Targeted demethylation of the SLC5A7 promotor inhibits colorectal cancer progression

Cell lines incubation and antibodies

Human CRC cells (HCT116 and RKO) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and then cultured in high glucose (4.5 g/L) Dulbecco’s modified Eagle’s medium (DMEM, ThermoFisher, Massachusetts, USA) supplemented with 1% penicillin–streptomycin (ThermoFisher, Massachusetts, USA) antibiotics and 10% fetal bovine serum (FBS, ZETA Life, California, USA) at 37 °C in a moisturized atmosphere containing 95% air and 5% CO2. All cancer cells were authenticated periodically by DNA profiling on a common platform of the Scientific Research Center, West China Hospital, to ensure that they were mycoplasma-free. Azacitidine (5-azacytidine, AZA, Cat. No. S1782) and decitabine (5-aza-2′-deoxycytidine, DAC, Cat. No. S1200) were purchased from Selleck (Shanghai, China) and used for HCT116 and RKO treatment with a final concentration of 10 μM and 1 μM separately. At 24 h post AZA and DAC treatment, the cells were collected for further analysis.

The antibodies used in this study were as follows: SLC5A7 (MA1-46409, Invitrogen), p53 (10442-1-AP, Proteintech), phospho-p53 (Ser 37; 9289S, CST), p21 (2947, CST), IGFBP3 (10189-2-AP, Proteintech), CDK6 (13331, CST), Bax (2774, CST), and GAPDH (AC002, ABclonal).

Animal study

Five to six-week-old female BALB/c nude mice were obtained from Gempharmatech Co., Ltd. (Chengdu, China) and allowed to adapt in a SPF condition for a week before use. All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of West China Hospital, Sichuan University (No. 2021417A), and conformed to IACUC’s regulatory codes. Mice were raised in ventilated cages with a maximal number of 5 mice per cage and provided with adequate water and food. Housing environment was kept at a temperature between 20–24 °C and 30–70% humidified atmosphere. Twelve-hour light–dark cycles were applied to mimic natural day and night shift. To create a subcutaneous cancer model, control sgRNA and sgSLC5A7 HCT116 and RKO cells were subcutaneously implanted into the right flank of nude BALB/c mice (approximately 5 × 106 cells per mouse). Tumor volume (mm3) was calculated using the following formula: volume (mm3) = width (mm) × length2 (mm) × 0.52. Tumors from each mouse were collected after they were killed and weighed.

Clinical samples and HXCRC cohort

The HXCRC cohort used in the present study contained 69 pairs of malignant tissues and adjacent normal tissues, which were collected from patients with confirmed CRC attending the West China Hospital, Sichuan University, and stored at − 80 °C condition. The overall survival (OS) and disease-free survival (DFS) of patients were recorded for more than 5 years. This part of the study was approved by the Ethics Committee of West China Hospital, Sichuan University (approval number: 2018[280]).

dCas9-based demethylation vector construction

A vector system (Addgene plasmid 82559), which contains three fundamental components: Cas9 peptide sequence (linker length: 22aa), antibody-sfGFP-TET1CD, and gRNA [16], was used in this study. The gRNA insert fragment was cloned by linearizing the Addgene plasmid 82559 AflII site and Gibson assembly-mediated insertion. The total reaction system combined the AflII-digested plasmid 82559 (50 ng), 1 μL insert oligonucleotide mixture, 6 μL 2 × Gibson Assembly Master Mix, and H2O to meet 10 μL in volume. The reaction mixture was then incubated for 15 min at 50 °C. To select the stably transformed clones, competent Escherichia coli was combined with 5 μL reaction mixture indicated above and disseminated over a solid medium containing kanamycin. Finally, the plasmid was purified, and the colony was cultured. The targeted sequences were as follows:

sgSLC5A7-1: CACCGACACCCCTCGCGGTTCCAGG;

sgSLC5A7-2: CACCGGATACCATGGCTGGCAGCG;

sgSLC5A7-3: CACCGTGCGGGATGCGAAGGAAACG;

sgSLC5A7-4: CACCGCCTCGCACCCACACCCCTCG;

sgSLC5A7-5: CACCGAAAAGTCCCCTTTATAAGGG;

sgSLC5A7-6: CACCGATGCTCTCCCGGCCCCCTG.

Cell transfection with sgRNA and small-interfering RNA (siRNA)

For transfection, HCT116 and RKO cancer cells were cultured in 6-well dishes (Corning, New York, USA) and X-tremeGENE HP DNA Transfection Reagent (Roche, Basel, Switzerland) was used following the manufacturer’s protocols for plasmid transfection into cells. G418 (1000 μg/mL) was added to the culture to select successfully transfected cells with a demethylation system. Four days after transfection, the cells were collected to extract genomic DNA and RNA to detect target gene expression and DNA methylation levels. For selecting the stable transfected cells used for animal study, G418 was added to the transfected cells and the concentration was increased from 1000 to 3000 μg/mL, last for 12 days.

