The fresh clinical CC tissues and adjacent normal tissues were obtained from patients who underwent surgical treatment for CC at Affiliated Hospital of Changchun University of Traditional Chinese Medicine (Changchun, China) between August 2014 and December 2016. All tissues were immediately placed in liquid nitrogen in a freezing tube following surgery and were stored at −80°C until the extraction of total RNA and protein were performed. All experiments in the study were performed in accordance with guidelines and regulations, and were approved by the Institutional Ethics Committee of Tumor Hospital of Jilin Province (Changchun, China); written informed consent was obtained from all participants. None of the patients received preoperative chemotherapy or radiotherapy. All samples were blindly confirmed by two experienced pathologists. Clinical stage was conducted in accordance with the protocols of the American Joint Committee on Cancer Staging System.

The human colon cancer cell lines (HT29, HCT116, SW480, SW620, and LoVo), and the human normal colonic epithelial cell line (NCM460) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). All cells were cultured in Roswell Park Memorial Institute 1640 medium (RPMI-1640) supplemented with 10% fetal bovine serum (FBS; both Gibco; BRL, Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin, and 100 mg/ml streptomycin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at 37°C in a humidified incubator containing 5% CO₂.

A short hairpin RNA (shRNA)-targeted CASC15 was designed by GenePharma (Shanghai, China) and cloned into pRNAT-U6.1/Neo plasmid (Biovector, Beijing, China) to generate a pRNAT-U6.1/Neo-shCASC15 plasmid. The empty pRNAT-U6.1/Neo plasmid was used as the negative control (shNC). All constructs were confirmed by DNA sequencing. To knockdown CASC15 expression, CC cells were transfected with shNC or shCASC15 by using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's instructions. For establishing a cell line with stable silence of CASC15, the plasmids carrying shCASC15 or shNC were co-transfected with packaging vectors, namely, pMDLg/pRRE, pRSV-REV, and pCMV-VSVG, to produce pseudotyped lentiviruses designated as Lv-shSUMO1P3-1 and Lv-shNC. The lentiviruses were concentrated by ultracentrifugation and then infected CC cells. After 2 weeks of screening using 200 µg/ml neomycin (Sigma-Aldrich; Merck KGaA) for CC cells transfected with shCASC15 or shNC plasmids (in vitro assays) single clones were collected.

Five-week-old male athymic nude BALB/c mice were maintained under specific pathogen-free conditions and manipulated according to protocols approved by the Medical Experimental Animal Care Commission at Tumor Hospital of Jilin Province; all experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals. For subcutaneous xenograft assay, 5×10⁵ SW480 cells infected with shNC or shCASC15 were subcutaneously inoculated in the flanks of the mice (n=10 mice/group). A caliper was used to examine the tumor volumes once a week. At 5 weeks post-inoculation, the mice were sacrificed by euthanasia. The tumors were then removed and weighed.

2000 HCT-116 or SW480 cells were seeded in 96-well plates for 3 days. Cell Counting kit-8 (CCK8) (Beyotime, Shanghai, China) was used to assess the cell proliferation. In general, 10 µl CCK8 solution was added to each plate and cells were incubated for 2 h in 37°C. The cell viability was revealed by the absorbance which was measured at 450 nm.

SW480 and HCT-116 cells transfected with shCASC15 were harvested after 48 h. The cells were resuspended in serum-free medium, and 3×10⁴ cells (100 µl) was plated into the upper chambers of Transwell inserts (8.0-µm pore size; Corning Inc., Corning, NY, USA) for invasion (with Matrigel) or migration (without Matrigel) assays. The inserts were placed in 24-well plates containing 600 µl media with 10% FBS as a chemoattractant. After incubation for 24 h for migration and 48 h for invasion, cells were fixed with 4% triformol for 20 min and stained with 1% crystal violet. The cell numbers were calculated and imaged under a microscope from five random fields.

The CC cells were trypsinized and washed with cold phosphate-buffered saline (PBS). The cells were then fixed with ice-cold 70% ethanol at 4°C overnight. After washing with PBS, the cells were treated with RNAase (Takara, Dalian, China) for 30 min at 37°C. Intracellular DNA was labeled with propidium iodide (50 µg/ml; Sigma-Aldrich; Merck KGaA) at 4°C for 30 min and then analyzed using BD FACSCalibur flow cytometry (BD Bioscience, San Jose, CA, USA). The proportions of cells in the G0/G1, S, and G2/M phases were calculated using the ModFit software (Verity Software House Inc., Topsham, ME, USA).

After transfection, total RNA were prepared by TRIzol extraction (Invitrogen; Thermo Fisher Scientific, Inc.) and reversed transcribed into cDNA using a PrimeScript RT reagent kit (Takara). RT-qPCR analyses were performed with SYBR-Green (Takara). Results were normalized to the expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Relative gene expression levels were calculated using the 2^−∆∆Cq method (20).

Cells were co-transfected with plasmid containing target genes [wild type (WT)- or mutant (Mut)-CASC15 and WT- or Mut-LGR5-3′UTR)] and 50 nM miR-4310 mimic. Forty-eight hours after transfection, cells were collected and luciferase activity was detected by Dual-Luciferase Reporter Assay kit (Promega Corp., Madison, WI, USA) in accordance with manufacturer's instructions.

All statistical analyses were performed using SPSS 20.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism version 5.0 software (GraphPad Software, Inc., La Jolla, CA, USA). Student's t-test and one-way analysis of variance followed by Tukey's post hoc test were used to analyze 2 or multiple groups, respectively, for statistical significance. Pearson correlation coefficient analysis was used to determine the correlations. P<0.05 was considered to indicate a statistically significant difference.

