MeCP2 inhibits proliferation and migration of breast cancer via suppression of epithelial‐mesenchymal transition

Abstract Methyl‐CpG‐binding protein 2 (MeCP2) is an important epigenetic regulator for normal neuronal maturation and brain glial cell function. Additionally, MeCP2 is also involved in a variety of cancers, such as breast, prostate, lung, liver and colorectal. However, whether MeCP2 contributes to the progression of breast cancer remains unknown. In the present study, we investigated the role of MeCP2 in cell proliferation, migration and invasion in vitro. We found that knockdown of MeCP2 inhibited expression of epithelial‐mesenchymal transition (EMT)‐related markers in breast cancer cell lines. In conclusion, our study suggests that MeCP2 inhibits proliferation and invasion through suppression of the EMT pathway in breast cancer.

and the loss of epithelial properties, including the loss of tight junctions and E-cadherin proteins. 6,7 EMT is one of the key processes for cancer metastasis in which epithelial cells assume a mesenchymal cell phenotype, thus enhancing migratory and invasive properties of cancers. 8 EMT is also considered to be the most important and critical step in the process of chemotherapy resistance, local recurrence and distant metastasis. 9,10 Many studies have found that activating EMT can promote the proliferation and migration in breast cancer cell, while the proliferation and migration also can be inhibited by suppressing EMT. 11,12 EMT also is an important biological process that is closely related to the dynamic phenotypic plasticity of cells during embryonic development and the progression of tumours. 13 Recent research revealed that MeCP2 inhibits the expression of HIF-1 via promoting its methylation in basal-like breast cancer cells. 14 And MeCP2 suppresses the progression of pancreatic cancer. 15 So we speculate that MeCP2 plays a vital role in breast cancer.
The methyl-CpG-binding protein 2 (MECP2) gene, located on the long arm region 2 band 8 of the X chromosome (Xq28), is mutated or dysregulated in many nervous system diseases. 16 As a methylation binding protein, MeCP2 plays important roles in epigenetics. 17 In recent years, MeCP2 has been shown to regulate genes in vivo in myelomas, as well as in breast and other tissues. 18 MECP2 has been found to be related to many kinds of cancers including breast and hepatic. 19 Many reports indicate that MECP2 is a tumour suppressor gene. An early report suggested that MeCP2 promotes the methylation of BRCA1 promoter region and inhibits the expression of BRCA1 in breast cancer. 20 What's more, a study revealed that MeCP2 transcription level is higher in oestrogen receptor-positive (ER+) breast cancer than in oestrogen-negative(ER−) breast cancer, and the activation of ERα inhibits EMT of breast cancer. 21,22 MeCP2 also can be considered as a novel regulator of EMT, which knockdown inhibits EMT process of glioma cells. 23 However, in breast cancer, the relationship between MeCP2, EMT and cell proliferation and migration is rarely reported. So we propose the hypothesis that MeCP2 may inhibit the proliferation and migration in breast cancer cell by suppressing EMT. In our study, we demonstrate that MeCP2 plays a tumour suppressor role in breast cancer cells. The loss of MeCP2 expression is an important feature of TNBC, and MeCP2 can inhibit EMT in breast cancer cell lines. MDA-MB-231, SKBR3, MCF-7, BT-549 and T47D cell lines were   obtained from the Committee on Type Culture Collection of the   Chinese Academy of Science and cultured in DMEM supplemented with 10% foetal calf serum (FCS), 1% penicillin/streptomycin and 1% glutamine. During the culture period, the medium was changed with fresh medium and passaged once every 3 or 4 days. After cells reached confluence, they were harvested and used for subsequent experiments. The overexpression pCMV-MeCP2 plasmid used in MDA-MB-231 cells and the control vector pCMV were purchased from Addgene. For MECP2 knockdown experiments in MCF-7 cells, shMeCP2-#1, shMeCP2-#2 and control shMeCP2-#NC were synthesized by GeneCopoeia.

| Cell culture and transfection
Cells were cultured in 6-well plates at a density of 3 × 10 5 cells per well in 2 mL medium supplemented with serum. 2 µg DNA and 5 µL P3000 were diluted to 125 µL with serum-free medium, and 6 µL Lipo3000 was diluted to 125 µL with serum-free medium for transient transfection. The two solutions were mixed gently, incubated at room temperature for 30 minutes, and then, cells were washed once with 2 mL serum-free medium. The medium was replaced with fresh medium after 6-8 hours following transfection.
Gene activity of the cell extracts was measured 24-72 hours after transient transfection, depending on cell type and promoter activity.

| RNA purification and quantitative real-time PCR (qPCR) analysis
Purification of RNA was performed using the Trizol reagent, and contaminated DNA was removed using the TURBO DNA-free Kit (Ambion Inc). cDNA was synthesized from 1 µg of total RNA using the PrimeScript RT reagent kit (Takara Bio) in a 20 µL reaction mixture. For detection of mRNA expression, the SYBR Select Master Mix (Thermo Fisher) and CFX96 Real-time PCR Detection System (Bio-Rad) were used to perform qPCR. Primers were designed online using IDT SciTools. Primers used for qPCR are shown in Table 1. In RCS-p + rates relative to the control rate, the expression change of a target gene was calculated as fold change = 2−(ΔCT, Tg-ΔCT, control).

