CRC tissues along with the adjacent non-cancerous tissues were obtained from 60 CRC patients (age: 58±9.4 years old who underwent surgery at the First Affiliated Hospital of Kunming Medical University between June 2015 and August 2019. All patients had not received any treatment and had no comorbidities. The detailed characteristics of the patients are shown in Table I. Specimens were collected and stored at −80°C. This study was approved by the Ethics Committee of the First Affiliated Hospital of Kunming Medical University, and conducted following the Helsinki Declaration. All patients had signed a written informed consent.

CRC cell lines (HT-29, HCT-116, HCT-8, LoVo and RKO) as well as the normal colonic epithelial cell line (NCM460) and 293T cells were purchased from American Type Culture Collection (ATCC, USA). We authenticated the cell lines used in this study by STR profiling. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (FBS) at 37°C, and humidified with 5% CO₂ atmosphere.

The N13.3 overexpression vector pcN13.3 and pcDNA3 (negative control), short hairpin N13.3 (sh-N13.3–1 and sh-N13.3–2) and its negative control were purchased from GenePharma. The mimics and ASO of miR-4722-3p (miR-4722-3p mimics and miR-4722-3p ASO) and the respective negative control (mimics NC and ASO NC) were purchased from RiboBio. The transfection was performed with Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) following the method described by the manufacturer.

The total RNA of tissues and cell lines were extracted with Trizol Reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the method described by the manufacturer. The total RNA was then reversely transcribed into complementary DNA (cDNA) using RT-PCR kit obtained from Promega Corp. Real-Time qPCR kit purchased from Qiagen was used to estimate the expression of N13.3, miR-4722-3p and P2RY8 through the instrumentation of an ABI 7500 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). Thermocycling conditions consisted of 95°C for 5 min, 40 cycles of 95°C for 5 sec and 60°C for 20 sec, 95°C for 15 min. The primer sequences used in the study are as follows: lncRNA RP11-400N13.3-forward, 5′-TCACAGTAAGCTGCCTTCTAAGGAG-3′ and lncRNA RP11-400N13.3-reverse, 5′-TGGTAAGATCCCTCGGACTAAAACA-3′; miR-4722-3p-forward, 5′-TGCGGTGGCTGGACGTCCCTCCA-3′ and miR-4722-3p-reverse, 5′-CCAGTGCAGGGTCCGAGGT-3′; U6-forward, 5′-TGCGGGTGCTCGCTTCGGCAGC-3′ and U6-reverse, 5′-CCAGTGCAGGGTCCGAGGT-3′; P2RY8-forward, 5′-CGCACCGATCTCACCTACC-3′ and P2RY8-reverse, 5′-GATGGTGGCCGTGTAACAAG-3′; GAPDH-forward, 5′-CTTCTACAATGAGCTGCGTG-3′ and GAPDH-reverse, 5′-TCATGATTGAGTCAGTCAGG-3′. U6 or GAPDH was used as an endogenous control. The relative levels of expression were calculated by the 2^−ΔΔCq method (20).

CRC cells were collected after the different treatments and were seeded into a 6-well plate having a density of 1,000 cells per well, and were cultured for 2 weeks ensuring the replacement of the culture medium at 3-day intervals. Colonies were fixed with 10% formaldehyde and stained with 0.4% crystal violet in 20% ethanol for 5 min. Colonies were photographed and counted using a inverted microscope with ×4 magnification (Olympus Corp.).

Cell proliferation assay was performed with the Cell Counting Kit-8 (CCK-8) Dojindo) following the method described by the manufacturer. In brief, transfected cells were seeded into a 96-well plate at a density of 1×10⁵ cells per well for specific time intervals (0, 24, 48 and 72 h) following the method described by the manufacturer. Absorbance was measured at 450 nm using a microliter plate reader, and in turn, the cell viability was calculated.

The cell migration and invasion capacities were ascertained by Transwell assays in Transwell chamber plates (24-well plate, 8-mm pores; BD Biosciences). For invasion assay, the Transwell chamber was pre-coated with 50 µl of Matrigel (1:7 dilution; BD Bioscience) before the experiments. Cells (5×10⁴ cells) following the different treatments were placed in serum-free medium and were added into the top chambers, with the lower chamber filled with DMEM containing 10% FBS. After incubation for 24 h, the chambers were fixed using 4% paraformaldehyde and stained with 0.1% crystal. Stained cells in the lower chambers were counted in three randomly selected fields with the aid of a inverted microscope with a magnification, ×4 (Olympus Corp.). For the migration assay, a Transwell chamber without pretreatment with Matrigel was used, while the other steps were the same as the invasion assay.

