The present study was reviewed and approved by the Ethics Committee of Medical Research, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China). Fresh paired human CRC tissues and the corresponding adjacent non-cancerous tissues were collected from 120 patients who were treated with surgical resection alone between June 2016 and May 2017 at Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology. These patients included 72 males and 48 females, and 67 of them were older than 60 years old. Written informed consents were provided from these patients.

Tissue microarrays with survival information were obtained from Shanghai Outdo Biotech Co., Ltd.

The human TP53-WT CRC cell lines, RKO, HCT116, Colo-678, LoVo, SW48, LS174T and human colonic fibroblast, CCD-112CoN were purchased from American Type Culture Collection (14). All cell lines were ensured to be free of mycoplasma contamination before use. All the cell lines were cultured in RPMI-1640 containing 10% fetal bovine serum, 1% penicillin and streptomycin (all from Invitrogen; Thermo Fisher Scientific, Inc.) and maintained at 37°C with 5% CO₂ in a humidified incubator (Thermo Fisher Scientific, Inc.).

Total RNA was extracted from cells using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and cDNA was synthesized from 1,000 ng total RNA with 5X PrimeScript RT master mix (cat. no. RR036A; Takara Bio Inc.) following the manufacturer's instructions. qPCR was performed using the SYBR Premix Ex TaqTM II kit (cat. no. DRR081A; Takara Bio Inc.) and performed using the LightCycler 96 system (Roche Diagnostics GmbH). β-actin was used as the internal control for normalization. The data were analyzed using the 2^−ΔΔCq method (15) and each experiment was repeated at least three times. The sequences of the primers used in qPCR were as follows: β-actin forward, 5′-GATTACTGCTCTGGCTCCTAGC-3′ and reverse, 5′-CAGCTCAGTAACAGTCCGCC-3′; SMAD7 forward, 5′-TTTGAGGTGTGGTG-3′ and reverse, 5′-GAGGCAGTAAGACAGGGATGA-3′; LINC00858 forward, 5′-CCCAGCTCCTTACACACGTT-3′ and reverse 5′-TTCAGAGGCCTGCATCACTG-3′; miR-25-3p forward, 5′-CAUUGCACUUGUCUCGGUCUGA-3′ and reverse, 5′-AGACCGAGACAAGUGCAAUGUU-3′. All the PCR primers were designed and synthesized by Genewiz, Inc.

CRC cells were seeded in 6-well plates and cultured to 60–70% confluence for transfection. miRNA mimics, inhibitors or corresponding controls, short hairpin (sh)RNAs and plasmid (Guangzhou RiboBio Co., Ltd.) were transfected using Lipofectamine^® 3000 (cat. no. L3000008; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

For the luciferase reporter assay, cells were cultured to 60–70% confluence for transfection. LINC00858-WT vector or LINC00858-mutant (MUT) vector were created with or without a 3′-untranslated region binding site for miR-25-3p using pmirGLO vector (Promega Corporation) and SMAD7-WT vector or SMAD7-MUT vector were constructed similarly. Cells were co-transfected with miRNA mimics or NC mimics along with luciferase reporter vector using Lipofectamine 3000 (cat. no. L3000008; Invitrogen; Thermo Fisher Scientific, Inc.). After transfection for 48 h, luciferase activity was detected with a Dual-Luciferase Reporter Assay System (cat. no. E1910; Promega Corp.).

A Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) was utilized for measurement of RKO or SW48 cell viability. After cell transfection, the transfected cells were collected and seeded in 96-well plates (2×10³ cells/well). Cells were cultured for 24, 48 and 72 h at 37°C, the CCK-8 solution (10 µl) was added into the culture medium in 96-well plates. Subsequently, the mixture was incubated at 37°C for 1.5 h, and the absorbance at 450 nm was measured using a spectrophotometer (BioTek Instruments, Inc.).

For colony formation analysis, transfected cells were seeded into 6-well plates and incubated for 14 days at 37°C in a humidified incubator with 5% CO₂. Subsequently, the cells were washed in PBS and fixed with 4% paraformaldehyde. The cells were stained with 0.1% crystal violet at room temperature for 30 min (Sigma-Aldrich; Merck KGaA). The stained colonies were viewed using a fluorescence microscope.

For apoptosis analysis, following transfection for 48 h cells were washed twice with PBS and resuspended in buffer. Annexin-V-fluorescein isothiocyanate apoptosis detection kit (cat. no. 556547; BD Biosciences) was utilized according to the manufacturer's instructions. For cell cycle analysis, the transfected cells were collected and fixed with ice-cold 70% ethanol for 12 h at 4°C. Subsequently, the cells were stained with Cycle TEST DNA Reagent kit at room temperature for 10 min (cat. no. 340242; BD Biosciences). The cell cycle and apoptosis analysis were performed with flow cytometry using the BD FACSVantage™ SE System.

