Our study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. From July 2010 to December 2012, we collected 100 pairs of human CRC tissues and adjacent normal tissues from patients after they had signed an informed consent form. Of the patients, 64 were males and 36 were females. Ages of patients at the time of surgery ranged from 27 to 82 years (60.14±11.72 years). The tumor tissues and adjacent normal tissues were collected in liquid nitrogen within 5 min, and then immediately transferred to a −70°C freezer for long-term storage. The tumor-node-metastasis (TNM) stage was classified according to the National Comprehensive Cancer Network (NCCN). In this study, none of the patients received any preoperative treatments.

CRC cell lines LoVo, HCT116, DLD-1, and human colon epithelial mucosal cell line NCM460 were maintained in our laboratory. All cell lines were cultured with Dulbecco's modified Eagle's medium (DMEM; Winsent, Quebec, Canada) supplemented with 10% fetal bovine serum (Winsent), 100 U/ml penicillin, and 100 µg/ml streptomycin in a moist incubator (stabilized at 5% CO₂ and 37°C).

Total RNA was extracted from CRC tissues and adjacent normal tissues using Invitrogen™ TRIzol reagent (Thermo Fisher Scientific, Inc., Waltham, MA, USA) according to the manufacturer's instructions. The RNA was converted to cDNA using the PrimeScript RT reagent kit (Takara Biotechnology, Co., Ltd., Dalian, China). The qPCR experiment was performed using a SYBR Green PCR kit (Roche Diagnostics, Indianapolis, IN, USA) in a volume of 20 µl, and then the final reaction was carried using athe Applied Biosystems Step-One Plus Real-Time PCR System (Thermo Fisher Scientific, Inc.). The qPCR cycling was performed as follows: Hot-start DNA polymerase activation at 95°C for 10 min; 40 cycles of 95°C for 15 sec, and 60°C for 1 min; followed by 1 cycle of melting curve analysis of 95°C for 15 sec, 60°C for 1 min, and 95°C for 15 sec. The primer sequences for uc.338 were as follows: 5′-AGCGACAGTGCGAGCTTT-3′ (forward) and 5′-TTCCGAGTGAGTTAGGAAGG-3′ (reverse). The primer sequences for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were as follows: 5′-GTGGACATCCGCAAAGAC-3′ (forward) and 5′-AAAGGGTGTAACGCAACTA-3′ (reverse).

Small interfering RNA (siRNA) targeting uc.338 and a negative control (NC) siRNA were designed by Genepharma Corp. (Shanghai, China) and transferred into LoVo and HCT116 cells. The target sequence was as follows: siuc. 338, 5′-CCACAGGACAGGUACAGCA-3′. For uc.338 overexpression, 5 µg per-well (6-well plate) of the plasmids expressing uc.338 and negative control sequence designed by GeneCopoeia, Inc. (Rockville, MD, USA) were transferred into DLD-1 cells. All the above siRNA sequences and plasmids were transfected using Invitrogen™ Lipofectamine 3000 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. Transfection efficiency was analyzed after 48 h. Additionally, a lentivirus expressing a small hairpin RNA against uc.338 (shuc.338) for an in vivo study was designed according to the sequence of siuc.338. The phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) inhibitor LY294002 was purchased from Cell Signaling Technology (Danvers, MA, USA).

Cell proliferation was measured using a Cell Counting Kit-8 assay (CCK-8; Dojindo Laboratories, Tokyo, Japan) according to the manufacturer's protocol. After 24, 48, 72 and 96 h, 10 µl CCK-8 assay solution mixed with 90 µl of serum-free medium was added in each well containing the cells to be tested. The absorbance was measured 2 h later using a microplate reader at a test wavelength of 450 nm and a reference wavelength of 630 nm.

The EdU assay kit (RiboBio, China) was used to detect the number of cells that were in the DNA replication period, which could indirectly reveal the cell proliferation rate. The steps were as follows. Before the addition of EdU (50 µM), the cells were cultured with DMEM (10% FBS) for 24 h in 24-well plates (2×10⁴ cells/well). The cells were then incubated for 2 h at 37°C, fixed in 4% formaldehyde for 30 min, and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. After washing with phosphate-buffered saline (PBS) for 5 min, 400 µl of 1X ApolloR reaction cocktail was added to react with the EdU for 30 min. Finally, 400 µl of Hoechst 33342 was added for 30 min to visualize the cell nuclei. The cells were then observed under a Nikon microscope (Nikon TI-DH; Nikon, Tokyo, Japan).

