Dna methylation of mir 138 regulates cell proliferation and emt in cervical cancer by targeting ezh2

(2022) 22:488 Chen et al BMC Cancer https://doi.org/10.1186/s12885-022-09477-5 Open Access RESEARCH DNA methylation of miR‑138 regulates cell proliferation and EMT in cervical cancer by targeting EZH2 Rui Chen1†, Qiyu Gan1†, Shuting Zhao1, Dongrui Zhang1, Shunli Wang2, Lili Yao3, Min Yuan3* and Jingxin Cheng1*  Abstract  Background:  Emerging evidence has identified miR-138 as a tumor suppressor that can suppress the proliferation of various cancers Meanwhile, the cause of abnormal miR-138 expression in cervical cancer remains uncertain This study clarified the mechanism by which miR-138 regulates proliferation, invasion, metastasis, and EMT in cervical cancer cells Results:  miR-138 expression in human cervical cancer and adjacent normal tissue was measured using qPCR SiHa and C33A cells were used to determine the function of miR-138 via miR-138 mimic or inhibitor transfection, followed by wound healing, Cell Counting Kit-8, flow cytometry, and Transwell assays Epithelial and mesenchymal marker expression was analyzed using Western blotting DNA methylation in the miR-138 promoter was examined using bisulfite sequencing PCR The downstream target genes of miR-138 were identified via bioinformatics analysis and luciferase reporter assays A tumor xenograft model was employed to validate DNA methylation-induced miR-138 downregulation and tumor growth inhibition in cervical cancer in vivo miR-138 levels were significantly lower in cervical cancer tissues than in adjacent control tissues Furthermore, lower miR-138 expression and higher CpG methylation in the miR-138 promoter were identified in lymph node-positive metastatic cervical cancer tumors versus that in non-metastatic tumor tissues Upon miR-138 overexpression, cell proliferation, metastasis, invasion, and EMT were suppressed miR-138 agomir transfection and demethylating drug treatment significantly inhibited cervical tumor growth and EMT in tumor xenograft models DNA methylation inhibited miR-138 transcription, and enhancer of zeste homolog (EZH2) downregulation mediated the tumor suppressor function of miR-138 in cervical cancer Conclusion:  We demonstrated that miR-138 suppresses tumor progression by targeting EZH2 in cervical cancer and uncovered the role of DNA methylation in the miR-138 promoter in its downregulation These findings demonstrated the potential of miR-138 to predict disease metastasis and/or function as a therapeutic target in cervical cancer Keywords:  Carcinoma of cervix, miRNA, Epigenetic regulations, Metastasis, Invasion *Correspondence: yuanmin003@126.com; 13899899061@163.com † Rui Chen and Qiyu Gan contributed equally  to this work and share first authorship Department of Obstetrics and Gynecology, Shanghai East Hospital, Tongji University School of Medicine, Shanghai 200120, People’s Republic of China Department of Gynecology, Tumor Hospital Affiliated to Xinjiang Medical University, Urumqi 830011, People’s Republic of China Full list of author information is available at the end of the article Introduction Cervical cancer has become the fourth most common malignant tumor in females worldwide, and it carries a high mortality rate and poor prognosis Although immunization against human papillomavirus (HPV) and the improvement of cervical cancer screening have led to significant decreases of the incidence of cervical cancer, the prognosis of patients remains poor because of lymph © The Author(s) 2022 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder To view a copy of this licence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/ The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data Chen et al BMC Cancer (2022) 22:488 node metastasis and pelvic invasion [1–4] Thus, identification of the key factors that regulate epithelial-mesenchymal transition (EMT) and metastasis in cervical cancer is urgently required MicroRNAs (miRNAs) are short RNAs (20–22 nucleotides) that not encode proteins [5] The first miRNA was found from caenorhabditis elegans in 1993 and the mammalian miRNA was discovered in 2000 [6, 7] The genes encoding miRNA in the nucleus are transcribed into primary miRNA (pri-miRNA) Pri-miRNA