MeCP2 Promotes Colorectal Cancer Metastasis by Modulating ZEB1 Transcription

Background: Recurrence and distant organ metastasis is a major cause of death in colorectal cancer (CRC); however, the underlying molecular mechanisms regulating this phenomenon are poorly understood. MeCP2 is a key epigenetic regulator and is amplified in many types of cancer. Its role in CRC and the molecular mechanisms underlying its action remain unknown. Methods: We used western blot and immunohistochemistry to detect MeCP2 expression in CRC tissues, and then investigated its biological functions in vitro and in vivo. Chromatin immunoprecipitation, co-immunoprecipitation, and electrophoretic mobility shift assays were used to detect the associations among MeCP2 (Methyl-CpG binding protein 2), SPI1 (Spi-1 Proto-Oncogene), and ZEB1 (Zinc Finger E-Box Binding Homeobox 1). Results: Using the Cancer Genome Atlas and Oncomine databases, we found MeCP2 expression was upregulated in CRC tissues and this upregulation was related to poor prognosis. Meanwhile, MeCP2 depletion (KO/KD) in CRC cells significantly inhibited stem cell frequency, and invasion and migration ability in vitro, and suppressed CRC metastasis in vivo. Mechanistically, we show MeCP2 binds to the transcription factor SPI1, and aids its recruitment to the ZEB1 promoter. SPI1 then facilitates ZEB1 expression at the transcription level. In turn, ZEB1 induces the expression of MMP14, CD133, and SOX2, thereby maintaining CRC stemness and metastasis. Conclusions: MeCP2 is a novel regulator of CRC metastasis. MeCP2 suppression may be a promising therapeutic strategy in CRC.


Patient ID Age (years) Gender Location
The right parts represent the mutation, the right with Strikethrough represent deletion sequence.

Cell culture
The CRC cell lines HCT116, SW480, and HT29 were purchased from the National Infrastructure of Cell Line Resource (China, Beijing). HCT116 cells were cultured in IMDM supplemented with 10% FBS. SW480 cells were cultured in DMEM supplemented with 10% FBS. HT29 cells were cultured in DMEM/F12 supplemented with 10% FBS. All cells were cultured at 37ºC under 5% CO2 in a highhumidity atmosphere.

Immunoblotting
Cells were lysed in RIPA buffer containing 1X protease inhibitor cocktail. Protein concentration was quantified using the BCA protein concentration assay kit (Pierce). Cell lysates were electrophoresed on SDS-polyacrylamide gels and transferred to a PVDF membrane (Millipore). Membranes were incubated with primary antibodies in TBST overnight at 4°C. The membranes were then incubated with HRPconjugated secondary antibodies for 1 h at room temperature, and visualized using an ECL kit (Millipore). The antibodies used for immunoblotting were as follows: MECP2 (

Immunohistochemistry (IHC) analysis
Fresh 4-μm sections were cut from paraffin-embedded tissue samples. After the sections were baked (65ºC, 30 min), they were deparaffinized in xylene, then rehydrated in graded ethanol solutions. The sections were boiled with citrate buffer for 5 min for antigenic retrieval. The sections were incubated with a primary antibody overnight at 4°C. As a negative control, PBS was used to verify the antibody specificity. After washing, anti-rabbit or anti-mouse secondary antibodies (Zhongshan Biotech, Beijing, China) were used. The sections were incubated with DAB (3,3-diaminobenzidine), counterstained, dehydrated, and mounted in permanent mounting medium. IHC stained sections were reviewed and scored independently by two superior pathologists. A final score was calculated by multiplying the proportion of positively stained tumor cells (0-100%) with the staining intensity (0,1,2,3).

RNA extraction and qRT-PCR
Total RNA from the cell lines was extracted using TRIzol Reagent (Invitrogen, USA) according to the manufacturer's instructions. cDNA was synthesized via reverse transcription (RT) according to the manufacturer's protocol (Takara, Dalian, China). PCR amplification was performed in triplicate using the SYBR Green PCR kit (Takara, Dalian, China). GAPDH was used as a control. The following primers were used: GAPDH (forward:

Cell migration and invasion assays
A total of 1 × 105 cells/well were loaded into an insert with serum-free medium and allowed to adhere to a polycarbonate filter that was either pre-coated with 50 μl of Matrigel for the invasion assay or uncoated for the migration assay. The lower chambers were filled with DMEM or IMDM and 10% FBS. Cells on the upper surface of the filters were wiped out after 48 h. Cells that had migrated and invaded through the Matrigel were then fixed and stained with crystal violet. The membranes containing migrated and invaded cells were counted in five randomly selected microscopic views.