In order to determine the existence of mutual interaction between N13.3 and miR-4722-3p, RNA pull-down assay was performed with a T7 Megascript kit (Thermo Fisher Scientific, Inc.) following the method described by the manufacturer. In addition, expression of miR-4722-3p was detected by RT-qPCR.

The interaction between miR-4722-3p-N13.3 and miR-4722-3p-P2RY8 was determined by dual-luciferase reporter assay. In brief, sequences of N13.3 or P2RY8 3′UTR containing wild-type or mutated sites of miR-4722-3p were synthesized. Cells were co-transfected with luciferase reporter vectors and miR-4722-3p mimic/ASO, alongside their respective negative controls using Lipofectamine 2000. After transfection for 48 h, the luciferase activity was measured by the dual-luciferase reporter assay system (Promega Corp.).

Total protein was extracted from cells or tissues with RIPA buffer (Sigma-Aldrich; Merck KGaA). Protein concentration was determined by the Bicinchoninic Acid (BCA) protein assay (Beyotime Biotechnology). The 30 µg protein extracts were separated by 10% SDS-PAGE, and then transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked by dipping them in 5% non-fat milk at room temperature for 1 h, and incubated with primary antibodies against caspase-3 [product no. 9662S, Cell Signaling Technology, Inc. (CST)], Bcl2, (ab32124; Abcam), Bax (product no. 5023S; CST), P2RY8 (cat. no. ABP52106; Abbkine) and GAPDH (product no. 51332S; CST) overnight. All primary antibodies were diluted with 5% non-fat milk dissolved in 1X TBST. Furthermore, the membranes were incubated with secondary antibodies anti-rabbit (product no. 7074S; CST) and anti-mouse (product no. 7076S; CST) at room temperature for 1 h; the secondary antibody was combined with horseradish peroxidase. All secondary antibodies were also diluted with 5% non-fat milk dissolved in 1X TBST. The generated signal for the protein-antibody complex was detected by enhanced chemiluminescence (ECL) substrate (Millipore). Protein quantification was completed with ImageJ 1.8.0 (National Institute of Mental Health).

All animal experiments were approved by the Ethics Committee of the First Affiliated Hospital of Kunming Medical University. A total of 48 males BALB/c nude mice (age: 4-weeks; weight: 17–23 g) were obtained from Beijing HFK Bioscience. Mice were housed in an SPF environment with 40–60% relative humidity at 22–24°C with a 12-h light/dark cycle and free access to water and food. The mice were randomly divided into two groups (six per group), and then injected subcutaneously with 5×10⁶ HT-29 cells in 200 µl PBS transfected with sh-NC/sh-N13.3, pcDNA3/pcN13.3, mimics NC/miR-4722-3p mimics and ASO NC/miR-4722-3p ASO, respectively. Tumor size was measured at an interval of 4 days, and tumor volume (V) was calculated as V=length × width²/2. After 24 days, the nude mice were sacrificed and tumors were excised for further investigation.

HT-29 cells (1×10⁶) infected with sh-NC or sh-N13.3 were injected into the tail vein of the nude mice (n=6). After 7 weeks, mice were sacrificed and lung metastasis was assessed by counting the number of tumor nodules, and the weight of the lung was subsequently weighed. In addition, H&E staining was used to evaluate lung metastasis.

Cells were transfected for 48 h and collected in binding buffer. Cells were stained with Annexin V-FITC for 10 min, and PI for 5 min (Beyotime Institute of Biotechnology), following the method described by the manufacturer. The rate of apoptosis in cells was estimated by flow cytometry (FACScan; BD Biosciences).

Multiple lncRNA expression in CRC patients and normal individuals was analyzed with NONCODE database (http://www.noncode.org/index.php). Refseq database (https://www.ncbi.nlm.nih.gov/refseq/) was also used to analyzed the expression of lncRNAs in CRC patients. RegRNA 2.0 database was used to predict the target of N13.3 (http://regrna2.mbc.nctu.edu.tw/). P2RY8 was found to be a potential target of miR-4722-3p by Targetscan online software (http://www.targetscan.org/vert_71/).

