The study was performed on 84 fresh CRC tissues (colon and rectal cancers) confirmed by histopathology at the Qingdao West Coast New Area Central Hospital (Qingdao, China) from May 2017 to January 2019. All patients with CRC did not receive any treatment before surgery. The study was approved by the Institutional Ethics Committee of the Qingdao West Coast New Area Central Hospital. Patients who participated in this research had complete clinical data. Signed written informed consents were obtained from the patients and/or guardians.

Normal human intestinal epithelial cells HIEC-6 and CRC cell lines HCT-116 and SW620 were purchased from ATCC. The cells were incubated in DMEM (10% FBS, 5% CO₂, 37°C) for subsequent experimentation.

miR-758 mimics, miR-758 inhibitor, and PAX6 overexpression plasmid were purchased from Shanghai GenePharma Co., Ltd. HCT-116 cells were transfected using Lipofectamine^® 2000 (Invitrogen, Thermo Fisher Scientifc, Inc.), respectively. HCT-116 cells with human homologous sequences were used as control (miR-NC). According to the manufacturer's protocol, the cells were plated in 6-well plates (at 70–90% confluence) and were transfected with 100 pmol miR-758 mimics, inhibitor and their control or 2.5 µg PAX6 overexpression plasmid. After 6 h of incubation at 37°C with 5% CO₂, the transfection efficiency was detected and subsequent assays were performed.

Total RNA isolation was performed using TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The cDNA solution was then obtained using a PrimeScript Reverse Transcription kit (Qiagen China Co., Ltd). The temperature conditions for reverse transcription were as follows: 37°C for 15 min and 85°C for 5 sec. RT-qPCR was performed using SYBR Green I (Takara Biotechnology Co., Ltd.) following the manufacturer's instructions. The thermocycling conditions for PCR amplification were as follows: 5 min at 95°C, followed by 40 cycles of 95°C for 30 sec and 60°C for 45 sec. U6 and GAPDH were used as the controls for miR-758 and PAX6, respectively. The expression of miR-758 and PAX6 was quantified using the 2^−ΔΔCq method (15). Primers were designed as follows: miR-758 forward, 5′-ACACTCCAGCTGGGTTTGTGACCTGGTCCA-3′ and reverse, 5′-TGGTGTCGTGGAGTCG-3′; U6 forward, 5′-CGCTTCGGCAGCACATATACTA-3′ and reverse, 5′-GCGAGCACAGAATTAATACGAC-3′; PAX6 forward, 5′-TCTTTGCTTGGGAAATCCG-3′ and reverse, 5′-CTGCCCGTTCAACATCCTTAG-3′; and GAPDH forward, 5′-GGCACTGAGAAGCGGGGCCG-3′ and reverse, 5′-GAAGATGGTGATGGGATTTC-3′.

An 8.0-µm pore polycarbonate membrane insert (Corning, Inc.) was used to determine the invasion and migration of CRC cells. The upper chamber was coated with Matrigel (BD Biosciences) for invasion assay. Next, the transfected HCT-116 cells (3×10³ cells/well) were placed in the upper chamber. DMEM with 10% FBS was added to the lower chamber. After 24 h, the cells were fixed and stained with 0.5% crystal violet at room temperature for 30 min. For cell migration assay, Matrigel was not required and the procedure followed was identical to that of the cell invasion assay. Observation and image capturing were performed using a light microscope (magnification, ×200).

The prepared HCT-116 cells were incubated in a 96-well plate for 24 h (at 37°C, 5% CO₂). HCT-116 cells (4×10³/well) were then incubated for 24, 48, 72 and 96 h. Next, the cells were incubated for 4 h with 10 ml of CCK-8 (Dojindo Molecular Technologies, Inc.) solution. The absorbance at 450 nm was observed with a microplate reader (Molecular Devices, LLC).

Protein samples were obtained using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was quantified using a bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) and 40 µg protein/lane were separated via SDS-PAGE on a 10% gel. The separated proteins were transferred onto polyvinylidene fluoride membranes (Thermo Fisher Scientific, Inc.) and blocked for 3 h at room temperature with 5% non-fat milk in PBS (Thermo Fisher Scientific, Inc.) containing 0.1% Tween-20 (Sigma-Aldrich; Merck KGaA). The membranes were then incubated with E-cadherin (rabbit monoclonal; dilution, 1:1,000; cat. no. ab1416; Abcam), N-cadherin (rabbit polyclonal; dilution, 1:1,000; cat. no. ab18203; Abcam), Bcl-2 (rabbit monoclonal; dilution, 1:1,000; cat. no. ab185002; Abcam), Bax (mouse monoclonal; dilution, 1:1,000; cat. no. ab77566; Abcam), PI3K (rabbit monoclonal; dilution, 1:1,000; cat. no. ab32089; Abcam), p-PI3K (rabbit monoclonal; dilution, 1:1,000; cat. no. ab154598; Abcam), AKT (rabbit polyclonal; dilution, 1:1,000; cat. no. ab8805; Abcam), p-AKT (rabbit monoclonal; dilution, 1:1,000; cat. no. ab81283; Abcam) and GAPDH (rabbit monoclonal; dilution, 1:1,000; cat. no. ab181602; Abcam) primary antibodies overnight at 4°C. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (dilution, 1:5,000; cat. no. ab7090; Abcam) for 1 h at room temperature. Protein bands were visualized using ECL kit (Beyotime Institute of Biotechnology).

