Cell culture and treatment
Cell lines Caco2, HT-29, Hct-8, Hct-116(human colon cancer cell lines) and FHC (normal human intestinal epithelial cell line) were obtained from Shanghai Cell Collection, Chinese Academy of Sciences. All above cell lines were maintained in DMEM (Hyclone, USA) containing 10% fetal bovine serum (FBS; Hyclone) and cultured at 37℃ in a humidified atmosphere with 5% CO2. All the tumor cell lines had been tested by short tandem repeat (STR) method in ABI 3500 Genetic Analyzer(USA Life 3500). EBSS purchased from Life Technologies (14155-063, Gibco, USA) and 3-methyladenine(3-MA, KeyGEN, China) were used in inducing and inhibiting autophagy.
Sphere-formation Assay
In order to form CSCs spheres, 1 × 104 cells were plate donto polyHEMA (Sigma)-coated 6-well-plates, medium with B27 supplement 20 ng/mL, EGF20 ng/mL, basic fibroblast growth factor and 20 ng/mL in serum free DMEM-F12 (HyClone, UT). The spheres were collected and dissociated into single cells, and then re-suspended in the above medium to culture next-generation spheres for 7 days. CSC spheres were defined as cell colonies with a diameter > 50 µm[13] and area > 50% showing three-dimensional structure and blurred cell margins. Percentage of sphere forming efficiency was calculated as (number of actual spheres/number of cells plated × 100).
Clinic Samples
Tumor tissues and matched adjacent normal tissues were obtained from enrolled CRC patients who underwent surgery at Nanjing First Hospital Affiliated to Nanjing Medical University, between 2018 and 2019. All specimens were immediately frozen in liquid nitrogen after surgery and stored at -80℃. No patients received chemotherapy or radiotherapy at pre-operation. The medical ethics committee of the Nanjing First Hospital Affiliated to Nanjing Medical University approved the study. Animal experiments were performed in strict accordance with the guide for the Care and Use of Laboratory Animals of the Nanjing First Hospital Affiliated to Nanjing Medical University.
Allelic Expression Of IGF2
Exon 9 of the human IGF2 gene is used to evaluate the IGF2 polymorphism by PCR which is known as an ApaI digestion single nucleotide polymorphism (rs680). The primer sequences are as follows: IGF2Fw: 5'-CCTTGGACTTTGAGTCAAATT-3' and Rev: 5'-GGTCGT GCCAATTACATTTCA-3'. The PCR conditions were initial denaturation for 5 min at 94 ºC, followed by 35 PCR cycles of denaturing at 94ºC for 30 s, annealing at 55ºC for 30 s, and elongation at 72ºC for 30 s. The RT-PCR products were also digested by Apa I and the amplifed products were sequenced. Cells that maintain normal imprinting express a single parental allele, while the LOI showed biallelic expression of IGF2.
Methylation Analysis Of IGF2 DMR
Genomic DNA from informative samples was treated with bisulfite (EpiTect Plus DNA Bisulfite Kit 59124) to convert unmethylated cytosines to uracils, whereas methylated cytosines are unaffected. The region corresponded to GenBank were listed in supplementary table 1. The PCR condition was as follows: denatured at 95℃ for 15 min, followed by 95℃ for 30sec, 57℃ for 30sec and 72℃ for 20sec respectively for 50 cycles, and enlongated at 72℃ for 5 min. Each PCR product (5 ul) was analyzed on 2% agarose gel and the remaining 20 ul were purified using QIAquick purification kit (Qiagen) to be sequenced using the forward primer, and the sequencing results were analyzed using CpG viewer, a software tool for DNA methylation available by free download from http://dna.leeds.ac.uk/cpgviewer/download.php.
Reverse Transcriptase-quantitative PCR
Total RNA were extracted simultaneously from paraffin-embedded tissues using E.Z.N.A.FFPE RNA Kit (omega, USA) following the manufacturers' protocol. To avoid the possible DNA contamination, RNA was digested with RNase free DNaseI and cleaned with the RNeasy MinElute Cleanup Kit (Qiagen). The primers of IGF2, H19, CD133, CD44, KLF4, SOX4, OCT4, MYC were showed in supplementary table 1. Quantitative PCR was performed in duplicates using the Takra qPCR Master Mix in ABI Prism 7700 SDS. Results in all figures show mean ± standard error.
Flow Cytometry Analysis And Isolation Of CRC Stem Cells
As previously reported [14], expression of theCSC marker CD133 was finally used to detect and isolate CSCs from CRC. Briefly, nonspecifc binding of cell membranes was blocked by incubating with 5% BSA for 30 min at room temperature. Cells were then incubated with CD133 antibody (MACS,Miltenyi Biotec, Germany) for 30 min before being subjected to flow cytometry analysis (FACSAria, BD,CA). After being washed with phosphate-buffered saline (PBS), ten thousand events per sample were acquired and the cells were counted and collected which were designated as CD133 + cells (CSCs) and CD133 − cells(non-CSCs).
