All the specimens of 54 patients from the Second Affiliated Hospital of Nanchang University were diagnosed as CRC by pathological test from June 2018 to December 2018. Total proteins and mRNAs from these patients’ clinical tissues were immediately obtained when tissues was still fresh. Moreover, formalin-fixed paraffin-embedded 130 CRC patients’ tissues from December 2012 to December 2017 were randomly selected to observe the expression levels by immunohistochemistry and their corresponding follow-up data about survival was obtained from the hospital database.Informed consent was obtained from all patients, those patients’ clinical data was recorded during hospitalization and the research program was approved by the Ethics Committee of the Second Affiliated Hospital of Nanchang University.
According to what the manufacturer's protocol described, 20μM BrdU was put into CRC cells for 4h at 37°C. Then washed with PBS for three times, the cells were mixed with apollo reaction for 1h. The cells were stained with 100 ul of Hoechst 33342 (5 ug/ml) for 30 min to visualize the nuclei and observed under a fluorescence microscope (Olympus, Tokyo, Japan) .
Real-time proliferation assay
The xCELLigence real-time cell analysis (RTCA) system (ACEA Bioscience) was used to analyze cell proliferation. Cells were seeded on a 96-well plate (E-plate, Germany). After treated with different approaches for 12h, the growth rates were recorded every 10 min by the instrument. Under the same xCELLigence RTCA program, continue to monitor the changes of cell index for 36 hours. We used mean cell index values in real-time to present cell proliferation changes. For each experiment, we tested three biological replicates.
In vivo tumorigenicity study
After construction of a DLD-1 cell line that stably interfered with JARID1B expression, 5 × 106 cells in 200 ul of PBS were injected subcutaneously into the flanks of nude mice (male athymic BALB/c nude mice,4-6 weeks). We used the random number table as a random method to determine the experimental animals, making sure more than 40 mices. Five mice were randomly selected every 5 days to get tumor tissues, and then tumor volumes were measured according to the protocol: V=1/2 (largest diameter)×(smallest diameter)2. In vivo imaging showed tumor growth on day 30, and fluorescent pictures were taken. After 40 days, tumors tissues from 5 mices were harvested and individually weighed after the mice were anesthetized. The data was presented as tumor weight (mean ± SD).
Commercialized CRC cell lines SW620, HCT116, LOVO, SW480 and DLD-1 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). SW620 and SW480 were cultured in L-15 medium (Gibco). LOVO was cultured in F12K medium (Gibco). DLD-1 and HCT116 were cultured in DMEM medium (Gibco). All cell lines were cultured at 37 ℃, 5% CO2 condition. Moreover, 10 uM XVA-939 from American AbMole was used to inhibite β-catenin degradation. Over-expression plasmids and shRNA interference fragments are transfected into cells through lipofectamine 2000 and lipofectamine LTX (Catalog:11668019, Invitrogen, Carlsbad, CA, USA).
Plasmids and reagents
SiRNA of JARID1B and CDX2 was synthesized by InvivoGen. The target sites of shRNA are detailed in Supplementary Table S1. The stable knockdown and overexpressed JARID1B CRC cells according to the Manufacturer’s protocol. the shJARID1B and shCDX2 of CRC cells was selected based on resistance to hygromycin. The pcDNA3.1(+)-JARID1B-expressing CRC cells were selected using G418. The following reagents were used: Lipofectamine 3000 (Catalog: L3000001, Invitrogen, Carlsbad, CA, USA), dual-luciferaseassay kit (Catalog: E1910, Promega), SimpleChipTM Enzymatic Chromatin IP KIT (Catalog: #9003, CST, USA), EdU kit (Catalog: C10310, RiboBio, China).