The siRNAs for DNMT1, DNMT3a, DNMT3b, and siNC were purchased from Riobio, Co., Ltd., Guangzhou, China. All procedures were consistent with the manufacturer’s protocol, and 48–72 h later, cells were obtained to test the transfection efficiency.

Quantitative real-time PCR

Whole RNA was extracted from cancer cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription of the extracted RNA into cDNA was achieved using the PrimeScript™ II 1st Strand Synthesis Kit (Takara, Kusatsu, Japan). The acquired complementary DNA replicates were further analyzed using SYBR Green Master Mix (Takara, Kusatsu, Japan) following the reaction conditions of 95 °C (300 s), followed by 45 cycles of 95 °C for 5 s, 58 °C for 30 s, 95 °C for 10 s, 65 °C for 60 s, and 97 °C for 1 s. Quantitative real-time PCR was performed using the QuantStudio3 PCR system (ThermoFisher, Massachusetts, USA), and relative mRNA abundance was calculated using the 2−ddCt method.

Western blot analysis

Cancer cells were collected after transfection with sgRNA or siRNA. Then, using RIPA lysis buffer (Beyotime, Nanjing, China) containing 1% protease inhibitor cocktail (Merck Millipore, Massachusetts, USA), total protein was extracted on ice for 30 min. Next, the BCA Protein Assay Kit (Thermo Scientific, Massachusetts, USA) was used to determine the protein concentration. Further, using SDS-PAGE gel electrophoresis, 10 μg of extracted proteins with loading buffer was separated and transferred to a PVDF membrane. The PVDF membranes were blocked with 5% skim milk, followed by incubation at 4 °C overnight with antibodies against SLC5A7 (MA1-46409, Invitrogen), p53 (10442-1-AP, Proteintech), phospho-p53 (Ser 37; 9289S, CST), p21 (2947, CST), IGFBP3 (10189-2-AP, Proteintech), CDK6 (13331, CST), Bax (2774, CST), and GAPDH (AC002, ABclonal). The next day, secondary antibodies labeled with horseradish peroxidase (Zsbio, Beijing, China) were incubated for 1 h, and visualization was performed using an ECL western blotting substrate kit (ThermoFisher, Massachusetts, USA).

Analysis of DNA methylation

A Tissues & Cell Genomic DNA Purification Kit (DP021, GeneMark) was used to extract genomic DNA from cells and samples, and the modified DNA was used for time-of-flight mass spectrometry, which was performed by Oebiotech Co., Ltd., Shanghai, China.

Cell proliferation and colony formation assays

Cell Counting Kit 8 (CCK-8; Dojindo, Kumamoto, Japan) was used to measure cell viability. In brief, approximately 1000 cancer cells per well, with five replicates, were seeded in 96-well plates and incubated. After 72 h of incubation, 10 μL of CCK-8 reagent was added to the culture and incubated for 3–4 h. Then, the absorbance at 450 nm was measured for each well. In the colony-formation assay experiments, approximately 1000 cells per well were seeded in triplicate in six-well plates and incubated for 8–10 days. When visible cell colonies were detected, each well was gently washed twice with phosphate-buffered saline (PBS) and then fixed with 4% paraformaldehyde. Finally, after staining with 0.1% crystal violet, the visible colonies were counted under an inverted microscope. Each experiment was repeated at least three times.

Immunohistochemistry (IHC) staining

IHC staining of paraffin-embedded tissues of subcutaneous tumors was performed with primary antibodies against SLC5A7 (MA1-46409, Invitrogen), p53 (10442-1-AP, Proteintech), and phospho-p53 (Ser 37; 9289S, CST) in a moist dark chamber at 4 °C for 8–10 h. Then, these specimens were incubated with secondary antibody for approximately 2 h and stained with DAB (Maixin, Fuzhou, China). Positive cells were deemed to be cells with moderate to strong brown staining in both the nucleus and cytoplasm.

Statistical analysis

All experiments were conducted 3–5 times to ensure a consistent outcome. Kaplan–Meier survival analysis of disease-free survival (DFS) and overall survival (OS) was performed using the median value as the cutoff point, and significance comparison was examined using the log-rank test. All data are presented as the mean ± SD. Two-tailed unpaired Student’s t and Chi-square tests were used for further comparisons, and Pearson’s correlation analysis was performed to determine the correlation between SLC5A7 expression and methylation level. Statistical significance was set at p ≤ 0.05. All statistical analyses were performed using the GraphPad Prism 5 (California, USA).

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