Data on the relative expression of HDAC1, HDAC2, HDAC3 and HDAC8 were downloaded from the Oncomine public database (www.oncomine.org). The dataset contains 330 primary sites and 43 metastatic CRC tissue samples.
Survival analysis of HDAC1, HDAC2, HDAC3 and HDAC8
We labeled TCGA samples as “high” or “low” according to whether the expression of HDAC1, HDAC2, HDAC3 and HDAC8 was higher or lower than the corresponding median value among all samples. The log-rank test was used to measure whether the survival time was significantly different between the “high” and “low” expression groups. Kaplan-Meier plots were made by Gene Expression Profiling Interactive Analysis (GEPIA: http://gepia.cancer-pku.cn/).
CRC tissue sample and immunochemistry
Commercially available tissue microarray (TMA) slides (HLin-Ade075Met-01, Shanghai Biochip Co., Ltd., Shanghai, China) containing histologically confirmed tissues from CRC patients were purchased for immunohistochemistry (IHC) analysis. Specific primary antibodies against HDAC2 (Cell Signaling Technology) were used for IHC with a 2-step protocol.
DLD1, HCT116, SW480 and SW620 cells were obtained from ATCC. DLD1 and HCT116 cells were cultured in RPMI 1640 medium, while SW480 and SW620 were cultured in high glucose DMEM. All media were supplemented with 10% FBS, 100 µg/m L penicillin and 100 U/m L streptomycin. The DLD1HDAC2 KO cell line was constructed in the lab of Professor Run-lei Du, who is our collaborator in this study25.
The total RNA from DLD1 and DLD1HDAC2 KO cells was prepared for microarray analysis (n = 3 each). The Affymetrix microarray was used to detect mRNA and long noncoding RNA expression profiles. Microarray data were normalized using the RAM (robust multiple-array average) normalization method. The differentially expressed genes were determined with a threshold cutoff of 2-fold (p < 0.01).
Transwell migration assays
Tumor cell migration assays were performed according to the manufacturer’s instructions. Briefly, cells were harvested and resuspended in serum-free medium and then seeded onto Transwell inserts at a density of 100,000 cells/well. Then, the inserts were placed in a lower chamber filled with 600 μl culture media containing 10% FBS. Transwells were incubated for 24 h at 37°C. Cells on the inside of the Transwell inserts were removed with a cotton swab; then, cells that migrated to the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Photographs were taken from five random fields, and the cells were counted to calculate the average number of cells that had transmigrated.
Vector construction and luciferase reporter assay
The H19 promoter containing intact SP1 recognition sequences was PCR-amplified and subcloned into the KpnI and HindIII sites of the pGL-3-basic vector, and the vector was named pGL-3-H19. The pGL3-H19 vector with point mutations in the SP1 binding sites was synthesized by GenScript (Nanjing, China) and named pGL3-H19-mut (SP1). For the luciferase reporter assay, HEK293 cells cultured in 24-well plates were cotransfected with luciferase reporter plasmids and HDAC2 plasmids. Twenty-four hours posttransfection, HEK293 cells were lysed in lysis buffer. After centrifugation at 12,000 rpm for 5 min, the supernatant was transferred to a new tube. The luciferase activity was monitored by mixing 10 μl supernatant with 30 μl luciferase assay buffer and using a GloMax 20/20 Luminometer (Promega).
siRNA and gene transfection
The siRNAs were synthesized by RiboBio Company (Guangzhou, China). Oligonucleotide transfection was conducted by using Lipofectamine™ RNAiMAX transfection reagent (Life, USA) following the protocol recommended by the manufacturer. After 48 h posttransfection, the cells were collected and used for further investigations.