WT-PAX6-3′UTR or MUT-PAX6-3′UTR was inserted into the pmirGLO luciferase reporter vector (Promega Corporation). HCT-116 cells were transfected with the luciferase vector and miR-758 mimics using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientifc, Inc.) according to the manufacturer's protocol. After 24 h, the Renilla and firefly luciferase activity were detected using a dual-luciferase reporter assay system (Promega Corporation). Firefly luciferase activity was normalized to Renilla luciferase activity.

Data were analyzed using SPSS 17.0 (IBM Corp.) or GraphPad Prism 6 (GraphPad Software, Inc.) software and were expressed as the mean ± SD. The differences were analyzed using Student's t-test or one-way ANOVA followed by Tukey's post hoc test. The univariate Kaplan-Meier method with log-rank test and the Chi-square test were used to analyze the association between miR-758 and patient survival or clinical features. Spearman's rank correlation analysis was performed to analyze the correlation between miR-758 and PAX6 expression levels. P<0.05 was considered to indicate a statistically significant difference.

RT-qPCR was performed to assess the expression of miR-758 in CRC. miR-758 expression was decreased in CRC tissues compared with that in normal tissues (P<0.01, Fig. 1A). The downregulation of miR-758 was also detected in HCT-116 and SW620 cells in comparison with miR-758 expression in HIEC-6 cells (P<0.01, Fig. 1B). Additionally, the low expression of miR-758 was found to be associated with differentiation, TNM stage and lymph-node metastasis in CRC patients (P<0.05, Table I). Moreover, the poor prognosis in CRC patients was found to be associated with the downregulation of miR-758 (P<0.05, Fig. 1C). These results suggest that miR-758 may be involved in CRC progression. Since the difference of miR-758 expression between the HCT-116 and HIEC-6 cells was more significant than that between SW620 and HIEC-6 cells, HCT-116 cells were selected for the following experiments.

To investigate the function of miR-758 in CRC, a gain-loss function experiment was performed in HCT-116 cells with miR-758 mimics or inhibitor. The expression of miR-758 was increased by its mimics and decreased by its inhibitor (P<0.01, Fig. 2A). Functionally, the overexpression of miR-758 restrained cell proliferation (P<0.01), whereas the downregulation of miR-758 accelerated the proliferation of HCT-116 cells (P<0.05) (Fig. 2B). In addition, miR-758 mimics inhibited cell migration, whereas miR-758 inhibitor promoted cell migration in HCT-116 cells (P<0.01, Fig. 2C). Consistently, miR-758 mimics also inhibited cell invasion, whereas miR-758 inhibitor promoted HCT-116 cell invasion (P<0.01, Fig. 2D). Based on these results, miR-758 restrained CRC progression by inhibiting cell viability and metastasis.

For the prediction of the downstream target of miR-758, the TargetScan database was used (http://www.targetscan.org). It was predicted that miR-758 has a site that binds to the 3′-UTR of PAX6 (Fig. 3A). A luciferase reporter assay was then designed to confirm this prediction. miR-758 mimics were identified to reduce the luciferase activity of WT-PAX6 (P<0.01, Fig. 3B). However, miR-758 mimics had no effect on MUT-PAX6 luciferase activity. To further confirm the relationship between miR-758 and PAX6, the PAX6 expression was observed in HCT-116 cells with miR-758 mimics or inhibitor. RT-qPCR showed that the overexpression of miR-758 reduced the expression of PAX6, whereas the downregulation of miR-758 promoted the expression of PAX6 (P<0.01, Fig. 3C). In addition, PAX6 was shown to be upregulated in CRC tissues compared with normal tissues (P<0.01, Fig. 3D). Moreover, miR-758 was negatively correlated with PAX6 expression in CRC tissues (P<0.01, R²=0.7269; Fig. 3E). The results suggest that miR-758 directly targets PAX6 and negatively regulates PAX6 expression in CRC.

To explore the interaction between PAX6 and miR-758, PAX6 overexpression vectors were transfected into HCT-116 cells with miR-758 mimics. The results showed that the decreased expression of PAX6 induced by miR-758 mimics was restored by PAX6 overexpression vector in HCT-116 cells (Fig. 4A). Functionally, the overexpression of PAX6 attenuated the inhibitory effect of miR-758 on the cell proliferation of HCT-116 cells (P<0.01, Fig. 4B). Consistently, miR-758-mediated inhibition of cell migration and invasion was also weakened by PAX6 overexpression (P<0.01, Fig. 4C and D). These results suggest that the upregulation of PAX6 impairs the inhibitory effect of miR-758 on CRC.

To further illustrate the molecular mechanism of miR-758 in CRC, the way miR-758 regulates EMT (E-cadherin, N-cadherin), apoptosis (Bcl-2, Bax) and PI3K/AKT pathway (PI3K, p-PI3K, AKT, p-AKT) in HCT-116 cells was investigated. Fig. 5 shows that the overexpression of miR-758 promoted E-cadherin and Bax expression levels, whereas miR-758 overexpression inhibited N-cadherin and Bcl-2 expression levels in HCT-116 cells. In addition, miR-758 mimics inhibited the expression of p-PI3K and p-AKT, whereas miR-758 inhibitor promoted their expression in HCT-116 cells (Fig. 5). However, miR-758 mimics did not affect the expression of PI3K and AKT (Fig. 5). Moreover, downregulation of miR-758 showed opposite effect on the expression of these genes (Fig. 5). Taken together, miR-758 overexpression blocked EMT and the PI3K/AKT pathway in CRC.