Five hundred cells were treated with siuc.338 and NC and cultured in wells of a 6-well plate to perform the colony formation assay. All cells were cultured with DMEM + 10% serum + 100 U/ml penicillin and 100 µg/ml streptomycin under the same conditions. One week later, the cells were washed with cold PBS three times at room temperature. The cells were immediately fixed with ethyl alcohol for 30 sec, and stained with crystal violet dye for 20 min. Cell colonies (≥50 cells/colony) were observed and imaged using a digital camera (Canon DS126211; Canon, Tokyo, Japan) in each plate after washing with PBS.

All the CRC cells were collected and saved in 75% ethyl alcohol at 4°C overnight. Before detection, the treated cells were fixed with 500 µl of propidium iodide (PI) staining solution and incubated for 30 min in the dark. The analysis of cell cycles was performed using a fluorescence-activated cell sorting (FACS) Calibur flow cytometer with BD CellQuest software (version 3.0; BD Biosciences, Franklin Lakes, NJ, USA).

Our animal experiments were approved by the Animal Ethics Committee of Nanjing medical university (NJMU). Fifteen male BALB/c nude mice (aged 3–4 weeks, 13–15 g weight) were purchased from the Animal Center of NJMU and were randomly divided to two groups (the LoVo cell group and the HCT116 cell group). Then, 2×10⁶ of CRC cells mixed with 200 µl PBS were subcutaneously injected into the anesthetized mice. Each mouse was randomly injected with cells treated in two different ways (shuc.338 and NC) in their right and left armpits. The mice were maintained under the following conditions: room temperature, 20–26°C; relative humidity, 40–70%; light/dark cycle, 12 h; food and water, 5 g food and 100 ml water per 100 g body weight per day. Four weeks after injection, all mice were sacrificed by cervical dislocation and all the tumors were surgically removed. The maximum tumor size was as allowable by IACUC guidelines (diameter, 1.5 cm; area, 1.8 cm²; and volume 1.8 cm³).

Protein was extracted from CRC cells by using a Radioimmunoprecipitation assay (RIPA) kit (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacturer's protocols. The concentration was determined using the Bicinchoninic Acid Protein Assay kit (BCA). Proteins (40 µg) with different molecular weights were separated on 10% SDS-PAGE gels in running buffer and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA) in transfer buffer. The membranes were then blocked in 5% non-fat milk at room temperature for over 2 h and incubated in specific primary antibodies at 4°C overnight. After washing in TBST three times (10 min each), the membranes were incubated with secondary antibodies (anti-rabbit or anti-mouse) at room temperature for 2 h. Immunoreactive protein bands were visualized using ECL Plus (Millipore, Billerica, MA, USA) with a Bio-Imaging System. The specific primary antibodies recognized the following proteins: p-PI3K (diluted 1:1,000; cat. no. ab182651; Abcam), PI3K (diluted 1:1,000; cat. no. ab86714; Abcam), p-AKT (diluted 1:500; cat. no. ab38449; Abcam), AKT (diluted 1:500; cat. no. ab8805; Abcam), p21 (diluted 1:5,000; cat. no. ab109520; Abcam) and cyclin D1 (diluted 1:10,000; cat. no. ab134175; Abcam). GAPDH (diluted 1:5,000; cat. no. ab8245; Abcam) was used as an internal control. The secondary antibody was horseradish peroxidase (HRP)-goat anti-rabbit (diluted 1:5,000; cat. no. GAB007; Hangzhou Multi Sciences Biotech, Co., Ltd., Hangzhou, China) or anti-mouse (diluted 1:5,000; cat. no. GAM007; Hangzhou Multi Sciences Biotech, Co., Ltd., Hangzhou, China) IgG.

SPSS (version 15.0) (SPSS, Inc., Chicago, IL, USA) and Graphpad Prism5 (GraphPad Software, Inc., La Jolla, CA, USA) were used for the statistical analysis. Data were derived from at least three repeated experiments. The statistical analyses were performed using t-tests, Pearson's χ² tests and ANOVA. Differences were considered to be statistically significant at P≤0.05. The Kaplan-Meier method was used to perform the cumulative survival analysis.

To reveal the role of uc.338 in CRC progression, we first detected its expression level in 100 pairs of CRC tissues and adjacent normal tissues using qPCR. The results showed that uc.338 expression was significantly upregulated in CRC tissues compared with that in normal tissues (Fig. 1A). According to the expression level of uc.338 in CRC tissues, we further verified that higher uc.338 expression predicted a larger tumor size, deeper tumor invasion, and increased lymph node metastasis (P<0.05, Table I). However, we did not find any association between uc.338 expression and other clinical features, including sex, age, tumor stage, liver metastasis, location and CEA. We then examined whether the expression level of uc.338 could predict the overall survival (OS) of patients diagnosed with CRC. Using Kaplan-Meier curves, we found that higher uc.338 expression predicted poorer overall survival (OS) in patients with CRC (Fig. 1B). The P-value was 0.012 (P<0.05).

