Bioinformatics analysis
The University of California, Santa Cruz (UCSC) database was used to find the sites 2000 bp upstream and 100 bp downstream of Ap-1 TSS [17]. PROMO program has projected possible TFs capable of binding to the HMGCS1 promoter [18,19]. The cBioPortal results from The Cancer Genome Atlas (TCGA) have been used to analyze HMGCS1 and AP-1 expression levels in patients with or without recurrences of CRC [20,21]. The GEPIA2 server was used to study mRNA expression and the association between the two genes in HMGCS1 and Ap-1 in CRC and normal tissues [22].
Cell culture & treatment
The CRC cell lines used in the experiment include HCT-116, Caco-2, RKO, LAS174T, HT-29, and HEK293T were grown in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100mU/ml of Penicillin (Gibco) and 100µg/ml of Streptomycin (Gibco) at 37ºC in a Humidified incubator of 5% CO2. The medium was changed every 24hr. All cell lines were purchased from American Tissue Culture (ATCC).
Reagents included 50 ng/ml recombinant human stem cell factor (rhSCF, 255-SC, R&D Systems, USA) added for 15 minutes, 2 µmol/L Imatinib (STI571, S1026, Selleck, Shanghai, China) added for 2.5 hr and 30 µmol/L ISCK03 (ab145066, Abcam) added for 2.5 hr.
Over-expression of AP-1 by plasmid transfection
AP-1 over-expression and its negative control plasmid were constructed by GeneChem Technology Co., Ltd (Shanghai, China). We co-transfected the reporter plasmids with AP-1 overexpressing plasmid into Caco-2 cells using Lipofectamine TM 2000 (Thermo Scientific). The vector scheme is shown in Figure S1.
Over-expression of KIT by lentivirus infection
Lentivirus used for KIT stable over-expression was constructed by Shanghai GeneChem Technology Co., Ltd (Shanghai, China) and transfected into CRC cells using Lipofectamine TM 2000 (Thermo Scientific).
Knock-down of HMGCS1 by siRNA
Both siRNA siHMGCS1 and siNC were purchased from The RiboBio (Guangzhou, Guangdong, China). According to the manufacturer's protocol, all the transfection was performed using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Inc.). Cells were cultured into a 60mm dish when the cells were in 80% to 90% confluency. Then, the complete DMEM into antibiotic and FBS-free DMEM for 24 hours. To prepare Lipofectamine reagent, take 250µl of Opti-MEM and 10µl Lipofectamine reagent per well and put them for 5 minutes at room temperature. For the preparation of siRNA solution taken, 250µl of Opti-MEM and 2500-5000ng siRNA per well. Then gently mix Lipofectamine reagent and DNA at room temperature for 20 minutes. After this, discarded DMEM and added Lipofectamine reagent DNA mixture into the dish. Add 1.5ml of Opti-MEM to make the volume up to 2ml per well. After 24/48 hrs., check the inflorescence intensity to evaluate transfection efficiency. If transfection was successful, then harvest cells. The vector scheme is shown in Figure S2.
Chromatin immunoprecipitation (ChIP)
ChIP assay was performed according to the manufacturer's instructions (EZ-Magna ChIPTM A Kit, #17-408, Merck-Millipore, USA). Chromatin was immunoprecipitated for 24hr at 4 ̊C using rabbit monoclonal anti-Ap1. Primers are shown in Table 1.
Table 1. Primers sequences
Genes
|
qRT-PCR Primer Sequences
|
KIT
|
Forward: 5’-CAGGCAACGTTGACTATCAGT-3’
Reverse: 5’-ATTCTCAGACTTGGGATAATC-3’
|
HMGCS1
|
Forward: 5’-GCTCTTGGGATGGACGGTAT-3’
Reverse: 5’-GCGGTCTAATGCACTGAGGT-3’
|
c-Jun
|
Forward: 5’-TATGAAGTGAGTCATGGGCAA-3’
Reverse: 5’-TATGAAGCAGACACTGGGCAA-3’
|
GAPDH
|
Forward: 5’- CCTGCACCACCAACTGCTTA -3’
Reverse: 5’- AGTGATGGCATGGACTGTGG -3’
|
ChIP- qPCR primer sequences
|
Ap1 promoter
|
Forward:5’-TATGAAGTGAGTCATGGGCAA-3’
Reverse: 5’-TATGAAGCAGACACTGGGCAA-3’
|
Real-time cellular analyzer (RTCA)
RTCA (ACEA Biosciences, USA) was used to monitor cell proliferation, and invasion as previously described [23].
