Human CRC tissues and corresponding adjacent non-neoplastic tissues were collected between October 2014 and March 2016 from surgical specimens from 28 patients with CRC at Xiangyang Central Hospital (Xiangyang, China). None of these patients were treated with chemotherapy or radiotherapy before surgery. These specimens were frozen immediately after resection and stored at −80°C until further use. The study was approved by the Ethical Review Committees of Xiangyang Central Hospital, and all patients gave informed consent prior to specimen collection according to institutional guidelines.

Five human CRC cell lines (HCT116, HT29, LoVo, SW480, SW620) and normal human colon epithelium cell line FHC were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% heat-inactivated foetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.) 100 U/ml streptomycin and 100 U/ml penicillin and grown at 37°C in a humidified incubator with 5% CO₂.

MiR-212 mimics and miRNA mimics negative control (miR-NC) were obtained from RiboBio Co., Ltd. (Guangzhou, China). PIK3R3 overexpression plasmid without the 3′-UTR of PIK3R3 (pcDNA3.1-PIK3R3) and blank plasmid pcDNA3.1 were acquired from GeneCopoeia (Guangzhou, China). Cells were seeded into 6-well plates with a density of 60–70% confluence each well. Cells were transfected with miR-212 mimics (100 pmol), miR-NC (100 pmol), or cotransfected with miR-212 mimics (100 pmol) and pcDNA3.1-PIK3R3 (1 µg) using Lipofectamine 2000 transfection reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

Total RNA was extracted from tissue samples or cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. The quantity and the quality of total RNA was assessed using a Nanodrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). To analysis miR-212 expression, cDNA of miRNA was synthesized from total RNA using TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Carlsbad, CA, USA). Real-time quantitative PCR was performed using a TaqMan MicroRNA PCR kit (Applied Biosystems). U6 was used as an internal control for miR-212. To quantify PIK3R3 mRNA expression, total RNA was reversed transcription into cDNA using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA) and qPCR was conducted using SYBR Premix Ex Taq™ (Takara Biotechnology Co., Ltd., Dalian, China). β-actin served as an internal control for PIK3R3 mRNA expression. The primers were designed as follows: miR-212, 5′-CGCTAACAGTCTCCAGTC-3′ (forward) and 5′-GTGCAGGGTCCGAGGT-3′ (reverse); U6, 5′-CTCGCTTCGGCAGCACATATACT-3′ (forward) and 5′-ACGCTTCACGAATTTGCGTGTC-3′ (reverse); PIK3R3, 5′-CTTGCTCTGTGGTGGCCGAT-3′ (forward) and 5′-GACGTTGAGGGAGTCGTTGT-3′ (reverse); and β-actin, 5′-TGGCACCCAGCACAATGAA-3′ (forward) and 5′-TAAGTCATAGTCCGCCTAGAAGCA-3′ (reverse). The relative expression of miR-212 and PIK3R3 mRNA were calculated using the 2^−ΔΔCt method (21).

MTT assay was utilized to determine cell viability. Briefly, cells were seeded into 96-well plates at a density of 3×10³ cells per well. After incubation overnight at 37°C, cell transfection was performed and grown for 0, 24, 48, and 72 h. At indicated time point, 20 ul MTT solution (5 mg/ml; Sigma, St. Louis, MO, USA) was added into each well and incubated at 37°C for additional 4 h. Subsequently, culture medium was removed and 200 µl DMSO (Sigma) was added to each well. After incubation at 37°C incubator for 5 min to dissolve crystals, the optical density (OD) was examined at a wavelength of 490 nm using a microplate reader (Bio-Rad, Richmond, CA, USA). All experiments were performed in triplicate and repeated three times.

