Human tissues collection and cell culture
A total of 78 human primary CRC tissues without metastasis and 46 human primary CRC tissues with matched hepatic or lung metastasis were collected between 2006 and 2015 at Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, and Fudan University Shanghai Cancer Center. HCT116 (human colon, CRC) and LoVo (human colon, Dukes’ type C, grade IV, colorectal adenocarcinoma) cells were purchased from the ATCC (Manassas, USA), and cultured in RPMI-1640 and F12K medium, respectively. Both mediums were supplemented with 10% heat-inactivated FBS, 100 U ml− 1 penicillin, and 100 μg ml− 1 streptomycin. These cells were incubated under 37 °C, 5% CO2 conditions. All the experimental procedures were approved by the Institutional Review Board of Shuguang Hospital, Shanghai University of Traditional Chinese Medicine.
Plasmids construction and lentivirus infection
Three shRNA fragments for human JMJD2C (Gene ID:23081) were synthesized by the Sangon Biotech company (Shanghai, China), sub-cloned into the pLKD shRNA vector, and named pLKD-CMV-G&PR-U6-JMJD2C-shRNA1/2/3, respectively. The full length of JMJD2C gene was amplified by PCR, following by gene sequencing, and the right full fragments was cloned into the plenti overexpression vector, named plenti-CMV-EGFP-P2A-JMJD2C-3FLAG. These above plasmids were used to transfect well-growing state HCT116 and LoVo cells by using Lipofectamine 3000 transfection reagent. After 48 h transfection, the transfected cells were selected with neomycin (G418, 1 mg ml− 1, Sigma, USA). Recombinant lentiviruses containing pLV4-shRNA/NT, pLV4-shRNA/JMJD2C, pLV4-empty vector, and pLV4-JMJD2C+/+ were prepared by GeneChem (Shanghai, China), respectively. HCT116 or LoVo cells were infected with 2 × 106 transducing units of corresponding lentiviruses, and were selected with 2 μg/ml puromycin for 2 weeks. The efficiencies of knockdown or overexpression of JMJD2C were determined by real-time PCR and western blot.
Real-time PCR
Total RNA was extracted using the TRIzol reagent (Takara) according to the manufacturer’s instructions. The RNA concentrations were determined using a NanoDrop ND-1000 (NanoDrop). cDNA was synthesized with the PrimeScript RT Reagent Kit (TaKaRa) using 500 ng total RNA as template. Real-time PCR analyses were conducted to quantitate KDM4C mRNA and lncRNA-MALAT1 relative expression using SYBR Premix Ex Taq (TaKaRa) with GAPDH as an internal control. The Real-time PCR results were defined from the threshold cycle (Ct), and relative expression levels were calculated by using the 2-△△Ct method. PCR was performed using an ABI 7500 instrument (Applied Biosystems, USA). The primers used for real-time PCR analysis were as follow: KDM4C, forward primer: 5-AGGCCTAAGGCTGATGAGGA-3, reverse primer: 5-TTGGCCATGAAAGCTCGGAT-3; MALAT1, forward primer: 5-GCTCTGTGGTGTGGGATTGA-3, reverse primer: 5-GTGGCAAAATGGCGGACTTT-3, GAPDH, forward primer: 5-GGTGGTCTCCTCTGACTTCAACA-3, reverse primer: 5-CCAAATTCGTTGTCATACCAGGAAATG-3.
Luciferase reporter assay
To test MALAT1 promoter activity, HCT116 or LoVo cells were co-transfected with the recombinant plasmid pGL3-basic-MALAT1 promoter with a control positive plasmid pRL-SV40 as previously described [18]. The promoter activity was analyzed using a commercial dual-luciferase assay kit (Promega, USA) according to the manufacturer’s instructions.
Immunofluorescence staining
To observe the expression and location of JMJD2C and β-catenin, HCT116 and LoVo cells after transfection were plated at a density of 2.0 × 104/ml in 6-well plates, fixed with methanol, blocked with 5% BSA. The cells were first stained with β-catenin mouse antibody followed by Cy3-conjugated goat anti-mouse IgG (Millipore). After the cells were washed four times with PBS, the JMJD2C rabbit antibody was added, followed by FITC-conjugated goat anti-rabbit IgG (Millipore). Nuclear staining was done with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) solution. Cells were imaged using TCS SP2 spectral confocal system (Leica, Germany). All experiments were conducted according to instructions from the antibody manufacturer.
Western blot analysis
Protein lysates from cells were prepared in lysis buffer and centrifuged at 12000 rpm at 4 °C. The primary antibodies used were JMJD2C (Santa Cruz, USA), β-catenin (CST, USA), c-Myc (CST, USA), ITGBL1 (Abcam, USA), PCNA (CST, USA) and GAPDH (CST, USA). The secondary antibody used was HRP-labeled goat anti-rabbit/mouse IgG (H + L) (Beyotime, China). Each band was quantitatively analyzed using quantity one Software and normalized to the expression of GAPDH in the same lane.
