All 31 CRC tissue specimens and pericarcinomatous tissues were collected from the Third Affiliated Hospital of Central South University between January 2017 and May 2017 (Changsha, China). There are 15 female and 16 male patients and their age ranged from 35 to 70 years. All tissues were immediately flash-frozen in liquid nitrogen after resection and were then stored in liquid nitrogen. All cases were diagnosed as colorectal cancer (CRC) by pathological sections, and the patients received no chemo-, radio- or hormone therapy. All the patients signed consent forms. The present study was approved by the Institute Research Medical Ethics Committee of Central South University (Changsha, China).

BALB/C nude mice (n=24, female, 5-weeks-old and 16–20 g) were purchased from the SJA Laboratory Animal Company (Changsha, China) and housed under specific pathogen-free conditions with a 12-h light/dark cycle and autoclaved food/water were provided freely. The LV-PLAGL2-Homo-808 or LV–Vector (Shanghai GenePharma Co., Ltd., Shanghai, China) was stably transfected in SW480 and HCT116 cells following the manufacturer's instructions, and a suspension of transfected-cells [5×10⁶ in 100 µl phosphate-buffered saline (PBS)] was injected into nude mice (n=6 for each group). Three groups were injected with HCT116 cells (mock, LV-Vector and LV-PLAGL2-Homo-808). The tumor volume was assessed every three days beginning 10 days after the injection and was calculated with the following formula: Tumor volume (mm³) = (length × width²)/2. All mice were sacrificed 30 days later by cervical dislocation after the mice were anaesthetized, and all animal protocols in this study were approved by the Animal Ethics Committee of Central South University (Changsha, China).

The human CRC cell lines SW480 and HCT116 were purchased from Wuhan Boster Biological Technology Ltd. (Wuhan, China). SW480 cells were cultured in L15 medium (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China) containing 10% fetal bovine serum (FBS) (Biological Industries, Kibbutz Beit-Haemek, Israel). HCT116 cells were cultured in McCoy's 5A medium (Nanjing KeyGen Biotech Co., Ltd.) containing 10% FBS. All cells were cultured in a humidified incubator at 37°C and 5% CO₂.

The human CRC cell lines SW480 and HCT116 were transfected with three different vectors, which were transferred by lentivirus, targeting PLAGL2 to knock down PLAGL2 protein expression. Three short hairpin RNAs targeting human PLAGL2 and a non-targeting RNA sequence serving as a negative control were cloned into the pGPU6 vector (Shanghai GenePharma Co., Ltd., Shanghai, China). The details of the RNA sequences are presented in Table I. Virus packaging was performed in 293T cells. SW480 and HCT116 cells were cultured in 6-well plates with normal medium containing 10% FBS the day before transfection (5×10⁵ cells/plate). After 24 h, the cells were transfected with 50 µl lentivirus with 5 µl Polybrene (5 µg/ml) and 1.5 ml medium. The cells were selected for 5 days and then used for various assays. The lentivirus and vectors were purchased from Nanjing KeyGen Biotech Co., Ltd.

Full-length human WNT6 cDNA (NM_006522.3) was cloned into the pCMV vector (Shanghai GenePharma Co., Ltd.). Virus packaging and transduction were performed as aforementioned. WNT6 was ovexpressed in HCT116 cells as PLAGL2 was knocked down. In addition, WNT6-overexpressed-cells were cultured in McCoy's 5A medium (Nanjing KeyGen Biotech Co., Ltd.) without FBS.

Total protein was extracted by lysis in RIPA buffer (Nanjing KeyGen Biotech Co., Ltd.) containing protease inhibitor cocktail (Nanjing KeyGen Biotech Co., Ltd.) for 15 min at 4°C and centrifuged at 16,666 × g for 15 min at 4°C. The proteins were measured by BCA assay. Proteins (40 µg) were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes.