Cell apoptosis detection by flow cytometry (FCM)
The cells were fixed with 70% ethanol in PBS and routinely kept at -20C overnight. The cells were washed with PBS and permeabilized with PBS, 4% fetal bovine serum, and 0.1% Triton X-100 for 10 min ice. Then cells were incubated within 1:200 dilutions in PBS, 4% fetal bovine serum for 2hr. Cells were washed twice with PBS-T and incubated with 1:200 dilutions of fluorescein-tagged goat anti-mouse secondary antibody. After washing with PBS-T, cells will be suspended in PBS and analyzed using a flow cytometer.
Western blot
A mixture of Pierce RAPI Lysate (Applygen, Beijing, China) and Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, Inc.) was used to lyse cells at a ratio of 100:1. The protein concentration was measured using the BCA method using NanoDrop 2000c (Thermo Scientific, Gene Company Limited). The proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, MA, USA). The membranes were blocked with 5% bovine serum albumin (BSA) and incubated with primary antibodies against HMGCS1 (1:1000), p-c-JUN (1:1000), C-JUN (1:1000), C-KIT (1:1000), P-KIT (1:1000), and GAPDH (1:5000) for overnight at 4ºC. Then membranes were incubated with goat anti-rabbit secondary antibody (1:5000) for 2 hours at 37ºC. Super Signal West Pico Chemiluminescent substrate (Thermo Fisher Scientific, Inc.) visualized the protein bands. ImageJ 1.50i (National Institutes of Health, Bethesda, MD, USA) was used to quantify the protein bands.
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated from cell lines or tissues using TRIzol (Invitrogen, Thermo Fisher Scientific, Inc.) and reverse transcribed into cDNA using the 5x All In One RT Master Mix (ABM, Canada). The expression was measured using the SYBR Green Script miRNA RT-PCR kit (Takara) with the AB1 7500 Real-time PCR system (Bio-Rad Laboratories Inc., Hercules, CA, USA). All of these steps were operated according to the manufacturer's protocol. The oligonucleotide sequences of the qRT-PCR primers are listed in Table 1.
Cell proliferation measured by CCK8 cell viability assay
Following the manufacturer's protocol, cell proliferation was measured using the CCK8 assay Kit (Dojindo Laboratories, Japan). The cell was plated in 96-well plates and treated with drugs. After 72 hrs. Incubation of 10µl CCK8 was added to each well for an additional 2 hrs. Then absorbance was measured by a Multiskan FC photometer at 450nm.
c-Kit loss of functional mutant mice (Wadsm/m)
Five c-kit loss of functional mice (Wadsm/m) and %WT littermates were used on the C57BL/6 background and used in a previous study [24]. Animal studies were performed under strict protocols approved by the Animal Care and Use Committee of Capital Medical University.
Tissue specimen and clinicopathological information
Specimen of tumor and adjacent normal tissues of the colon were obtained from 30 patients who had been pathologically diagnosed with colorectal cancer and underwent surgical resection. Tissue specimens were ground then sonicated in the lysis buffer (50 mM Tris-HCL, pH 7,4, 1mM EGTA, 150 mM Nack, 5% Triton X-100) with protease inhibitors. The samples were micro centrifuged to remove the large debris and subjected to western analysis. All patient-derived specimens were collected from Beijing Friendship Hospital, Capital Medical University Beijing, and archived under complete protocols approved by the Research Board of Capital Medical University.
Statistical analysis
The student's t-test (two-tailed) was performed for comparisons between groups in cell proliferation assay and gene expression analysis by GraphPad Prism 5. Significance was presented as a p-value <0.005 (*), <0.01 (**) and <0.001 (***); non-significant differences were presented as NS.