Chromatin immunoprecipitation assay
ChIP assays were performed using a ChIP assay kit (Millipore, USA). Chromatin extracts were immunoprecipitated using 10 μg anti-JMJD2C (Abcam, USA), 4 μg anti-H3K9 (Abcam, USA), and 4 μg anti-H3K36 (Abcam, USA), respectively. IgG (Merck, Germany) were used as mock ChIP controls. Fold enrichments were calculated by determining the ratios of the amount of immunoprecipitated DNA to that of the input sample, and were normalized to the level observed at a control region, which was defined as 1.0. The ChIP primers for MALAT1 promoter are as follow: forward primer: 5-GGTCAGCCTGAGACCACTTC-3, reverse primer: 5-CTGTGCCTGTTCTGGGGAAT-3.
Transwell assay
To measure cell migration, the transfected HCT116 and LoVo cells (2 × 105) were seeded to the upper chambers and cultured with 100 μl serum-free F12K or RPMI1640 medium, whereas the lower chamber was filled with 600 μl F12K or RPMI1640 medium containing 15% FBS and 10 μg ml− 1 fibronectin. After incubation for 48 h at 37 °C and 5% CO2, the chambers were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The cells in the upper chamber were carefully removed with a cotton swab. The numbers of cells were analyzed by counting five independent visual fields under a DMI3000 B inverted microscope (Leica, USA) with a 20 × objective. All assays were performed in triplicate and independently repeated three times.
Wound healing assay
Cells (4.5 × 105) were seeded on a six-well plate to form a confluent monolayer in 10% FBS-containing medium. The monolayer cells were scratched by a plastic tip and washed with PBS to remove cell debris; 0.5% FBS-containing F12K or RPMI1640 were then added to each well, and the scratched monolayer was incubated in a 37 °C incubator with 5% CO2 for 24 h. Wound closure was measured in five random fields at × 200 magnification using Image J software and a DMI3000B inverted microscope (Leica, USA). Percentage of wound healing was calculated as follows: migrated cell surface area/ total surface area × 100, in which, migrated cell surface area = length of cell migration (mm) × 2 × length of defined areas, total surface area = beginning width × length of defined areas.
MTT assay
Cells were cultured at a density of 2.5 × 103 cells per well in a flat-bottomed 96-well plate. MTT [3-(4,5)-dimethylthiahiazo(−z-y1)-3,5-di-phenytetrazoliumromide] was used to determine the cell viability by measuring the absorbance at 490 nm. All assays were performed in triplicate and independently repeated three times.
Flow cytometry
Cells were harvested by trypsinization, washed once with cold PBS. Then, the cells were stained with PI (propidium iodide) and anti-Annexin-V antibody (Becton Dickinson, USA) at 4 °C for 1 h. Subsequently, cells were washed once with PBS and analyzed by FACS (BD, USA).
In vivo analysis
All the animal experiments were performed under the approval of the Institutional Committee for Animal Research and were in accordance with national guidelines for the care and use of laboratory animals. HCT116-shRNA/NT, HCT116-shRNA/JMJD2C, HCT116-empty vector, and HCT116-JMJD2C+/+ cells (2 × 106 cells in 100 μl) were injected into female BALB/c nude mice (6–8 weeks old, obtained from SLAC Laboratory Lab, Shanghai, China) by tail intravenous to establish the lung metastasis mice model, respectively. Mice were euthanized 42 days post injection, and the lung metastases images were observed and quantified by LB983 NIGHTOWL II system. The numbers of lung metastatic nodes were measured, and the metastatic tumor tissues were collected for immunohistological and western blot analysis with JMJD2C, β-catenin, c-Myc, and ITGBL1 antibody.
HE staining and IHC staining
Paraffin-embedded tissues were sectioned for HE (hematoxylin and eosin) and IHC (immunohistochemistry) staining. For IHC analysis, the experiments were performed using the first antibody, HRP-conjugated secondary antibody, and DAB (diaminobenzidine) detection reagents. The DMI3000B microscope connected to the digital imaging system was applied for taking photographs and the following analysis. All the data were evaluated and classified blindly by two investigators (ZYW and LLW) from the pathology department of Shuguang Hospital, Shanghai University of Traditional Chinese Medicine.
Statistical analysis
All data are presented as means with standard deviation (SD) or median with 95% confidence interval (95% CI). Statistic comparison was performed using the Student’s t-test, one-way ANOVA analysis, Mann-Whitney test, or Kruskal-Wallis test, as appropriate, with the significance level at P<0.05. All statistical analyses were completed with SPSS 22 software.