Data are expressed as the mean ± standard deviation (SD) of at least three separate experiments, and analyzed using GraphPad Prism 5 software (GraphPad Software, Inc.) and SPSS 20 software (IBM, Corp.). Differences between the two groups were assessed with Student's independent t-test. The relationship between N13.3, P2RY8 and miR-4722-3p was determined by Spearman's correlation. Chi-square test was employed to assess the clinical relationship between N13.3 expression and tumor features. One way ANOVA was used to analyze data from multiple groups. The overall survival curve was analyzed using the Kaplan-Meier method and log-rank test. The expression of N13.3, miR-4722-3p, and P2RY8 was analyzed by paired t-test in cancer tissues of CRC patients. P<0.05 was considered to indicate a statistically significant difference.

We firstly analyzed the level of expression of multiple lncRNAs in CRC patients and normal individuals with NONCODE database (http://www.noncode.org/index.php). The relationship between N13.3 expression and clinical features of the CRC patients is shown in Table I. The heatmap indicated that N13.3 was highly expressed in CRC patients compared with normal individuals (Fig. 1A). Further investigation revealed that 32 lncRNAs were overexpressed in CRC in NONCODE and Refseq database (https://www.ncbi.nlm.nih.gov/refseq/) (Fig. 1B). In addition, the expression of N13.3 in CRC 60 cancer tissues and corresponding adjacent normal tissues was detected by RT-qPCR. We found that the expression of N13.3 was upregulated in CRC tissues when compared with that in corresponding adjacent normal tissues (Fig. 1C). Similarly, high expression of N13.3 was found in CRC cell lines when compared with that in the normal colonic epithelial cells (Fig. 1D). We also found that the overexpression of N13.3 was correlated with poor overall survival of CRC patients (Fig. 1E). This results indicate that N13.3 may play a pivotal role in CRC.

To further investigate the role of N13.3 in CRC cells, HT-29 and HCT-116 cells were transfected with sh-N13.3 or sh-NC and transfection efficiency was measured by RT-qPCR (Fig. 2A). CCK-8 assay revealed that the proliferative ability of CRC cells was significantly suppressed by the knockdown of N13.3 (Fig. 2B and C). Consistently, we observed that the rate of apoptosis in cells was significantly elevated following the knockdown of N13.3, as compared with sh-NC group (Figs. S1A and 2D). We also analyzed the expression of apoptotic proteins. The results showed that caspase-3 and Bax were significantly increased, and Bcl2 was significantly decreased in the HT-29 and HCT-116 cells transfected with sh-N13.3 when compared with the sh-NC group (Fig. 2E). The role of N13.3 in the inhibition of cell apoptosis was confirmed by detecting the expression of caspase-3, Bax and Bcl2 in HT-29 and HCT-116 cells having highly expressed N13.3 (Fig. 2F). In addition, colony formation and Transwell invasion assays revealed that the knockdown of N13.3 greatly reduced the colony formation, migration and invasion capacity of HT-29 and HCT-116 cells (Fig. 2G-L).

To further validate the function of N13.3 in CRC cells, we upregulated the expression of N13.3 and ascertained the effect on proliferation, migration and invasion of CRC cells. We found that the expression of N13.3 was successfully increased in CRC cells transfected with pcN13.3 (Fig. 3A). As expected, the cell proliferation, colony formation, migration and invasion capacity were significantly enhanced following transfection of pcN13.3 into the CRC cells (Fig. 3B-I).