Proteins were extracted using RIPA buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) supplemented with EDTA-free protease inhibitor cocktail (cat. no. 04693159001; Roche Diagnostics GmbH) as previously described (16). The suspension was collected after centrifugation at 12,000 × g for 15 min at 4°C, followed by protein concentration determination using BCA assay. Equal amounts of proteins (20–40 µg) were separated on 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred to PVDF membranes using a Bio-Rad semi-dry transfer system. Then the membrane was blocked with 5% skimmed milk. After blocking, the membranes were incubated with primary antibodies (dilution ratio of 1:1,000 with PBST containing 5% BSA) overnight at 4°C, followed by horseradish peroxidase-labeled secondary antibodies (dilution ratio of 1:5,000 with PBST containing 5% non-fat milk; cat. no. 7074; Cell Signaling Technology, Inc.) for 1 h at room temperature. The protein bands were visualized using the Tanon 4600 imaging system (Tanon Science and Technology Co., Ltd.) with enhanced chemiluminescence (cat. no. 407207; EMD Millipore; Merck KGaA). The primary antibodies used included Smad7 (cat. no. sc-11392; Santa Cruz Biotechnology, Inc.) and GAPDH (product code ab128915; Abcam).

EZ-Magna RIP RNA-Binding Protein Immunoprecipitation kit (cat. no. 17-701; EMD Millipore; Merck KGaA) was used to perform the RNA-binding protein immunoprecipitation assay. Cells were cultured to 80–90% confluence and then harvested. Subsequently, 3 µg of purified antibodies and corresponding IgG were added to the cell lysate, and the mixture was then incubated overnight at 4°C with rotation. Anti-protein argonaute-2 (AGO2; cat. no. 2897; Cell Signaling Technology, Inc.), and normal rabbit IgG (cat. no. PP64B; EMD Millipore; Merck KGaA) were used for the RIP assay.

TargetScan V7.0 (http://www.targetscan.org/) provides information concerning humans, rats and other species and predicts target genes by searching the conserved 8 and 7mer sites that match each miRNA seed region (17). StarBase database (http://starbase.sysu.edu.cn) also includes information on the prediction of microRNA targets in the genomes of human and other species, as well as miRNA expression profiles in different tissues and diseases (18). With the two forecasting tools, comparisons of the relative mRNA expression levels among the different groups of samples was performed.

Data are presented as the mean ± SEM from three independent experiments. All data were analyzed using the GraphPad Prism v6.0 (GraphPad Software, Inc.). Survival curves were generated using the Kaplan-Meier method, and a log-rank test was used for comparison. Student's t-test, χ² test or one-way ANOVA was utilized as appropriate. Both paired and unpaired Student's t-tests were used correspondently. ANOVA was performed to evaluate the differences followed by Tukey's post hoc test. The correlation between the expression levels of two genes was analyzed using Pearson's correlation analysis. P<0.05 was considered to indicate a statistically significant difference.

Dysregulation of LINC00858 has been previously associated with CRC, however, the mechanisms involved remain elusive (12). To validate the dysregulated expression in CRC tissues, the levels of LINC00858 in 60 colorectal tumor tissues and 60 adjacent normal tissues were detected. In accordance with the aforementioned study (13), the expression level of LINC00858 was significantly higher in colorectal tumor tissues compared with that in adjacent normal tissues (Fig. 1A). The relationship between LINC00858 expression level and clinical outcomes of patients with CRC was investigated further (Table I). High expression of LINC00858 was significantly associated with poor overall survival time (Fig. 1B; P<0.001).

To further clarify the functional role of LINC00858 in CRC, the following assays were performed. Firstly, LINC00858 expression levels were evaluated in six TP53-WT CRC cell lines (RKO, HCT116, Colo-678, LoVo, SW48 and LS174T) and human normal colonic fibroblast (CCD-112CoN) cells and high expression levels were revealed in all CRC cell lines compared to CCD-112CoN. RKO and SW48 cells were markedly higher compared with the other cell lines (Fig. 1C) (14). Therefore, these two cell lines were selected for further experiments and were subsequently transfected with sh-LINC00858 to knockdown the target lncRNA (Fig. 2A). Subsequently, viability and colony formation assays were performed. Cell proliferation was revealed to be significantly suppressed after transfection (Fig. 2B-D). To investigate further, the cell cycle and cell apoptosis analysis was performed. Cell cycle assays revealed that sh-LINC00858 transfection increased the cell population in the G₀/G₁ phase and decreased the cell population in S phase compared with that in the control groups in both CRC cell lines (Fig. 2E and F). Cell apoptosis analysis indicated that downregulation of LINC00858 induced a significant increase in apoptosis of CRC cells compared with that in the control groups (Fig. 2G and H). Collectively, these results revealed that downregulation of LINC00858 suppressed CRC cell proliferation potentially through induction of G₀/G₁ phase arrest and apoptosis.