Immunofluorescence(IF)
Cells were plated onto sterile cover slips at a concentration of 5 × 105 cells in fully supplemented keratinocyte media and allowed to adhere for approximately 4 h. Cells were then washed twice with PBS and fixed with 10% neutral-buffered formalin for an hour at room temperature. Cover slips with fixed cells were incubated overnight at 41℃ with antibodies (1:50, supplementary table 1). FITC and Rhodamine B-conjugated anti-rabbit secondary antibody (1:100; Sigma, Canada) was used for detection and cells were counterstained with propidium iodide. Nuclear staining was with DAPI (Molecular Probe) diluted1:1000 and incubated at RT for 30 min. After washing with PBS, the cells were dyed green or red fluorescent and examined by fluorescence microscopy (Axioplan, Zeiss).
Transfection And Establishment Of Cell Lines
Three different specifc siRNAs of IGF2, IR-A and IGF1R were designed and formed (Genepharma, China). Following manufacturer’s protocol (Genepharma, China), transfection reagent was used for transient transfection in cells. Then the siRNA of each gene which showed most inhibition efficiency was used in the next experiments. The list of siRNAs and negative control were showed in supplementary table 1.In order to overexpressed IGF2 mRNA in cells. The synthetic IGF2 DNA fragment and lentiviral vector PGMLV-6395 were enzyme digested with BamH I/EcoRI, Ligation was performed to construct IGF2 overexpressed lentivirus recombinant plasmid. The following lentivirus package was performed by Auragene biotech (Changsha, China). In order to overexpressed miRNA in cells, miRNA mimics was formed (Genepharma, China), and transfected with lip2000(Invitrogen, USA).
Co-immunoprecipitation (CO-IP)
Following cell lysis by RIPA lysis solution, supplemented with protease and phosphatase inhibitors, proteins were obtained by centrifugation at 4 °C. The protein concentration was established using a BCA kit. Equivalent quantities of protein were incubated with an antibody overnight at 4 °C. The next day, they were incubated with Protein G/A agarose beads on a horizontal rotator. Beads-antibodies compound was harvested and freed out the antigen and antibody for Western blot analysis with antibodies against IR-A and IGF1R.
Western Blot Analysis
Samples were lysed using a lysis buffer. Then, the protein concentration was quantified using a BCA protein assay kit (Beyotime, Shanghai, China). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses were performed according to the standard procedures. Primary antibodies used were showed in supplementary table 1. Secondary antibodies were goat anti-rabbit HRP secondary antibody (System Biosciences, Mountain View, CA). Image J was used to detect the gray value of each strip.
mRFP-egGFP-LC3 Fluorescence Assay
Cells with mRFR-eGFR-LC3 constitutive expression were treated with earle's balanced salts solution (EBSS)purchased from Life Technologies (14155-063, Gibco) for 6 hours. Then fixed with 4% PFA for 30 min and washed with PBS for 3 times. The immunofluorescent images were obtained by using a confocal laser scanning microscope (Leica TCS SP8, Solms, Germany) and typical images were presented. For quantitative assay, the green-puncta and red-puncta numbers were counted
Apoptosis Assay
AnnexinV-FITC/PI kit (Keygentec, China) was used in this study. According to the kit instructions, Cells were digested using 0.1% tryps in without EDTA and centrifuged at 1000 rpm. Then binding buffer was used to suspend cells, keeping cell concentration at 5 × 105 cells/mL. Annexin V and PI were added, and the mixture was further incubated for 15 min. After the incubation, cell apoptosis was detected using flow cytometry within 1 h. In the results with 4 quadrants, the fourth quadrant represented the number of early apoptotic cells, and the apoptosis rate was calculated.
Tumorigenicity In Nude Mice
Animal experiments were performed in strict accordance with the guide for the Care and Use of Laboratory Animals of the Nanjing First Hospital Affiliated to Nanjing Medical University. 20 Female BALB/c nude mice aged 6–8 weeks were used. Then CD133+ cells were resuspended in serum-free medium and mixed with matrigel at the ratio of 1:1. NOD/SCID mice were randomly divided into 2 groups. 1 × 105 indicated cells were inoculated subcutaneously into the inguinal folds of NOD/SCID mice. 7 days after inoculation, tumor formation was evaluated by palpation of injection sites, then the mice that developed palpable tumors were injected with or without IR-A mRNA knockdown lentivirus at the injection site every 3 days for 3 times. At the end of experiment (21 days), the mice were sacrificed under deep anesthesia with pentobarbital and eosin (HE) staining. The tumors were then dissected and captured.
Computational Analysis Of TCGA RNA-Seq Datasets
We downloaded the CRC RNA-Seq datasets (Illumina HiSeq2000) from The Cancer Genomics Atlas (TCGA) and The Human Protein Atlas, processed and analyzed using R statistical software package “ggplot2”.
Statistical analysis
Results from at least three independent experiments were expressed as mean ± SD. Statistical analysis of data from two groups was compared by two-tail t-test. Data from multiple groups was performed by one-way ANOVA, followed by Tukey post test. Statistical significance was determined as P < 0.05.