The membranes were blocked with 5% non-fat milk in PBS for 2 h at room temperature and were then incubated with primary antibodies at 4°C overnight. Subsequently, the membranes were incubated with IRDye 800CW conjugated goat (polyclonal) anti-rabbit IgG (H+L) (dilution 1:15,000; cat. no. 926-32211; LI-COR Biosciences, Lincoln, NE, USA) for 2 h at room temperature. The protein bands were visualized by Infrared fluorescence with Odyssey CLx (LI-COR Biosciences) and the excitation wavelength was 778 nm. The primary antibodies used were as follows: Anti-PLAGL2 (cat. no. ab139509) and anti-Wnt6 (cat. no. EPR9244) (both from Αbcam, Cambridge, UK), anti-β-catenin (cat. no. 8480) (Cell Signaling Technology, Danvers, MA, USA) at a 1:1,000 dilution; anti-GAPDH (cat. no. ab181602; Abcam) at a 1:3,000 dilution at a 1:3,000 dilution.

Total RNA was extracted by TRIzol reagent (Invitrogen; Thermo Fisher, Scientific, Inc., Waltham, MA, USA). Real-Time Quantitative PCR (RT-qPCR) was performed with the Toyobo RT kit and KOD SYBR^® qPCR kit (Toyobo Life Science, Osaka, Japan) according to the manufacturer's protocol. The primers for mRNA were produced by Sangon Biotech Co., Ltd. (Sangon Biotech, China) and the primer sequences are listed in Table II. The thermocycling conditions are listed in Table III. All mRNA expressions were standardized to GAPDH, and the mRNA levels were determined by the 2^−ΔΔCq method. The method of quantification employed by Livak and Schmittgen (13).

Cell proliferation was assessed by a Cell Counting Kit-8 assay (CCK-8 Kit; Dojindo Laboratories, Kumamoto, Japan). After cells were stably transfected with lentivirus, which included the vectors to knock down the expression of PLAGL2, the cells (4×10³ cells/well) were incubated in normal medium without serum at 37°C overnight the day before and incubated in 96-well plates with 100 µl normal culture medium at 37°C. At the indicated time-points (12, 24, 48, 72 and 96 h), 10 µl of CCK-8 solution was added into each well and then cultured for 3 h at 37°C. The absorbance of each well at 450 nm (A450) was detected with an EnVision microplate reader (PerkinElmer, Inc., Waltham, MA, USA). All experiments were performed in triplicate.

Cells were plated into a 6-well plate at a density of 3×10⁵ cells/well for 24 h and fixed with 2 ml 1% paraformaldehyde and stored in 70% ethanol at −20°C. The cells were stained with 0.5 ml PI/RNase (BD Biosciences, Fraklin Lakes, NJ, USA) staining buffer for 15 min at room temperature and analyzed by flow cytometry.

A total of 3×10⁵ cells were plated into the 24-well plates for 24 h at −20°C and each well was covered by a glass coverslip. Cells were fixed in 4% paraformaldehyde for 24 h at room temperature and blocked in 10% BSA for 2 h at room temperature. Cells were labeled with anti-PLAGL2 antibodies (dilution 1:100; cat. no. 139509; Abcam) at 4°C for 12 h. The following day, the cells were stained for 1 h with cy3-labeled goat anti-rabbit IgG (dilution 1:100; cat. no. KGIF010; Nanjing KeyGen Biotech Co., Ltd.) at 37°C and slides were washed three times for 5 min with PBS. Nuclei were stained with DAPI (Nanjing KeyGen Biotech Co., Ltd.) for 5 min at 37°C. PLAGL2 immunofluorescence was imaged using a Zeiss LSM 800 confocal microscope (Carl Zeiss AG, Oberkochen, Germany).

The overexpression of PLAGL2 was analyzed by the GEPIA database (14) and COSMIC database (http://cancer.sanger.ac.uk/cosmic/). The NCBI gene browser (https://www.ncbi.nlm.nih.gov/gene/) was used to find the human Wnt6 locus (Chr2: 218,858,382-218,875,674) and the Wnt6 promoter region (Chr2: 218,858,821-218,860,072). Candidate transcription factor binding sites were predicted by means of the JASPAR database (http://jaspar.genereg.net/).

