cyclin D1 monoclonal antibody (ab134175; 1:1,000); rabbit anti-p27 monoclonal antibody (ab32034; 1:1,000) overnight at 4°C, and anti-α-tubulin antibody (ab7291; 1:5,000; all from Abcam, Cambridge, MA, USA) was used as a reference protein. The next day, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (ab205718; 1:10,000; Abcam) for 2 h at room temperature and bound antibodies were visualized using an enhanced chemiluminesence kit (Beyotime Institute of Biotechnology) with the SuperSignal West PICO chemiluminescent detection system (Pierce; Thermo Fisher Scientific, Inc.).

Cell growth was measured by MTT assay (Sigma-Aldrich; Merck KGaA) according to the manufacturers' protocol. Transfected SW480 cells were seeded in 96-well plates at 5×10³ cells/well. Cells were allowed to grow for 0–5 days. A total of 20 µl, 5 mg/ml MTT solution was then added to each well and incubated for 4 h, and then 150 µl DMSO (Sigma-Aldrich; Merck KGaA) was added. Absorbance was measured at 490 nm using a microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA).

The soft agar colony-forming assay was performed using transfected SW480 (2,000 cells/well), which were seeded into each well of a 6-well plate with 0.5% agar (Sigma-Aldrich; Merck KGaA). After 14 days, three fields were randomly selected and cell colonies >0.1 mm² were counted with an inverted microscope at a magnification of ×200.

Following 48 h of incubation post-transfection, SW480 cells were removed from culture plates by trypsinization and were washed with PBS. Cells were fixed with 75% ice-cold ethanol at 4°C. Following overnight incubation at 4°C and washing with PBS, the cells were treated with RNase A (300 µg/ml) at 37°C for 30 min and stained with propidium iodide (PI) at 4°C for 30 min in the dark, and the cell cycle distribution was analyzed using a flow cytometer (BD FACSCalibur^™; BD Biosciences, Franklin Lakes, NJ, USA). The percentages of cells in G₀/G₁, S and G₂/M phases were counted by BD CellQuest™ Pro Software version 3.3 and compared. Experiments were repeated three times.

The INPP5E open reading frame with the 3′-untranslated region (UTR) was cloned into pGL3 vectors were purchased from GeneCopoeia, Inc. (Guangzhou, China). SW480 cells (5×10⁴/well) were cultured in 24-well plates. Transient co-transfection with either the INPP5E 3′-UTR wild type and miR-598 or miR-598- in or control mimics were performed using Lipofectamine 2000 reagent. A total of 48 h following transfection, firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega Corporation, Madison, WI, USA) according to manufacturer's protocol.

Following transfection cells were grown on cover slips (Thermo Fisher Scientific, Inc.) and were incubated with BrdU for 1 h and then stained with an anti-BrdU antibody (20-BS17; 1:500; Upstate Biotechnology, Inc., Lake Placid, NY, USA) according to the manufacturer's protocol. Gray level images were acquired using a laser-scanning microscope (Axioskop 2 plus; Carl Zeiss AG, Oberkochen, Germany).

All statistical analyses were performed using SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA). All data are expressed as the mean ± standard deviation. Student's t-test was used to evaluate the significance of the differences between two groups of data and one-way analysis of variance followed by Tukey's post hoc test was used for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.

To investigate the potential roles of miR-598 in CRC, the expression data downloaded from TCGA and GEO (accession number GSE30454) was analyzed and indicated that miR-598 expression was notably upregulated in CRC tissues (n=218) compared with normal colorectal tissue (n=8; Fig. 1A). The results of GEO analysis demonstrated that compared with normal colorectal tissue (n=20), the expression of miR-598 was significantly upregulated in CRC samples (n=54; P<0.05; Fig. 1B). In addition, RT-qPCR was performed to analyze the expression of miR-598 in CRC and demonstrated that compared with the matched tumor adjacent tissues, miR-598 expression was upregulated in the CRC tissues (Fig. 1C) and in all six tested CRC cell lines (SW620, COLO320DM, SW403, SW480, HT-29 and COLO205) had significantly upregulated expression of miR-598 compared with the normal colonic cell line FHC (P<0.05; Fig. 1D). These results indicated that miR-598 was upregulated in CRC cell lines and tissues. miR-598 expression in SW480 cells was decreased compared with COLO320DM, HT-29, SW403 and COLO205, but it was increased compared with SW620. SW480, therefore appeared to be a suitable cellular model for processing up and downregulation of miR-598.