We firstly analyzed the expression of CASC15 in CC tissues by RT-qPCR. The results showed that CASC15 was overexpressed in CC tissues compared to matched normal tissues (Fig. 1A; P<0.05). Then we analyzed the correlation between CASC15 expression and clinicopathological features in CC patients. We divided these CC samples into two groups based on TNM stage. Through RT-qPCR, we found that the expression of CASC15 was higher in CC samples of stage III/IV than I/II (Fig. 1B; P<0.05). Moreover, the expression of CASC15 in metastatic CC patients was upregulated compared to those non-metastatic patients (Fig. 1C; P<0.05). We then analyzed the expression of CASC15 in CC cell lines. RT-qPCR results indicated that CASC15 expression was higher in CC cell lines (HT29, HCT116, SW480, SW620, and LoVo) than in the human normal colonic epithelial cell line NCM460 (Fig. 1D; P<0.05). These data indicated that CASC15 was overexpressed in CC cells.

Among all CC cell lines, the expression of CASC15 was highest in HCT116 and SW480 cells. Thus, we chose these two cell lines for experiments. To determine the function of CASC15, we knocked down CASC15 in HCT116 and SW480 cells. RT-qPCR results showed that the expression of CASC15 was significantly down-regulated in HCT116 and SW480 cells transfected shCASC15 (Fig. 2A; P<0.05). Then we performed CCK8 assay to evaluate cellular proliferation. As shown in Fig. 2B, knockdown of CASC15 significantly inhibited cellular proliferation. Moreover, FACS analysis indicated less HCT116 and SW480 cells entered into S phase and G2/M phase after transfection with shCASC15, and more shCASC15 cells were arrested in G0/G1 phase (Fig. 2C; P<0.05). In consistence, knockdown of CASC15 decreased the protein levels of Cyclin D1 and Ki67 in HCT116 and SW480 cells (Fig. 2D; P<0.05), which suggested that CASC15 knockdown inhibited CC cell proliferation.

We above demonstrated a positive correlation between CASC15 expression and tumor metastasis. We then checked the effect of CASC15 on cell migration and invasion. Through Transwell assay, we found that knockdown of CASC15 significantly suppressed the migration and invasion of HCT116 and SW480 cells (Fig. 3A and B). Moreover, the expression of metastasis-related proteins, Twist and Snail, was significantly downregulated in HCT116 and SW480 cells transfected with shCASC15 (Fig. 3C).

LncRNAs have been demonstrated to serve as competing endogenous RNAs for miRNAs. Thus, we analyzed the predicted target miRNAs of CASC15 by informatics analysis. Among all potential targets, miR-4310 ranked first. We found that there were two potential binding sites of miR-4310 in CASC15 (Fig. 4A). Luciferase reporter assay indicated that overexpression of miR-4310 repressed the luciferase intensity in HCT116 and SW480 cells transfected with WT-CASC15 reporter vector (Fig. 4B). Moreover, overexpression of miR-4310 significantly suppressed the expression of CASC15 (Fig. 4C). MiRNAs could target the complementary sequence of target mRNAs to regulate gene expression. To further explore the downstream signal, we conducted another prediction and found that LGR5 was a potential target of mIR-4310. There was a complementary sequence of miR-4310 in the 3′-UTR region of LGR5 mRNA (Fig. 4D). Similarly, luciferase reporter assay indicated that overexpression of miR-4310 significantly inhibited the luciferase activity in HCT116 and SW480 cells transfected with WT-LGR5 reporter vector (Fig. 4E). And overexpression of miR-4310 suppressed the mRNA and protein level of LGR5 (Fig. 4F and G). LGR5 is acknowledged as an essential regulator for activation of Wnt/β-catenin pathway (21,22). Then we determined whether CASC15 regulated Wnt/β-catenin pathway by LGR5. Western blot results indicated that overexpression of CASC15 significantly upregulated the expression of LGR5 and increased the protein level of nucleus β-catenin and MYC (Fig. 4G), which suggested that CASC15 promoted the activation of Wnt/β-catenin. To further determine whether CASC15 promotes CC cell proliferation, migration and invasion in a Wnt/β-catenin pathway dependent manner, we inhibited the Wnt/β-catenin pathway with a specific inhibitor (IWR-1) in CASC15-overexpressed HCT116 and SW480 cells. Then we performed functional experiments. Through CCK8 and Transwell assay, we found that overexpression of CASC15 promoted the proliferation, migration and invasion of CC cells while inhibition of Wnt/β-catenin pathway reversed CASC15-mediated effects (Fig. 4H-J). Taken together, our results indicated that Wnt/β-catenin pathway is important for the function of CASC15 in CC.

We further investigated the function of CASC15 in CC cells in vivo. We subcutaneously injected shCASC15 or control SW480 cells into the flank of nude mice. We measured tumor volumes at indicative time points. As shown in Fig. 5A, knockdown of CASC15 significantly inhibited tumor growth in vivo. Moreover, the tumor tissues in shCASC15 group were smaller than control group (Fig. 5B). Then we measured the protein levels of proliferation- or metastasis-related genes in form tumor tissues. We found that knockdown of CASC15 significantly inhibited the levels of Cyclin D1, Ki67, Twist, Snail and LGR5 (Fig. 5C). These data indicated that CASC15 promoted CC progression in vivo.