Chromatin immunoprecipitation (ChIP) was performed using an EZ-ChIP^™ ChIP kit (Merck Millipore, Darmstadt, Germany) following the manufacturer's protocol, including anti-RNA polymerase II (Merck Millipore; cat. no. 05-623B), anti-IgG (mouse) (Merck Millipore; cat. no. 12-371B) and GAPDH primers (Merck Millipore; cat. no. 22-004). HCT116 cells (2×10⁷) were crosslinked with 37% formaldehyde. The chromatin was cleaved into fragments between 200 and 600 bp by ultrasound. Anti-PLAGL2 was purchased from Abcam and qPCR was performed with the KOD SYBR^® qPCR kit (Toyobo). Primer details are presented in Table IV. The levels of DNA were determined by the 2^−ΔΔCq method.

The promoter fragments of the WNT6 gene were generated by PCR using a forward primer with a KpnI (15) site inserted at the 5′ end and a reverse primer with a BglII (15) site inserted at the 3′ end. PCR products were digested with KpnI/BglII and cloned into a pGL3 firefly luciferase basic vector (Promega Corp., Madison, WI, USA) following the manufacturer's protocol.

HCT116 cells were transfected using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Cells were then incubated at 37°C for 48 h before assaying for luciferase activity using the Dual-Luciferase Reporter assay system (Promega Corp.) according to the manufacturer's instructions. Firefly luciferase activity was normalized relative to Renilla luciferase activity for each transfection and calculated as the fold increase over pGL3 firefly luciferase basic vector (pGL3Basic). At least two independent transfections and two replicates per assay were performed for each individual construct.

The statistical analysis was performed by using SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA). Data were imaged with GraphPad Prism 6 software (GraphPad Software, Inc., La Jolla, CA, USA) and expressed as the mean ± SD. Differences between groups were compared with Student's t-test (when 2 groups) and one-way ANOVA (when >2 groups) was used, followed by Student-Newman-Keuls (SNK) method for comparing each two groups. P<0.05 was considered to indicate a statistically significant difference.

Using the Catalogue of Somatic Mutations in Cancer database (COSMIC; http://cancer.sanger.ac.uk/cosmic/), overexpressed genes in colorectal cancer were screened and the top 20 were selected; PLAGL2 at position 16 was revealed (Fig. 1A). GEPIA analysis of the mRNA from 275 colon cancer specimens and 41 normal colonic tissues in TCGA revealed that the expression of PLAGL2 in cancer tissue was 1.235-fold that of PLAGL2 in normal tissue (Fig. 1B). The mRNA of 31 colorectal cancer tissue samples and 31 adjacent normal tissue samples was examined by RT-qPCR. The expression of PLAGL2 in cancer tissue was 2.247-fold that of PLAGL2 in the normal tissues (Fig. 1C). In the COSMIC database, ~295 gene fusion pairs exhibited PLAGL2 expression that was higher in cancer tissues than in normal tissues, accounting for ~48.36% of the total number of tissues. In the 31 pairs of tissues used in this experiment, PLAGL2 was found in cancer tissues. Approximately 19 pairs had higher expression in cancer tissues than normal tissues, accounting for ~61.3% of the total number of tissues (Fig. 1D and E). Eight of the 19 pairs of tissues in which PLAGL2 expression was higher in the cancer tissues than normal tissues were selected to detect the protein expression of PLAGL2 by western blot analysis. The expression of PLAGL2 in colorectal cancer tissue was significantly higher than that in the normal tissue adjacent to the tumor (Fig. 1F). These results indicated that PLAGL2 was overexpressed in colorectal cancer.