Cell invasive ability was evaluated with Transwell chambers (8-µm pores; Corning Costar Corp, Cambridge, MA, USA) coated with Matrigel (BD Biosciences, San Jose, CA, USA). Briefly, transfected cells were collected at 48 h post-transfection and suspended in FBS-free DMEM culture medium. 5×10⁴ transfected cells were seeded into the upper chamber. DMEM containing 10% FBS was placed in the lower compartment as a chemoattractant. After 24 h incubation at 37°C with 5% CO₂, non-invasive cells were wiped out carefully with cotton swab. The invasive cells on the underside of the filter membrane were fixed with 70% ethanol for 20 min and stained with 0.1% crystal violet for 10 min. Invasive cells were photographed and counted under a IX71 inverted microscope (Olympus, Tokyo, Japan) at magnification, ×200 in 5 randomly selected microscopic fields.

The bioinformatics software PICTA (http://pictar.mdc-berlin.de/) and TargetScan (http://www.targetscan.org/) were adopted to predict candidate targets of miR-212.

Luciferase reporter plasmid containing predicted miR-212 seed-matching sites in the 3′-UTR of PIK3R3 and corresponding mutant sites were constructed and confirmed by RiboBio. The constructed vectors were named as pMIR-PIK3R3-3′-UTR Wild-type (Wt) and pMIR-PIK3R3-3′-UTR mutant (Mut), respectively. For luciferase reporter assay, cells were seeded into 24-well plates and transfected with miR-212 mimics or miR-NC, and together with luciferase reporter plasmid using Lipofectamine 2000, according to the manufacturer's protocol. At 48 h after transfection, cells were harvested and luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega), following the protocol provided by the manufacturer. Firefly luciferase activities were used to normalize Renilla luciferase activity. All experiments were performed in triplicate and repeated at least three times.

Total protein was extracted from cells using RIPA lysis buffer (Beyotime, Shanghai, People's Republic of China) supplemented with a protease inhibitor cocktail (Sigma), according to the manufacturer's instruction. Bicinchoninic Acid Protein Assay kit (Beyotime, Shanghai, People's Republic of China) was performed to detect concentration of total protein. Equivalent amounts of proteins were separated on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After that, the membranes were blocked with 5% fat-free milk for 2 h at room temperature and incubated with primary antibodies at 4°C overnight. The primary antibodies used in this study include mouse anti-human monoclonal PIK3R3 antibody (sc-376615; 1:1,000 dilution), mouse anti-human monoclonal p-AKT antibody (sc-271966; 1:1,000 dilution), mouse anti-human monoclonal AKT antibody (sc-56878; 1:1,000 dilution), mouse anti-human monoclonal p-mTOR ser 2481 antibody (sc-293132; 1:1,000 dilution), mouse anti-human monoclonal mTOR antibody (sc-293089; 1:1,000 dilution) and mouse anti-human monoclonal GAPDH antibody (sc-32233; 1:1,000 dilution; all Santa Cruz Biotechnology, CA, USA). Then, the membranes were washed three times with Tris-buffered saline containing Tween-20 (TBST) and further incubated with goat anti-mouse horseradish peroxidase-conjugated secondary antibody (sc-2005; 1:5,000 dilution; Santa Cruz Biotechnology) at room temperature for 1 h. ECL Protein Detection kit (Millipore) was used to visualize the proteins. Optical densities were analyzed with ImageJ software (NIH, Bethesda, MD, USA).

Data are presented as the mean ± standard error and analyzed with SPSS 17.0 (SPSS, Inc., Chicago, IL, USA) using Student's t-test or one way ANOVA test. Student-Newman-Keuls (SNK) was used to compare between two groups in multiple groups. P-value <0.05 was considered to indicate a statistically significant difference.

To investigate the status of miR-212 in CRC, RT-qPCR was used to analyse the expression levels of miR-212 in 28 pairs of CRC tissues and corresponding adjacent non-neoplastic tissues. MiR-212 was obviously downregulated in CRC tissues compared with adjacent non-neoplastic tissues (Fig. 1A, P<0.05). Then, the expression levels of miR-212 in CRC cell lines (HCT116, HT29, LoVo, SW480 and SW620) and normal human colon epithelium cell line (FHC) were examined. The expression levels of miR-212 significantly reduced in all CRC cell lines compared with that in the FHC cell line (Fig. 1B, P<0.05). Among the five CRC cell lines, HCT116 and SW620 showed the lowest miR-212 expression. Thus, HCT116 and SW620 cells were selected for further investigation.