In this study, the RegRNA 2.0 database (http://regrna2.mbc.nctu.edu.tw/) predicted N13.3 as a potential downstream target of miR-4722-3p (Fig. 4A). To ascertain the potential binding between miR-4722-3p and N13.3, the luciferase reporter assay was performed. In briefly, HT-29 cells were transfected with N13.3 [wild-type (wt) and mutant (mut), respectively] luciferase reporter vector and miR-4722-3p (miR-4722-3p mimics and miR-4722-3p ASO, respectively). Results showed that miR-4722-3p mimics inhibited luciferase activity (Fig. 4B). However, it was enhanced by miR-4722-3p ASO (Fig. 4C). Furthermore, we detected the expression of miR-4722-3p in CRC cell lines and tissues. The results revealed that miR-4722-3p was significantly downregulated in the CRC cell lines and cancer tissues as compared to the control groups (Fig. 4D and E). Furthermore, RNA pull down assay revealed that miR-4722-3p was enriched in the N13.3 overexpression group as compared to the pcDNA3 group (Fig. 4F). To further validate these results, HT-29 and HCT-116 cells were transfected with pcN13.3/sh-N13.3 and negative control, respectively. We found that the expression of miR-4722-3p was significantly decreased in cells transfected with pcN13.3 compared to the negative control group (Fig. 4G), while sh-N13.3 induced an upregulation of miR-4722-3p (Fig. 4H). To study the effect of miR-4722-3p on CRC cells, we first confirmed the overexpression or knockdown efficiency of miR-4722-3p in CRC cells transfected with miR-4722-3p mimics or ASO (Fig. S1D and E). Next, we found that the expression of N13.3 was significantly decreased or increased in the cells transfected with miR-4722-3p mimics or ASO, respectively (Fig. 4I and J). In addition, N13.3 was negatively correlated with miR-4722-3p in the clinical samples (Fig. 4K). Finally, we observed that miR-4722-3p mimics/ASO markedly suppressed/promoted the proliferation, colony formation, migration and invasion of HT-29 and HCT-116 cells, but these results were reversed in cells transfected with pcN13.3/sh-N13.3 (Figs. 4L-O and S1B and C).

We further predicted P2RY8 as a potential downstream target of miR-4722-3p by Targetscan online software (http://www.targetscan.org/vert_71/) (Fig. 5A). To determine whether miR-4722-3p effectively targets P2RY8, the luciferase reporter assay was employed. The results showed that the simulation of miR-4722-3p distinctly inhibited the luciferase activity of P2RY8-WT reporter (Fig. 5B), while miR-4722-3p ASO significantly increased luciferase activity of P2RY8-WT reporter (Fig. 5C). However, the miR-4722-3p mimics or ASO exhibited no function on the luciferase activity of cells transfected with mutated P2RY8 luciferase reporter (Fig. 5B and C). In addition, the RT-qPCR experiment revealed a significantly high level of expression of P2RY8 in the CRC cell lines and cancer tissues compared to the NCM460 cells and the normal tissue (Fig. 5D and E). In addition, miR-4722-3p mimics significantly downregulated P2RY8 expression in the HT-29 and HCT-116 cells (Fig. 5F). Correspondingly, miR-4722-3p ASO upregulated P2RY8 expression in the HT-29 and HCT-116 cells (Fig. 5G). Simultaneously, a negative and positive correlation was noted between miR-4722-3p and P2RY8, and between N13.3 and P2RY8, respectively, in the CRC tissues (Fig. 5H and I) and this was further investigated by western blot analysis (Fig. 5J and K).

To explore the effects of N13.3 and miR-4722-3p on CRC cell tumorigenesis in animal models, HT-29 cells were transfected with sh-N13.3/pcN13.3 or miR-4722-3p mimics/ASO, and were injected subcutaneously into male nude mice. The results showed that the knockdown of N13.3 inhibited tumor growth, but the tumors formed from the N13.3-overexpressing HT-29 cells showed faster growth rates when compared to the control group (Fig. 6A and F). The tumor weight was significantly lower in the sh-N13.3 group as compared to the control group. In contrast, a significantly higher tumor weight was observed in the N13.3-overexpressing group when compared to the control group (Fig. 6B and G). The expression of N13.3 and P2RY8 was decreased in the sh-N13.3 group (Fig. C and E) while N13.3 and P2RY8 was significantly increased in the HT-29 cells transfected with pcN13.3 (Fig. 6H and J). In addition, miR-4722-3p expression was assessed in the tumor tissues of the N13.3-knockout and overexpressing groups by RT-qPCR (Fig. 6D and I). In addition, we found that the tumor growth was suppressed in the miR-4722-3p mimics group and promoted in miR-4722-3p ASO group (Fig. 6K and M). Subsequently, the tumor weight was significantly lower in the miR-4722-3p mimics group and higher in the miR-4722-3p ASO groups (Fig. 6L and N). Low expression of N13.3 significantly inhibited the lung metastasis of HT-29 cells in vivo (Fig. 6O and P). H&E staining showed that low expression of N13.3 inhibited lung metastasis (Fig. 6Q).