was cleaved into stem-loop precursors(pre-miRNA) of approximately 70 nucleotides under the action of Drosha RNase Pre-miRNA are exported from the nucleus to the cytoplasm in Ran-GTP-dependent Exportin Under the action of Dicer enzyme (double-stranded RNA-specific RNA endonuclease), pre-miRNA is cleaved into 20–22 double-stranded miRNA [8, 9] Mature miRNA bind to their complementary sequences to form a double helix structure The double helix is then unwound, and one of them binds to the RNA-induced silencing complex (RISC) to form asymmetric RISC The complex binds to the target mRNA In most cases, the single stranded miRNA in the complex is not fully complementary to the 3′-untranslated region (UTR) of the target mRNA, thus blocking the translation process of the gene In addition, miRNAs have been demonstrated to play important roles in proliferation, apoptosis, invasion, differentiation, and other processes [10] Increasing numbers of studies have described miRNA dysregulation in many human cancers and the involvement of miRNAs in the regulation of cancer occurrence, development, and metastasis [11] Emerging evidence has identified miR-138 as a tumor suppressor capable of inhibiting cell proliferation in various cancers including glioma, ovarian cancer, liver cancer, lung cancer, kidney cancer, and prostate cancer [12–17] We previously found 18 differentially expressed miRNAs in HPV16-positive cervical cancer tissues, including miR-138, which was downregulated in cancer [18] However, few reports have described the regulatory roles of miR-138 in metastasis and EMT in cervical cancer EMT describes a biological process whereby epithelial cells lose their polarization and adherence and acquire mesenchymal-like migratory and invasive characteristics [19] The cellular epithelial status is mainly dependent on the calcium-dependent E-cadherin transmembrane adhesion molecule, which is responsible for sustaining connections between adjacent cells During EMT, cells undergo a series of changes in gene expression, functionality, and morphology that are associated with decreased E-cadherin expression and increased N-cadherin expression [20, 21] EMT is considered the pre-step of cancer cell metastasis Meanwhile, several studies have Page of 13 identified enhancer of zeste homolog (EZH2) as a positive upstream regulator of the EMT program EZH2 can combine with the CDH1 (encoding E-cadherin) promoter to decrease the expression of E-cadherin and promote the metastasis and invasion of cancer cells [22] DNA methylation, as one of the most common epigenetic regulations, is an important regulator of gene expression, cell apoptosis, tumorigenesis, and differentiation [23, 24] Accumulated data indicate that DNA methylation can promote the downregulation of miRNAs, thereby regulating tumor development and progression [25] The DNA methylation-mediated control of miRNA expression, such as miR-200b hypomethylation and miR124 hypermethylation, has been reported in cervical cancer [26, 27] In this study, we assessed the expression and function of miR-138 in cervical cancer Low miR-138 expression was associated with increased levels of CpG methylation in the miR-138 promoter in tumors from patients with lymph node positivity or lymphatic space invasion compared with the findings in non-metastatic tumor tissues miR-138 overexpression inhibited cell proliferation, invasion, migration, and EMT in cervical cancer miR-138 agomir and a demethylating drug significantly inhibited cervical tumor growth and EMT in xenograft models We identified EZH2 as an miR-138 target in this cancer type Methylation of the miR-138 promoter transcriptionally suppressed its expression These findings suggested the potential of miR-138 to predict disease metastasis and/or serve as a therapeutic target for the treatment of cervical cancer Materials and methods Clinical samples For this study, we utilized 15 pairs of cervical cancer and paracancerous tissues and 83 tumor tissues collected from patients diagnosed with cervical cancer who were treated at Affiliated Tumor Hospital of Xinjiang Medical University between 2012 and 2014 World Health Organization classifications were used for the histopathological diagnosis of these