Next, we sought to reveal the function of PLAGL2 in CRC. The CRC cell lines HCT116 and SW480 were selected and different vectors were applied to knock down PLAGL2. It was found that both in SW480 and HCT116 cells, the most efficient plasmid was PLAGL2-Homo-808 (LV3) (Fig. 2A and B). The PLAGL2-knockdown group (PL-LV3) was selected as the experimental group and the empty plasmid group (NC) was used as the control group for functional experiments. The results of the CCK-8 assay revealed that in SW480 cells, the growth rate of the experimental group cells was significantly lower than that of the control group cells after 48 h (P<0.05) and the difference among them was more pronounced with time (P<0.01). In HCT116 cells, from 72 h onwards, the growth rate of the experimental group of cells was significantly lower than that of the control group cells, with significant differences (P<0.05), and the differences among them were more obvious with time (P<0.01) (Fig. 2C and D). Flow cytometry was used to detect the cell cycle. The results indicated that in the HCT116 and SW80 cells, the cells in the experimental group exhibited a prolonged G1 phase and the S phase was shortened (Fig. 2E and F) when compared with the control cells. Tumor formation experiments in nude mice indicated that the tumor volume in the untreated and the control groups was greater than that in the experimental group (Fig. 2G), and the growth curve indicated that the growth rate of the control group was greater than that of the experimental group (Fig. 2H). These results indicated that PLAGL2 affected the development of colorectal cancer in vitro and in vivo.

PLAGL2 plays as an important role in other tumors, where it can always activate the Wnt/β-catenin pathway, and the Wnt/β-catenin pathway is one of most active pathways in colorectal cancer. Several key factors that are active in the Wnt/β-catenin pathway were selected and primers were designed. The mRNA of SW480 and HCT116 cells was extracted and the expression was detected with RT-qPCR. It was found that the expression of Wnt6 and Wnt11 was significantly decreased after PLAGL2 knockdown (Fig. 3A). While Wnt11 mainly regulates the Wnt/Ca²⁺ pathway, Wnt6 regulates the Wnt/β-catenin pathway. A western blot assay was used to detect changes in the expression of Wnt6 and β-catenin protein before and after PLAGL2 knockdown. The results revealed that the expression of Wnt6 and β-catenin proteins decreased with the knockdown of PLAGL2 (Fig. 3B). Wnt6 was overexpressed in HCT116 cells when PLAGL2 was knocked down and the two groups were compared. The WB assays revealed that β-catenin was increased when WNT6 was overexpressed but PLAGL2 was knock down. It revealed that PLAGL2 activated the Wnt/β-catenin pathway by promoting the expression of WNT6 (Fig. 3B). The sequence 1,000 bp upstream and 216 bp upstream of the WNT6 gene was used to make predictions using the JASPER database, indicating that the WNT6 upstream sequence was highly conserved among different species and that the conserved region had 7 possible binding domains for the PLAGL2 gene (Fig. 3C).

The three-ideal binding region mutation analyses predicted by the JASPAR database were selected; the horizontal lines represent possible binding sites and mutation regions of PLAGL2 and Wnt6 (Fig. 4A). The luciferase assay results revealed that after mutation of the predicted binding regions, the promoter activity was decreased. Compared to the activity of the original promoter region, mutation 1 resulted in a ~50% reduction in promoter activity, mutation 2 resulted in a ~35% reduction, and mutation 3 resulted in a ~10% reduction (Fig. 4B). In HCT116 cells, we applied ChIP assays to detect the binding of PLAGL2 to the Wnt6 promoter region. It was revealed that PALGL2 strongly bound to the Wnt6 promoter region in HCT116 cells, with a ~28 and ~12-fold enrichment compared to the GAPDH control (Fig. 4C). The results of immunofluorescence also revealed that the PLAGL2 proteins were located in the cell nucleus (Fig. 5A). These results indicated that PLAGL2 could activate Wnt6 expression in colorectal cancer cells, activating the Wnt/β-catenin pathway.