In order to investigate the role of miR-598 in CRC, an miR-598 mimic, miR-598-in or the relative controls were transiently transfected into SW480 cells. Relative miR-598 expression in SW480 cells was confirmed using RT-qPCR (Fig. 2A and B). Using MTT and anchorage-independent growth assays, it was observed that miR-598 promoted cell proliferation (Fig. 3A and C), while the growth rate of SW480 cells following transfection with miR-598-in was decreased (Fig. 3B and D), compared with respective NC-transfected cells. The results of the BrdU analysis indicated that miR-598 overexpression significantly increased the percentage of BrdU positive cells (P<0.05; Fig. 4A), while BrdU positive cells were significantly reduced in SW480 cells with miR-598-in (P<0.05; Fig. 4B). Additionally, flow cytometry indicated an increase in the percentage of cells in the S phase and a decrease in the percentage of cells in G₁/G₀ phase in miR-598 transfected SW480 cells (Fig. 4C). In SW480 cells transfected with miR-598-in, the number of cells in the S phase of the cell cycle decreased and the number in G₁/G₀ phase increased (Fig. 4D). These results suggested that miR-598 serves an important role in cell proliferation and cell cycle progression in CRC cells.

Using the miR target prediction website (www.targetscan.org), INPP5E was demonstrated to act as the potential target of miR-598. The wild type INPP5E 3-UTR mRNA contained a length of conserved sequence with miR-598 and a mutant miR-598 was constructed (Fig. 5A). Western blot analysis of the INPP5E expression levels demonstrated that INPP5E levels in the miR-598 mimic transfection group were significantly lower than those in the NC transfection group, whereas cells in the miR-598-in group demonstrated significantly increased INPP5E levels compared with the NC (P<0.05; Fig. 5B).

To confirm that INPP5E is directly targeted by miR-598, the present study investigated whether miR-598 recognizes the 3′UTR of INPP5E mRNA using a dual-luciferase reporter assay. The relative luciferase activity of the reporter containing the wild-type INPP5E 3′-UTR was significantly decreased following co-transfection with miR-598; however, this was notably increased following co-transfection with miR-598-in. The luciferase activity was not significantly affected by co-transfection with the miR-598-mut (P<0.05; Fig. 5C).

To analyze the effect of miR-598 on the expression levels of mRNA and proteins associated with cell proliferation and cell cycle progression in CRC cells, miR-598 or miR-598-in were transfected into SW480 cells and RT-qPCR and western blot analysis were performed. The results indicated that mRNA and protein expression of cyclin D1 expression was significantly upregulated and p27 was downregulated by miR-598 (P<0.05; Fig. 5D). In contrast, the expression level of cyclin D1 was downregulated and p27 was upregulated in SW480 cells transfected with the miR-598-in (Fig. 5E). These results confirmed that INPP5E is a target of miR-598.

To further investigate the role of INPP5E in CRC, specific siRNAs against INPP5E were designed and synthesized. Western blot analysis indicated that INPP5E protein level was decreased following treatment with siRNAs in SW480 cells transfected with miR-598-in (Fig. 6A). The results of anchorage-independent growth assays demonstrated that knockdown of INPP5E counteracted the growth arrest by miR-598-in (Fig. 6B). In addition, silencing of INPP5E also increased the percentage of BrdU positive cells in SW480 cells transfected with miR-598-in compared with the NC (Fig. 6C).