patients, with staging conducted using the criteria of the International Federation of Gynecology and Obstetrics The Ethics Committee of the Affiliated Tumor Hospital of Xinjiang Medical University approved the present study All patients have signed the written informed consent forms before surgical resection Following collection, tissue samples were snap-frozen and stored at − 80 °C prior to use Cell culture and reagents SiHa and C33A cells were obtained from Institute of Cell Research, Chinese Academy of Sciences (Shanghai, China) and grown in DMEM (SiHa) or MEM (C33A) Chen et al BMC Cancer (2022) 22:488 Page of 13 containing 10% FBS and penicillin/streptomycin at 37 °C in a 5% ­CO2 atmosphere All cell culture reagents were procured from Gibco (USA) progression was evaluated via flow cytometry (BD Biosciences, USA) Cell transfection Cells were collected at 24  h after transfection In total, 1 × ­105 cells in 200 µl of serum-free medium were seeded into the upper Transwell chamber, and the lower chamber was supplemented with 800 μl of medium containing 10% FBS Following incubation for 24 h, the upper chamber was washed thrice with PBS followed by incubation with paraformaldehyde (800  μl) for 10  and staining using crystal violet solution (800  μl) for 15–30  (all at room temperature) A cotton swab was utilized gently to scrape off cells attached to the upper surface of the filter membrane Invasive cells were then enumerated via microscopy (Olympus Corporation, Japan) Negative-control miRNA (miR-NC) and an miR-138 mimic and inhibitor were acquired from Shanghai Gene Pharmaceuticals Co., Ltd EZH2 siRNA was purchased from Shanghai USEN Biological Technology Co., Ltd These constructs were transfected into cells using Lipofectamine® 3000 (Thermo Fisher Scientific,USA) according to the product instruction manual At 24  h after transfection, cells were subjected to further experiments qRT‑PCR An miRNeasy Mini Kit (Qiagen, Germany) and Cell lysis buffer MZ (Tiangen, Beijing, China) were used to extract RNA from samples, after which PrimeScript™ RT reagent (Takara, Japan) and an miScript II RT Kit (Qiagen, Germany) were used to prepare cDNA based on the provided directions Next, an miScript SYBR Green PCR Kit (Qiagen, Germany) and SYBR® Premix Ex Taq™ II (Tli RNaseH Plus, Takara, Japan) were used to detect miRNA and mRNA expression levels The internal controls were U6 and β-actin (primers are listed in the Supplementary Table) Relative expression was quantified via the − ΔCt method Cell proliferation assay At 24 h after transfection, the Cell Counting Kit-8 (CCK8) assay (Beyotime, Beijing, China) was employed to assess the proliferation of cervical cancer cells using the manufacturer’s directions In total, 2000 cells were seeded into 96-well plates with three replicates and incubated with CCK-8 reagent (10 μl) for 24, 48, 72, or 120 h at 37 °C The relative cell viability was assessed at 450 nm using a microplate reader (Thermo Fisher Scientific, USA) Flow cytometry Transfected cells were plated into six-well plates (1 × ­106/ well) Then, apoptotic cells were identified using an Annexin V-FITC Cell Apoptosis Detection Kit (Beyotime Beijing, China) and measured using a BD FACSCanto™ II flow cytometer (BD Biosciences, USA), after which FlowJo software (v 10.4; FlowJo LLC) was used for data analysis For cell cycle analysis, at 24  h following transfection, cells were collected, washed thrice with PBS, and fixed overnight using 70% ethanol at 4  °C Then, cells were resuspended in PBS and incubated with propidium iodide staining solution at 37  °C for 30  Cell cycle Transwell assay Wound‑healing assay Transfected cells were seed in a six-well plate (8 × ­105 cells/well) After overnight incubation, cells were wounded using a pipette tip and incubated in serumfree medium for 0, and 24 h prior to imaging via inverted phase contrast microscopy (Olympus Corporation, Japan) Western blotting RIPA buffer (Beyotime, Beijing, China) supplemented with 1  nM benzylsulfonyl fluoride was employed to extract total protein from cells and tissue samples, after which a BCA assay kit (Beyotime, Beijing, China) was used to quantify protein levels in samples The protein (30  μg) was separated via 10% SDS-PAGE, followed