Human colorectal cancer tissues and adjacent tissues were collected from 32 patients at the First Affiliated Hospital of Kunming Medical University between December 2018 and June 2019. These patients included 18 males and 14 females. The age range of patients was 20–60 years old with an average age of 40.57±11.24 years. The present study was approved by the Ethics Committee of First Affiliated Hospital of Kunming Medical University (Ethical approval no. 2018-L-41) and all patients provided informed consent.

The human normal colonic epithelial cell line NCM460 (item no. MJ-550) was purchased from Shanghai Mingjing Biotechnology Co., Ltd. The colorectal cancer cell line HCT116 (item no. C4111) was purchased from Shanghai Guandao Biotechnology Co., Ltd. The colorectal cancer cell line DiFi (BSC-5023479758-01) was purchased from Shanghai Binsui Biotechnology Co., Ltd. The colorectal cancer cell line HROC18 (product no. CE18870) was purchased from Beijing Crisprbio Biotechnology Co., Ltd. The colorectal cancer cell line T84 (product no. YB-H3024) was purchased from Shanghai Yubo Biotechnology Co., Ltd. 293T cells (cell bank no. KCB200744Y) were obtained from the Chinese Academy of Sciences. Cells were cultured in RPMI-1640 culture medium (product code 0050001DJ; Invitrogen; Thermo Fisher Scientific, Inc.) with 10% fetal calf serum (item no. 21097; Cayman Chemical Company), 100 U/ml penicillin and streptomycin (product code DXT-11074440001; Roche Diagnostics). Cells were maintained at 37°C and 5% CO₂.

Small interfering (si)-circNSUN2 (5′-AUCAUAAGGUAUCCUGAAGAACUUG-3′), and circNSUN2 si-negative control (NC) (5′-UUCUCCGAACGUGUCACGUTT-3′), miR-181a-5p inhibitor (5′-ACUCACCGACAGCGUUGAAUGUU-3′), and miR-181a-5p inhibitor NC (5′-CAGUACUUUUGUGUAGUACAA-3′), si-ROCK2 (5′-GGATAAACATGGACATCTA-3′) and si-con (5′-CAGUACUUUUGUGUAGUACAA-3′), miR-181a-5p mimic (5′-AACAUUCAACGCUGUCGGUGAGU-3′) and miR-181a-5p NC mimics (5′-UUUGUACUACACAAAAGUACUG-3′) were constructed by Guangzhou RiboBio Biotechnology Co., Ltd. The HCT116 and T84 cells (2×10⁵) were seeded in a 6-well plate, and NC (50 nM), si-circNSUN2 (50 nM), NC mimics (50 nM), miR-181a-5p mimics (50 nM), miR-181a-5p inhibitor (100 nM), si-con (100 nM) and si-ROCK2 (100 nM), were transfected by Lipofectamine 2000 transfection reagent (item no. J44705; Duoxi) at 37°C for 24 h according to the manufacturer's protocol.

RT-qPCR was used to examine the expression of circNSUN2 and miR-181a-5p. TRIzol reagent (cat. no. 15596018; Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract tissue and cell RNA. The total RNA (1 µg) was used as template for cDNA synthesis using SuperScript III Reverse Transcriptase (cat. no. 18080093; Invitrogen; Thermo Fisher Scientific, Inc.). The tissue and HCT116 or T84 cell cDNA was diluted for PCR amplification using SYBR Green PCR Master Mix (product code SR1110; Solarbio Life Sciences). RT-qPCR was performed at 95°C for 1 min, 40 cycles at 95°C for 10 sec and 58°C for 40 sec. The following primer sequences were used: circNSUN2 forward, 5′-GGAUACCUUAUGAUGAGGCCGC-3′ and reverse, 5′-UGAGGAGCAGUGGUGGGAUC-3′; miR-181a-5p forward, 5′-CGGCAACATTCAACGCTGT-3′ and reverse, 5′-GTGCAGGGTCCGAGGTATTC-3′; ROCK2 forward, 5′-CTAGGCCGGGCGAAG-3′ and reverse, 5′-TCCAGCTTCCTCTGACGAC-3′; GAPDH forward, 5′-GGTGCTGAGTATGTCGTGGAGTCTA-3′ and reverse, 5′-TCTTGAGGGAGTTGTCATATTTCTC-3′; U6 forward, 5′-CTTCGGCAGCACATATAC-3′ and reverse, 5′-GAACGCTTCACGAATTTGC-3′. GAPDH and U6 were used as internal controls. The 2^−ΔΔCq method was used to normalize the fold change in expression (16).