qRT-PCR, western blot analysis and co-immunoprecipitation (Co-IP)
qRT-PCR and western blot analysis were made as previously reported . Whether it was tissue or cells, total RNA was extracted by Trizol reagent (Catalog:15596026, Invitrogen, USA) and were quantified by SYBR Green assays with RT primers and SYBR Green from Takara Biotechnology (Catalog: DRR041A, TAKARA, Dalian, China). Human GAPDH was amplified in parallel as an internal control. For western blot, total proteins of clinical samples were obtained through RIPA lysis buffer and prepared cells were harvested by RIPA lysis buffer containing protease inhibitor cocktail(Catalog: P8340-1ML, Sigma-Aldrich). All the proteins were fractionated by 10% SDS-PAGE. The antibodies anti-JARID1B(Catalog: ab198884, 1:2000, abcam), anti-GSK-3β(Catalog: ab93926, 1:1000, abcam), anti-Axin2(Catalog: ab109307, 1:1000, abcam), anti-CDX2(Catalog: ab76541, 1:1500, abcam), anti-c-MYC(Catalog: 10828-1-AP, 1:1000, proteintech), anti-phosphorylation-β-catenin(phospho Y142) (Catalog: ab27798, 1:2000,abcam), anti-β-catenin (Catalog: ab6302, 1:2000, abcam), anti-H3K4me3(Catalog: ab8580, 1:1500, abcam), anti-Histone3(Catalog: ab1791, 1:1500, abcam), anti-ubiquitin (Ub) (Catalog: ab7780, 1:500, abcam) and anti-tubulin(Catalog: 11224-1-AP, 1:1000, proteintech) were used. For co-immunoprecipitation, to deal with cells for 24 hours with 10 umol/L MG132, cell lysates were incubated overnight at 4 °C with anti-β-catenin, then conjugated to protein A/G agarose beads while rocking. Immunoprecipitates were washed with washing buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton-X), re-suspended in 2 × loading buffer, and resolved by SDS-PAGE followed by immunoblotting analysis.
Gene set enrichment analysis (GSEA)
GSEA was a statistical analysis method for assessing whether a particular gene set showed statistically significant and consistent differences between two biological states, such as tumor and non-tumor or low-expression and high-expression groups . Subsequently, mRNAs that were differentially expressed in CRC were imported into the GSEA software for Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.kegg.jp) assessment. Results from the GSEA software were visualized by the GOplot R software package. The GSEA software used a genome false discovery rate (FDR) of less than 25%, suggesting that FDR<25% indicated significantly enriched gene sets.
Luciferase reporter assay
Fragments of the CDX2 were amplified PCR using primers (Supplementary Table S2) and cloned into the luciferase reporter vector pGL3.0-Basic (Catalog: E1751, Promega, Madison, WI, USA) to generate CDX2 promoter reporter constructs. Plasmids containing firefly luciferase reporters and JARID1B plasmids were cotransfected into cells. For the TOP/FOP-Flash reporter assay , the TOP/FOP-Flash reporter and JARID1B plasmids were co-transfected into cells. After transfection for 48 h, the cells were harvested for analysis with the Dual-Luciferase Reporter Assay System (Catalog: E1910, Promega, Madison, WI, USA). Luciferase activity was measured using the PerkinElmer EnSpire Multilabel Reader 2300 (PerkinElmer Inc., Waltham, MA, USA). The luciferase intensity was normalized to the Renilla luciferase activity to normalize for transfection efficiency.
Chromatin immunoprecipitation Assay (ChIP)
ChIP assays were performed using the ChIP assay kit (Catalog: 8982, Cell Signaling Technology). Briefly, the cells were fixed with 1% formaldehyde for 10 minutes at room temperature, then washed with PBS for three times. After a series of processing according to the protocol, Digested DNA fragments was sonicated in the range of 150-300 bp. The immunoprecipitations were mixed with H3K4me3 antibody and agarose. The input and DNA were then subjected to qPCR.
Paired t test for the two groups and one-way ANOVA for more than two groups were used to analyze JARID1B expression data and its relationship with various clinicopathological factors.When data did not follow normal distribution, Mann-Whitney U test between two groups and Kruskal-Wallis H test for three or more groups should be used. Kaplan-Meier analysis and the logrank test were used for survival analysis. Correlation between two continuous values was analyzed by pearson’s correlation. Furthermore, univariate and multivariate analyses were performed using the logistic regression model. All of the data were analyzed using GraphPad Prism and SPSS 22.0. p-values of <0.05 indicated statistically significant changes. Each experimental design included more than three biological repeats.