To detect whether uc.338 is upregulated in CRC cell lines, we detected the mRNA expression level of uc.338 using qPCR. Compared with that in the human colon epithelial mucosa cell line NCM460, the mRNA expression of uc.338 was upregulated in LoVo, HCT116 and DLD-1 cells (Fig. 1C). To knockdown uc.338 expression, LoVo and HCT116 cells were transfected with a siRNA designed to inhibit uc.338 expression (siuc.338). The result revealed that the mRNA expression of uc.338 was significantly inhibited by siuc.338 in both LoVo and HCT116 cells (Fig. 1D and E). The interference efficiency of siuc.338 was 71 and 79% in LoVo and HCT116 cells, respectively (P<0.05).

To detect the effect of uc.338 on cell proliferation, we performed a CCK-8 assay, an EdU assay, and a plate colony formation assay in succession. In the CCK-8 assay, we found that the viability of LoVo and HCT116 cells was significantly decreased after knockdown of uc.338 (Fig. 2A and B). On the fourth day, the viability of LoVo and HCT116 cells had decreased by 33.9 and 41.1%, respectively (P<0.05). In the EdU assay, compared with the control cells, we found that the proportion of cells in the DNA replication phase decreased by 19.8 and 21.9% in the LoVo and HCT116 cells, respectively, after knockdown of uc.338 (Fig. 2C and D), which revealed that knockdown of uc.338 could inhibit DNA synthesis in CRC cells. In the plate colony formation assay, the result revealed that knockdown of uc.338 significantly inhibited the formation of cell colonies in the LoVo and HCT116 cells (Fig. 3A and B). After 7 days, we found that the number of cell colonies decreased by 46 and 42% in LoVo and HCT116 cells, respectively, after knockdown of uc.338 (P<0.05). According to the results of three assays, knockdown of uc.338 inhibited the cell proliferation in CRC LoVo and HCT116 cells.

To detect the role of uc.338 in the cell cycle of CRC cells, we performed cell cycle analysis using flow cytometry. After knockdown of uc.338, we found that the cell number in the G1 phase increased by 15.5 and 23.6% in LoVo and HCT116 cells, respectively, whereas the cell number in the S phase decreased by 11.4 and 15.5%, respectively, in these two cell lines (Fig. 3C and D). Our result indicated that knockdown of uc.338 caused marked cell cycle arrest in the G1/S phases in the LoVo and HCT116 cells.

To detect the role of uc.338 in the tumor growth in nude mice, LoVo and HCT116 cells were transfected with uc.338 inhibitor lentivirus (shuc.338) (Fig. 4B). Then, 2×10⁶ cells transfected with shuc.338 and a negative control sequence (control) were separately injected subcutaneously into the nude mice (Fig. 4A). After four weeks, we found that the size and weight of the tumors were significantly decreased in the nude mice transfected with the cells with knockdown of uc.338 (Fig. 4C and D). Compared with the tumors from the control groups, the weight of tumors formed by the uc.338-knockdown LoVo and HCT116 cells was decreased by 61.1 and 54.9% in the shuc.338 groups (P<0.05). Therefore, knockdown of uc.338 was able to suppress tumor growth in nude mice.

We revealed that uc.338 could cause cell cycle arrest in the G1/S phase in CRC cells. To further detect the potential mechanism of cell cycle arrest, we detected a series of cell cycle-related proteins that could regulate the G1/S phase in LoVo and HCT116 cells. The results showed that the level of cyclin D1 was significantly downregulated and the level of p21 was upregulated by uc.338 knockdown. Further investigation revealed that the levels of p-PI3K and p-AKT were downregulated by uc.338 knockdown, whereas no significant change was detected in the levels of total PI3K and AKT (Fig. 5A and B).

To further investigate the role of the PI3K/AKT pathway in uc.338-induced cell proliferation in CRC, a specific inhibitor of PI3K (LY294002) was used. uc.338 was overexpressed in the DLD-1 cell line (Fig. 6A), which expresses a relatively low level of uc.338, and then the cells were treated with LY294002. By western blotting, we found that the PI3K/AKT pathway was significantly inhibited by LY294002 (Fig. 6B and C). The colony formation assay showed that overexpression of uc.338 promoted cell proliferation in DLD-1 cells; however, this promotion of cell proliferation was inhibited by LY294002 (Fig. 6D and E). Taken together, the results demonstrated that knockdown of uc.338 inhibited the expression of cyclin D1 and promoted the expression of p21, possibly via the PI3K/AKT pathway.