Total protein was isolated from tissue and cells using RIPA lysis (cat. no. 28191; Norgen Biotek) was used to extract total protein. A BCA kit (cat. no. C-0018-30; Bioss) was used to determine the protein content. After denaturation, the proteins (5 µg) were separated by 10% SDS-PAGE and transferred to PVDF membranes. A solution of 5% skimmed milk was used to block the membranes at room temperature for 1 h. Then, the membranes were incubated with anti-caspase-3 (1:500 dilution; product code ab4051; Abcam), anti-cleaved caspase-3 (1:500 dilution; product code ab32042; Abcam), anti-ROCK2 (1:1,000 dilution; cat. no. orb251570; Biorbyt, Ltd.), anti-GAPDH (1:1,000 dilution; cat. no. G13-61M-100; SignalChem Biotech, Inc.) at 4°C overnight, followed by incubation with secondary antibodies IHC Select Streptavidin-HRP (1:1,000 dilution; cat. no. 20774; EMD Millipore) at room temperature for 2 h. The enhanced chemiluminescence kit (cat. no. orb90502; Biorbyt, Ltd.) was used to visualize the immunoreactive signals. GAPDH was used as an internal control. ImageJ 1.8.0 software (National Institutes of Health) was used to quantify the density of target bands.

CCK-8 (item no. R22305; Shanghai Yuanye Bio-Technology, Co. Ltd.) was used to examine cell proliferation. HCT116 cells and T84 cells (5,000 cells/well) /well were plated into a 96-well plate and then cultured in an incubator (37°C, 5% CO₂). After 48 h, 10 µl of CCK-8 was added into each well at 0, 24, 48, 72 and 96 h. Then, after 1 h, the optical density at 450 nm was measured by a microtiter plate reader.

Flow cytometry was used to examine the cell apoptosis rate. HCT116 cells and T84 cells (5,000 cells) were collected using trypsin and washed with PBS. Then, an Annexin V-FITC/PI apoptosis detection kit (cat. no. 556547; BD Biosciences) was used according to the manufacturer's instructions. The fluorescence intensity of the cells was quantified by flow cytometry (BD FACSCalibur™). Flow cytometry analysis was performed using FlowJo10.0.0 (FlowJo, LLC).

A Transwell assay was used to evaluate HCT116 and T84 cell migration ability using Transwell chambers (8-µm pore size; cat. no. MCEP06H48; EMD Millipore). Cell suspensions (100 µl cell suspension/well) were prepared in serum-free medium and plated in the upper chamber, while the lower chamber was filled with medium containing 10% FBS (cat. no. DXT-10099141, Gibco; Thermo Fisher Scientific, Inc.). The chamber was incubated for 24 h at 37°C. After 24 h, the cells in the upper chamber were removed with a swab, and the cells that migrated into the lower chamber were fixed in 75% methanol (item no. JKLN042113; Shanghai Jingke technology Co., Ltd.) for 30 min and stained with 1% crystal violet (item no. J12750; Duoxi) for 1 h at room temperature. The migrated cells were observed and imaged under a light microscope at a magnification of ×40 in 5 randomly selected visual fields in each group.