Previous studies proposed that lncRNAs modulate tumor growth and development by competitively binding to miRNAs (19). Therefore, the potential miRNAs which may have binding sites with LINC00858 were predicted using StarBase database (http://starbase.sysu.edu.cn). As revealed in Fig. 3A, the binding site of miR-25-3p on LINC00858 was predicted by StarBase. This interaction between LINC00858 and miR-25-3p was subsequently validated. Luciferase reporter assays revealed that miR-25-3p mimics significantly reduced the luciferase activity of LINC00858-WT vectors, with no marked change in LINC00858-Mut vectors (Figs. S1B and 3B and C). RIP assays revealed that LINC00858 and miR-25-3p were co-immunoprecipitated by the AGO2 antibody but not the IgG antibody (Fig. 3D). Next, the expression levels of miR-25-3p in human CRC tissues was evaluated. The expression level of miR-25-3p was significantly decreased in CRC tumor tissues compared with that in adjacent normal tissues (Fig. 3E). Pearson's correlation analysis revealed the negative correlation between the expression levels of LINC00858 and miR-25-3p (Fig. 3F). Furthermore, miR-25-3p was revealed to be downregulated in all the cell lines with high LINC00858 expression levels, while in the cell line with a low LINC00858 expression level, miR-25-3p was upregulated (Fig. 3G). In addition, knockdown of LINC00858 significantly increased the expression levels of miR-25-3p in RKO and SW48 cells (Fig. 3H and I). These results indicated that LINC00858 sponged miR-25-3p in CRC.

To further explore the role of miR-25-3p in the development of CRC, the following experiments were performed. sh-LINC00858 and miR-25-3p inhibitor were co-transfected into RKO and SW48 cells and the cell viability after transfection was determined. The data in the present study revealed that the cell viability was significantly recovered in the co-transfection group, compared with that in the cells transfected with sh-LINC00858 alone (Figs. S1C and 4A-C). Next, colony formation assay after the transfection as aforementioned, was performed. Colony formation assay indicated that colony formation was significantly increased after co-transfection of sh-LINC00858 and miR-25-3p inhibitor (Fig. 4D), which was consistent with the cell viability analysis. Cell cycle analysis revealed that concurrent downregulation of LINC00858 and miR-25-3p decreased the cell proportions arrested in the G₀/G₁ stage and increased the cell proportions in the S stage, compared with that in cells downregulated of LINC00858 alone (Fig. 4E and F). Furthermore, the apoptosis assay indicated that cell apoptosis induced by sh-LINC00858 was partially reversed by inhibition of miR-25-3p (Fig. 4G and H). Collectively, LINC00858 promoted the development of CRC, partially by binding to miR-25-3p.

To investigate the potential downstream target of miR-25-3p, TargetScan was used (http://targetscan.org/vert_72) to identify potential targets, which have binding sites with miR-25-3p. As revealed in Fig. 5A, SMAD7 may be the target of miR-25-3p. To investigate the interaction between SMAD7 and miR-25-3p further, luciferase reporter assays were performed. RIP assays revealed that miR-25-3p was co-immunoprecipitated by the SMAD7 antibody but not the IgG antibody (Fig. S1A). miR-25-3p significantly reduced the luciferase activity in SMAD7-WT vector but not in SMAD7-MUT vector (Fig. 5B). The correlation between miR-25-3p and SMAD7 expression levels were explored and a negative correlation between miR-25-3p and SMAD7 with Pearson's correlation analysis was identified (Fig. 5C). These results demonstrated that SMAD7 was a possible downstream target of miR-25-3p.

To verify the relationship between LINC00858, miR-25-3p and SMAD7, RT-qPCR and western blot analysis was performed. The results indicated that LINC00858 knockdown downregulated the expression of SMAD7, which was reversed with the addition of miR-25-3p inhibitor (Fig. 5D and E). Correlation analysis also revealed a positive correlation between LINC00858 and SMAD7 expression levels (Fig. 5F). These data indicated that LINC00858 may regulate proliferation of CRC through the LINC00858/miR-25-3p/SMAD7 axis. To verify our hypothesis, rescue assays were performed. Cell viability assays revealed that LINC00858 knockdown-induced inhibition of cell proliferation was partially reversed by SMAD7 overexpression in RKO and SW48 cells (Fig. 5G and H). In addition, cell apoptosis induced by LINC00858 knockdown was also rescued by SMAD7 overexpression (Fig. 5I and J). Collectively, the data in the present study demonstrated that the LINC00858/miR-25-3p/SMAD7 axis regulated the growth and development of CRC cells.