To observe the functional role of miR-212 in CRC cells, HCT116 and SW620 cells were transfected with miR-212 mimics or miR-NC. After transfection, RT-qPCR analysis demonstrated that miR-212 was remarkably upregulated in the HCT116 and SW620 cells transfected with miR-212 mimics compared with the cells transfected with miR-NC (Fig. 2A, P<0.05). The role of miR-212 in CRC cell viability was then evaluated using MTT assay. As shown in Fig. 2B, miR-212 overexpression decreased the viability of HCT116 and SW620 cells compared with the miR-NC groups (P<0.05). In addition, Transwell invasion assay showed that miR-212 upregulation inhibited the invasion capacities of HCT116 and SW620 cells (Fig. 2C, P<0.05). Overall, these data indicate the anti-viability and anti-metastasis roles of miR-212 in CRC.

MiRNAs exert functional roles mainly by base-pairing with the complementary sequence of their target genes (9). Hence, bioinformatics analysis was used to predict the potential target genes of miR-212. PIK3R3, which is associated with CRC tumorigenesis and development (22), was chosen for further confirmation (Fig. 3A). Then, PIk3R3 mRNA expression in CRC tissues and corresponding adjacent non-neoplastic tissues was detected using RT-qPCR. Results showed that the mRNA expression of PIK3R3 was dramatically upregulated in CRC tissues compared with adjacent non-neoplastic tissues (Fig. 3B, P<0.05). In addition, Spearman's correlation analysis revealed an inverse correlation between miR-212 and PIK3R3 mRNA expression (Fig. 3C, r=−0.6185, P<0.001).

The mRNA and protein expression levels of PIK3R3 in HCT116 and SW620 cells were measured after transfection with miR-212 mimics or miR-NC via RT-qPCR and Western blot analyses to investigate the negative regulation effects of miR-212 on endogenous PIK3R3 expression. Remarkable inhibition of PIK3R3 expression at both mRNA and protein levels was observed in the HCT116 and SW620 cells transfected with miR-212 mimics compared with those transfected with miR-NC (Fig. 3D and E, P<0.05). Moreover, luciferase reporter assay was conducted to identify the relationship between miR-212 and the 3′-UTR of PIK3R3. Results revealed that the restoration expression of miR-212 reduced the luciferase activities of pMIR-PIK3R3-3′-UTR Wt (Fig. 3F, P<0.05) but exerted no effect on the luciferase activities of pMIR-PIK3R3-3′-UTR Mut. Overall, these findings suggest that PIK3R3 is a novel target of miR-212 in CRC.

Rescue experiments were performed to investigate whether the tumor suppressive role of miR-212 in CRC is mediated by inhibiting the expression of PIK3R3. HCT116 and SW620 cells were transfected with miR-212 mimics in the presence or absence of pcDNA3.1-PIK3R3. Western blot analysis demonstrated that miR-212-induced PIK3R3 downregulation was rescued following co-transfection with pcDNA3.1-PIK3R3 (Fig. 4A, P<0.05). Moreover, PIK3R3 upregulation rescued the suppressive effects of miR-212 overexpression on the viability (Fig. 4B, P<0.05) and invasion (Fig. 4C, P<0.05) of HCT116 and SW620 cells. These results clearly show that miR-212 exerts tumor-suppressive roles in CRC, at least in part, by suppressing PIK3R3.

Previous studies demonstrated that PIK3R3 plays important roles in tumor formation and progression by regulating the AKT/mTOR signalling pathway (23–25). Hence, Western blot analysis was performed to determine p-AKT, AKT, p-mTOR and mTOR expression in the HCT116 and SW620 cells transfected with miR-212 mimics or miR-NC. MiR-212 overexpression decreased p-AKT and p-mTOR expression, whereas transfection with miR-212 mimics did not affect total AKT and mTOR protein levels (Fig. 5). These results indicate that miR-212 inhibits CRC progression by directly targeting PIK3R3 and regulating the AKT/mTOR signalling pathway.