The binding sites between circNSUN2 and miR-181a-5p and between miR-181a-5p and ROCK2 were predicted by starBase2.0 (http://starbase.sysu.edu.cn/) and TargetScan7.1 (http://www.targetscan.org/vert_71/). A dual-luciferase reporter assay was used to verify the relationship between circNSUN2 and miR-181a-5p and between miR-181a-5p and ROCK2. The circNSUN2 or ROCK2 region containing wild-type (WT) or mutant (MUT) binding sites of miR-181a-5p were cloned into pGL4.49 vector (cat. no. E4611; Promega Corporation). The 293T cells were plated and co-transfected with vectors by Lipofectamine 2000 according to the manufacturer's protocol. After 48 h, the luciferase activity was determined by a Dual-luciferase Reporter Assay kit (cat. no. K801-200, BioVision, Inc.). Firefly luciferase activity was normalized to Renilla luciferase activity.

An in vivo tumor growth assay was used to examine tumor growth. All animal experiments were approved by the Experimental Animal Ethics Committee of Kunming Medical University and adhered to international guidelines for proper animal care and maintenance. Nude mice (n=24; male; 3–4 weeks old; weight 19–25 g) purchased from the Animal Center of Kunming Medical University were randomly separated into four groups and housed in a pathogen-free facility (28°C; 55% humidity; 12-h light/dark cycle with food and water ad libitum). Approximately 1×10⁶ HCT116 and T84 cells with stable expression of si-circNSUN2 and NC were subcutaneously injected into BALB/C nude mice. Mice were under daily monitoring. The tumor volume was monitored at 4, 8, 12, 16, 20, 24, and 28 days using manual calipers after injection. Tumors were obtained at 4 weeks after injection. At the end of the experiment, six nude mice in each group were euthanized by anesthesia-mediated by overdose pentobarbital sodium via intravenous injection and tumors were harvested. The sacrifice of mice was confirmed by cessation of breathing and heartbeat which were monitored. The maximal tumor volume allowed in this experiment was 1.8 cm³. The tumor volume was measured according to the following equation: (width)²x(length/2).

TUNEL staining was used to evaluate tumor apoptosis. The tumors were removed from the mice, fixed in 4% formaldehyde at room temperature for 24 h and then embedded in paraffin. Then, the tumors were sectioned into 4-µm sections and adhered to slides. A TUNEL kit (cat. no. 4500-0121-1; Merck KGaA) was used to stain the sections at 37°C for 30–60 min according to the manufacturer's instructions. After washing the slides with PBS and air drying, the apoptotic cells were assessed in 10 randomly selected fields under a confocal microscope at a magnification of ×40.

The data are presented as the mean ± standard deviation (SD). Statistical analysis was performed using SPSS 17.0 software (SPSS, Inc.). He statistical significance of differences between two groups was evaluated by paired Student's t-test and significant difference between more than two groups was evaluated with one-way ANOVA by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

In the present study, the expression of circNSUN2 was examined in colorectal cancer tissues and cell lines by RT-qPCR. The results revealed that circNSUN2 was highly expressed in colorectal cancer tissues compared with adjacent tissues (Fig. 1A; P<0.01). Moreover, the expression levels of circNSUN2 were higher in CRC cell lines, especially in HCT116 and T84 cells (Fig. 1B; P<0.05 and P<0.01) compared with NCM460 cells. Therefore, HCT116 and T84 cells were selected for the following experiments. Notably, the aforementioned results indicated that circNSUN2 was highly expressed in CRC tissues and cell lines.

To determine the function of circNSUN2 in CRC, circNSUN2 was first knocked down in HCT116 and T84 cells by specific siRNA and the transfection efficiency was validated by RT-qPCR (Fig. 2A; P<0.05). Functionally, CCK-8 assays and Transwell assays revealed that inhibition of circNSUN2 attenuated cell proliferation and migration, while flow cytometric assays and western blot assays indicated that inhibition of circNSUN2 promoted cell apoptosis (Fig. 2B-E; P<0.05 and P<0.01). Moreover, the in vivo tumor growth assay results revealed that knockdown of circNSUN2 inhibited tumor growth in terms of both volume and weight (Fig. 2F; P<0.05). In addition, TUNEL staining results revealed that compared with the NC group, the apoptosis of tumor cells in si-circNSUN2 group increased (Fig. 2G). Hence, knockdown of circNSUN2 markedly inhibited the malignant biological behavior of CRC in vivo and in vitro.