Gene
Forward primer Reverse primer The PCR protocol used was as follows: 1 minutes at 95°C (10 seconds at 95°C, 15 seconds at 60°C) ×40 cycles and 4°C thereafter.

| Immunoblotting analysis
For detecting protein expression levels, cells were lysed with icecold RIPA lysis buffer (Beyotime). After centrifugation at 12 000 × g Proteins of interest were probed using primary antibodies (shown in  The staining intensity was multiplied by the percentage of positive tumour cells to obtain a total score. Sections with high expression were defined as a total score ≥ 6, and those with low expression were defined as a total score < 6. 24

| Wound healing assay
MDA-MB-231 or MCF-7 cells were cultured for 24 hours in serumfree medium; then, a line was made through the cells to simulate an injury using a 2 mm wide pipette-tip when cells reached 90% confluency. Subsequently, to remove detached cells, they were washed three times with phosphate-buffered saline, and then, they were allowed to migrate in serum-free medium. To measure cell migration,

| Statistical analysis
All experiments were performed in triplicate. Student's t-test was used to evaluate the statistical differences between the experimental and control groups. Data are shown as the mean ± standard error of the mean (SEM).
All the statistical analyses were performed using GraphPad Prism version 7.0. For all analyses, P < .05 was considered statistically significant.

| The expression of MeCP2 in luminal is higher than that in clinical breast cancer samples and TNBC cell lines
To investigate the potential role of MeCP2 in breast cancer, we examined the expression of MeCP2 in clinical breast cancer tissues and cell lines. Immunoblotting suggested that MeCP2 was mainly expressed in breast cancer cells of the luminal epithelial subtype, and E-cadherin expression was increased in T-47D and MCF-7 cell lines ( Figure 1A and B). The raw data as shown in supplemental Figure S1 and S2 and Table   S1 and S2. However, MeCP2 expression was not detected in either basal-like BT-549 or MDA-MB-231 cells, which express the stromal cell markers Slug, Snail and Vimentin ( Figure 1A and B). The raw data as shown in supplemental Figure S1 and S2 and Table S1 and S2. We also  Figure 1E and F). Overall, our results suggest that MeCP2 may be associated with the luminal epithelial subtype in breast cancer.

| Overexpression of MeCP2 increased the expression of epithelial markers but knockdown of MeCP2 increased the expression of mesenchymal markers
To   Figure 3J). Thus, these results confirm that MeCP2 inhibited proliferation, motility and migration in breast cancer cell lines.

| Longer survival curve for MeCP2 high expression
ONCOMINE analysis showed that the expression of MeCP2 mRNA in breast cancer was significantly lower than that in normal samples across a wide variety of datasets and in different cancer types ( Figure 4A). TCGA database was analysed for the expression of MeCP2 in various cancers, and the results showed that MeCP2 was down-regulated in breast-mammary tissue compared with other tissues ( Figure 4B). Additionally, we examined data from the human protein atlas and found that highly expressed MeCP2 mRNA was significantly related to the prolongation of overall survival (OS) in all breast cancer patients ( Figure 4C). Of note, the results indicated that the high expression of MeCP2 mRNA was significantly associated with prolongation of OS in patients, suggesting that MeCP2 may play a role in breast cancer targeted therapy.

| D ISCUSS I ON
Breast cancer mainly originates from mammary epithelial cells.
Of all malignant cancers in women, breast cancer has the highest All experiments were performed at least three times, and data were statistically analysed using a Student's t test (*P < .05, **P < .01 and ***P < .001 vs control) morbidity and mortality, primarily because of distant metastasis and its resistance to chemoradiotherapy or targeted therapy. 25 In summary, our research suggests that MeCP2 is highly correlated with the progression of breast cancer via suppression of EMT signalling pathways. These results suggest that MeCP2 is a promising prognostic marker and therapeutic target for breast cancer.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

AUTH O R CO NTR I B UTI O N S
Wei Jiang produced the initial draft of the manuscript and revised it critically for important intellectual content; Yan-Ling Liang con- All authors have read and approved the final submitted manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published paper.