Chromatin immunoprecipitation (ChIP) and q PCR
ChIP was performed using a SimpleChIP® Enzymatic Chromatin IP Kit (CST) following the manufacturer’s instructions. Briefly, genomic DNA-protein complexes were immunoprecipitated using anti-HDAC2 antibody or normal rabbit IgG as a control. After enzyme digestion and sonication, the precipitated DNA was amplified by SYBR Green-based quantitative real-time PCR using primers encompassing the promoter regions of the H19 gene. The ChIP PCR primers used were (the numbers in parentheses indicate the sequence regions corresponding to GenBank ID AF125183):
Primer 1: 5’-CCAGCCATGTGCAAAGTATG-3’ (9747-9766)
Primer 2: 5’-CCATCCTGGAATTCTCCAAA-3’ (9939-9920)
Primer 3: 5’-GCGGTCTTCAGACAGGAAAG-3’ (9468-9487)
Primer 4: 5’-CACGTTCCTGGAGAGTAGGG-3’ (9673-9654)
A co-immunoprecipitation assay was performed in the following steps. Briefly, the cells were washed with ice-cold PBS, lysed in NP-40 buffer containing cocktail and then centrifuged for 10 min at 12000 rpm and 4°C. Anti-SP1/HDAC2 antibody or normal rabbit IgG was added to the cell lysate and incubated at 4°C overnight. Then, 15 µl of protein A/G agarose beads was added to each tube, incubated at room temperature for 3 h and centrifuged for 3 min at 4000 rpm at 4°C. A total of 30 µl of 2× SDS-loading buffer was added to the antigen-antibody-protein A/G agarose bead complex, which was boiled for 10 min. The sample was collected for subsequent SDS-PAGE and Western blotting.
RNA-binding protein immunoprecipitation
The anti-Ago2 RIP assay was performed using a Magan RIPTM RNA-Binding protein Immunoprecipitation Kit（Milipore）following the manufacturer’s instructions. Briefly, DLD1 HDAC2 KO cells were washed with cold PBS and lysis by RIP Lysis Buffer. After that, the cell lysates were incubated with antibody against Ago2 (Milipore, USA). The normal Mouse IgG was used as negative control. For RNA immunoprecipitation, the supernatant was incubated with the antibody-coated Sepharose beads overnight. The RNA bound to Ago2 antibody was extracted with TRIzol reagent (Invitrogen, USA) and detected by qRT-PCR.
Fluorescence in Situ Hybridization (FISH)
The sequence of the H19 probe was 5’-FAM/GCTGCTGTTCCGATGGTGTCTTTGATGTTGGGC/FAM-3’; this probe was used for FISH of H19 from B-NDG mouse pulmonary metastases. After the metastatic lung tissues were removed and cleaned, they were immediately fixed in an ISH fixation solution (DEPC water preparation) for 12 h. After tissue fixation, they were dehydrated by gradient alcohol and then embedded in paraffin. The tissue sections were used for H19 probe hybridization, and the nuclei were restained by DAPI. The sections were observed under a fluorescence microscope, and images were collected. The H19 probe is shown in green, while DAPI staining is shown in blue.
B-NDG immunodeficient mice (with T cell, B cell and NK cell defects) were obtained from Beijing Biocytogen Biotechnology Co., Ltd. The B-NDG mice were randomly divided into two groups. A total of 5 × 106 luciferase-labeled CRC cells were injected intravenously into B-NDG mice via the tail vein. Four weeks later, the mice were anesthetized and injected intraperitoneally with fluorescein potassium salt (150 mg/kg), and 10 min later, the metastatic tumor was detected and photographed by a bioluminescent in vivo imager (VILBER Fusion FX7, France). The mice were sacrificed, and their lung tissues were removed for H&E staining, immunohistochemistry and FISH. The mean number of metastatic nodules in B-NDG mice with lung metastasis was calculated.
All experiments were repeated no fewer than 3 times. Experimental results are presented as the mean±S.E.M. The statistical significance of comparisons between two groups was determined with a two-tailed Student’s t-test. P＜0.05 indicated statistical significance.
Additional Materials and Methods
More details regarding the materials and methods can be found in the Supplementary information.