Wound healing assay
CRC cells were seeded into 6-well plates at a density of 1 × 105 cells/well in medium containing 10% FBS and cultured until ~80-90% confluence. The cells were scraped with a 10 μl pipette tip to generate scratch wounds. The cells were washed twice with serum-free DMEM to remove cell debris. To record scratch wound closure, images were captured at 0, 48, and 96 h time points in the same position.

In vivo metastasis assays
HCT116 cells were washed with 1X PBS. For the intravenous injection, a total of 1 × 106 cells in 0.1 mL of DMEM were injected into the tail vein of 6-week-old male nude mice. To assess the degree of tumor formation in the lung, imaging of living mice was performed on an Inveon small-animal SPECT/CT imaging system equipped with an isoflurane 2% anesthesia system at six weeks post-injection. After eight weeks, animals were sacrificed and the lungs were harvested. Collected lung tissues were fixed in 10% buffered formalin solution overnight. Fixed tissues were stained with hematoxylin and eosin (H&E).

DNA extraction and bisulfite pyrosequencing analysis
Genomic DNA from fresh-frozen tissues and cells was isolated using MiniBEST Universal Genomic DNA Extraction Kit Ver.5.0 (TaKaRa), according to the manufacturer's instructions. An EpiTect Bisulfite Kit (QIAGEN, 59104) was applied to conduct the bisulfite modification of DNA (1-2 μg). PyroMark Assay Design Software 2.0 (Qiagen) was used to design the bisulfite pyrosequencing primers. The PyroMark Q96 System and software (Qiagen) were utilized for the sequencing reaction and methylation level quantification.

CCK8 assay
Cell proliferation was evaluated using the CCK8 assay (MCE, NJ, USA) according to the manufacturer's instructions. The colon cancer cells were transfected with PLKO or shMeCP2, or transfected with Lenti-V2 or sgMeCP2, and plated into 96-well plates (2,000 cells per well) containing 100 μL media. Then, colon cancer cells were cultured for 0 h, 24 h, 48 h and 72 h and incubated with CCK-8 reagent at a final concentration of 10 μL/mL for 2 h at 37°C. The plate was mixed gently on an orbital shaker for 5 min before the absorbance was measured at 450 nm with a Multiskan FC microplate reader (ThermoFisher, USA). Each experiment was repeated three times.

Cell apoptosis
Cell apoptosis was assessed using FITC Annexin V Apoptosis Detection Kit with PI (BioLegend, CA, USA) according to the manufacturer's instructions. After labelling, the cells were quantified on a BD Accuri™ C6 Flow Cytometer (BD Biosciences). The FlowJo software (Tree Star, Inc.) was used for the post-acquisition analysis of dot plots and histograms. A total of 50,000 events were acquired from each sample.

Cell cycle
Cells were collected 48 h after transfection, and fixed in 75% ethanol at 4°C overnight. The cells were then resuspended in 500 μL PBS and mixed with 50 μg/mL propidium iodide (PI, BioLegend, USA). After incubation for 30 min at 37°C in the dark, the stages of the cell cycle were analyzed using a flow cytometer. Figure S1 A sgRNA-mediated MeCP2 knockout determined by Western blot (left). MeCP2 abrogation did not influence cell viability in HT29 cells (right). B sgRNA-mediated MeCP2 knockout determined by Western blot (left). MeCP2 abrogation did not influence cell viability in SW480 cells (right). C MeCP2 knockdown did not affect apoptosis of HT29 cells. D MeCP2 knockout did not influence apoptosis in SW480 cells. E MeCP2 knockout in HT29 cells did not influence the cell cycle distribution. F MeCP2 knockout in SW480 cells did not affect cell cycle distribution