by transfer to a polyvinylidene fluoride membrane (EMD Millipore, USA) The membrane was incubated for 2  h with non-fat milk followed by the primary antibodies, including those specific for EZH2 (1:1000; CST; #5246), E-cadherin (1:1000; CST; #3195), N-cadherin (1:1000; Abcam; No Ab18203), vimentin (1:1000; CST; #5741), β-actin (1:1000; Proteintech Group; No 66009–1-Ig), and GAPDH (1:1000; CST; #5174) overnight at 4  °C Blots were then washed with TBS-T and incubated with HRPconjugated polyclonal goat anti-rabbit immunoglobulin G (IgG, 1:2000; Beyotime; No A0208) or polyclonal goat anti-mouse IgG (1:1000; Beyotime; No A0216) for 1 h at room temperature An enhanced chemiluminescence kit (Beyotime, Beijing, China) was then used to detect protein bands, and protein expression was quantified using Quantity One software (v3.0; Bio-Rad Laboratories) Bioinformatics analysis TargetScan Software (v7.2; http://​www.​targe​tscan.​org/​ vert_​72/) was employed to detect putative miR-138 targets Chen et al BMC Cancer (2022) 22:488 Luciferase reporter assay Wild-type (WT) and mutant (Mut) EZH2 3′-UTR sequences were produced and inserted into luciferase reporter vectors (Obio Technology (Shanghai) Corporation) Cells (1 × ­105/well) were seeded into 24-well plates, after which vectors containing WT or Mut EZH2 3′-UTR regions were co-transfected into cells along with the miR-138 mimic or miR-NC using Lipofectamine® 3000 reagent (Thermo Fisher Scientific, USA) A Luciferase-Reporter Assay System kit (Promega, USA) was then employed to quantify luciferase activity at 48 h after transfection Page of 13 were anaesthetized by inhalation of 3% isoflurane and sacrificed by breaking the neck at 29  days after implantation, at which time tumors were isolated and weighed All experiments involving animals were approved by the Animal Care and Use Committee at Tongji University and followed by Guidelines for the ethical review of laboratory animal welfare People’s Republic of China National Standard GB/T 35,892–2018 Immunohistochemistry (IHC) IHC was performed with specific antibodies as described previously [28] Bisulfite Sequencing PCR (BSP) Statistical analysis A Genomic DNA Extraction Kit (GENERAY; GK1022, Shanghai, China) was employed to extract gDNA from all samples, after which an EpiTect Fast DNA Bisulfite Kit (QIAGEN; 59,824, Germany) was employed for bisulfite conversion according to the product manual Then, one microliters of the bisulfite modified DNA from each sample were subjected to PCR analysis in a 30 μL volume The reaction mixture was preheated at 95 °C for 10 min and amplified using a PCR program (i.e., 40 cycles of 94  °C for 30  s, 55  °C for 30  s, and 72  °C for 40  s; and a final extension of 5 min at 72 °C) The PCR products were then subjected to clone into the pTG19-T vector (Generay, Lot:GV6021) followed by sequencing analysis (after the cloning, 10 clones from each sample were randomly selected for DNA sequencing) SPSS 25.0 (SPSS, Inc.) and GraphPad Prism 8.0 (GraphPad Software, Inc.) were employed for all statistical testing Data are presented as the mean  ± standard deviation Relationships between miR-138 and patient clinicopathological parameters were analyzed using oneway ANOVA An independent-samples t-test or oneway ANOVA was used to analyze possible differences between the two groups The methylation status was analyzed comprehensively and comparatively using Biqanalyzer P  4 cm P value 0.940 Depth of Stromal Invasion 0.980 Lymph node Metastasis 0.046 Lymph Vascular Space Invasion 0.030 and Supplementary Fig.  1a) Contradictory results were detected in cells transfected with the miR-138 inhibitor (Fig.  2d and Supplementary Fig.  1a) In addition, we found that miR-138 was negative correlation with EZH2 expression in tumor tissues (Supplementary Fig. 1b) EZH2 mediates the regulatory activities of miR‑138 in cervical cancer We further examined whether EZH2 suppression is critical for miR-138–induced proliferation, apoptosis, and invasion in cervical cancer cells We determined that EZH2 silencing via siRNA significantly inhibited its expression (Fig.  3a and Supplementary Fig.  1c) Meanwhile, we observed that cell proliferation, invasion, and metastasis were inhibited upon EZH2 