Based on the results from the bioinformatics database starBase, miR-181a-5p was predicted to be a target gene of circNSUN2 (Fig. 3A). Dual-luciferase reporter assay results revealed that the luciferase activity of the circNSUN2 WT reporter was decreased in the miR-181a-5p mimics group, but the luciferase activity of the circNSUN2 MUT reporter was not significantly different from that of the NC group (Fig. 3B; P<0.05). Moreover, RT-qPCR results revealed that the expression level of miR-181a-5p was increased in the si-circNSUN2 group of HCT116 and T84 cells (Fig. 3C; P<0.05). In addition, RT-qPCR results revealed that the expression levels of miR-181a-5p in the CRC cell lines HCT116 and T84 were decreased compared with those in the normal colonic epithelial cell line NCM460 (Fig. 3D; P<0.05). Therefore, circNSUN2 targeted miR-181a-5p and downregulated its expression in the CRC cell lines HCT116 and T84. In addition, miR-181a-5p was downregulated in CRC cell lines.

In order to determine the role of miR-181-5p in circNSUN2-mediated malignant biological behavior of CRC cells, miR-181a-5p mimics transfection was performed. The transfection efficiency was determined by RT-qPCR (Fig. 4A; P<0.05). As revealed in Fig. 4B and C, miR-181a-5p overexpression resulted in decreased proliferation rates and migration abilities of HCT116 and T84 cells (P<0.05). Moreover, forced miR-181a-5p expression increased the apoptotic rate of HCT116 and T84 cells (Fig. 4D and E; P<0.05 and P<0.01). In addition, rescue experiments were performed to validate the relationship between circNSUN2 and miR-181a-5p. Transfection of HCT116 and T84 cells with si-circNSUN2 had a significant influence on miR-181-5p overexpression-mediated malignant biological behavior (Fig. 4A-E). Overall, these results indicated that overexpression of miR-181-5p attenuated the effects of circNSUN2 inhibition on HCT116 and T84 cell behaviors.

miRNAs function by modulating the expression of target genes (18). According to bioinformatics analysis, ROCK2 was revealed as a target of miR-181a-5p (Fig. 5A). Moreover, dual-luciferase reporter assay results revealed that overexpression of miR-181a-5p decreased the luciferase activity of the ROCK2 WT reporter but had no effect on the ROCK2 MUT reporter (Fig. 5B; P<0.05). Moreover, western blotting revealed that overexpression of miR-181a-5p downregulated the expression of ROCK2 in HCT116 and T84 cells (Fig. 5C; P<0.05). Therefore, these results indicated that ROCK2 is a direct target of miR-181a-5p in CRC.

Since ROCK2 was validated as a direct target of miR-181a-5p, we next focused on ROCK2 to investigate its role in the malignant biological behavior of CRC cells. As revealed in Fig. 6A and B, the expression levels of ROCK2 in CRC cells and tissues were upregulated (P<0.05). miR-181a-5p inhibitor transfection was determined by RT-qPCR (Fig. 6C; P<0.01 and P<0.001). Moreover, inhibition of circNSUN2 decreased the expression level of ROCK2 while miR-181a-5p knockdown increased the expression level of ROCK2 (Fig. 6D; P<0.01 and P<0.001), which indicated that circNSUN2 sponged miR-181a-5p to promote the expression of ROCK2. In order to determine the function of ROCK2 in CRC cell behaviors, HCT116 and T84 cells were transfected with siRNA targeting ROCK2 (Fig. 6E; P<0.001). As revealed in Fig. 6F-I, ROCK2 downregulation led to decreased proliferation rates and migration abilities and increased apoptosis rates (P<0.05, P<0.01 and P<0.001) in HCT116 and T84 cells. Thus, these results indicated that the circNSUN2/miR-181a-5p/ROCK2 axis played a critical role in CRC development.