suppression, in addition it promoted apoptosis The results were similar for miR-138 mimic transfection (Fig.  3b-e) We further explored the change of protein levels, observing increased E-cadherin expression (Fig.  3a) We next conducted rescue experiments by co-transfected miR-138 inhibitor and siEZH2 in SiHa and C33A cells, further elucidating the effects of miR-138 on the regulations of EZH2 As expected, miR-138 inhibitor relieved the suppression of siEZH2 on EZH2 expression (Fig.  3f and Supplementary Fig.  1d) Furthermore, miR-138 inhibitor markedly promoted metastatic potential, but was reversed by EZH2 knockdown (Fig. 3g) DNA methylation regulates miR‑138 expression at the transcriptional level blotting revealed decreases of EZH2 and N-cadherin expression and increases of E-cadherin expression at the protein level in miR-138 mimic-transfected cells (Fig. 2d Our data presented thus far indicated that the regulatory function of miR-138 is mediated by EZH2 expression However, the cause of abnormal miR-138 expression remained unclear Previous studies reported Chen et al BMC Cancer (2022) 22:488 Page of 13 Fig. 2  Enhancer of zeste homolog (EZH2) is a target gene of miR-138 in cervical cancer cells a Sequence alignment between miR-138 and the predicted binding site in the wild-type (WT) 3′-untranslated region (UTR) of EZH2 The sequence of the EZH2 3′-UTR carrying a mutation in the miR-138 binding site (Mut) was also employed for luciferase reporter analysis b Relative luciferase activity in SiHa and C33A cells transfected with the WT or Mut EZH2 3′-UTR-Luc with or without miR‐138 overexpression c Quantitative RT-PCR analysis was applied to determine the relative mRNA levels of EZH2 in SiHa and C33A cells transfected with the miR-138 mimic, miR-138 inhibitor, or negative-control miRNA (miR-NC) d Western blot analysis was applied to determine the protein levels of EZH2, E-cadherin, and N-cadherin in SiHa and C33A cells transfected with the miR-138 mimic, miR-138 inhibitor, or miR-NC β-actin served as a loading control that methylation is closely related to miRNA expression [25] Thus, we further assessed whether methylation also might contribute to the downregulation of miR-138 in cervical cancer Specifically, we measured the degree of methylation of CpG sites in the miR-138 promoter in cancer tissues and adjacent tissues in patients, as well as in cell lines The BSP results clearly confirmed the methylation of a single CpG site in the promoter region of miR-138 (Fig. 4a and Supplementary Fig. 2) We found that the extent of CpG-9 site methylation in the miR-138 promoter was higher in cancer tissues than in paracancerous tissues (Fig. 4b) To further verify our conjecture, we treated C33A cells with DAC, finding that miR-138 expression was increased after DAC treatment (Fig.  4c) (See figure on next page.) Fig. 3  Silencing of enhancer of zeste homolog (EZH2) mimics the function of miR-138 overexpression in regulating cervical cancer cell proliferation, migration, and invasion a Western blot analysis of EZH2 and E-cadherin expression in SiHa and C33A cells transfected with EZH2 or control siRNA β-actin served as a loading control b-e Analyses of cell proliferation (b), apoptosis (c), invasion (d), and migration (e) in SiHa and C33A cells transfected with EZH2 siRNA, miR-138 mimic, or negative-control miRNA (NC) f Western blot analysis of EZH2 expression in SiHa and C33A cells co-transfected with siEZH2 and/or miR-138 inhibitor g Transwell assay after miR-138 inhibitor and siEZH2 co-transfection Chen et al BMC Cancer (2022) 22:488 Fig. 3  (See legend on previous page.) Page of 13 ... of? ?miR? ? ?138 in? ?cervical cancer We further examined whether EZH2 suppression is critical for miR- 138? ??induced proliferation, apoptosis, and invasion in cervical cancer cells We determined that EZH2. .. space invasion compared with the findings in non-metastatic tumor tissues miR- 138 overexpression inhibited cell proliferation, invasion, migration, and EMT in cervical cancer miR- 138 agomir and. .. rescue experiments by co-transfected miR- 138 inhibitor and siEZH2 in SiHa and C33A cells, further elucidating the effects of miR- 138 on the regulations of EZH2 As expected, miR- 138 inhibitor relieved