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Aberrantly enhanced melanoma-associated antigen (MAGE)-A3 expression facilitates cervical cancer cell proliferation and metastasis via actuating Wnt signaling pathway
Biomedicine & Pharmacotherapy 122 (2020) 109710
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Aberrantly enhanced melanoma-associated antigen (MAGE)-A3 expression facilitates cervical cancer cell proliferation and metastasis via actuating Wnt signaling pathway
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Xinping Gaoa, Guobin Chenb, Huihua Caic, Xuefeng Wanga, Kaijing Songa, Limin Liub, Tianmei Qiua, Yuanli Hea,* a b c
Department of Obstetrics and Gynecology, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China Department of Obstetrics and Gynecology, Shenzhen Maternity and Child Healthcare Hospital, Shenzhen, Guangdong, China Department of Obstetrics and Gynecology, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, China
A R T I C LE I N FO
A B S T R A C T
Keywords: MAGE-A3 Knockdown Overexpression Proliferation Migration EMT
Background: The over-expression of melanoma-associated antigen (MAGE)-A3 in cervical cancer (CC) has been observed in our previous study, suggesting that it possibly take a vital role during the development and metastasis of CC. The present study aimed to investigate the biological function of MAGE-A3 in the progression of CC and explore how it executes its roles. Methods: The mRNA expression of MAGE-A3 in End1/E6E7 and CC cell lines (HeLa, SiHa and C33A) was measured by real-time quantitative reverse transcription PCR (qRT-PCR). Loss- and gain-of-function methods were used to assess the effect of MAGE-A3 on the proliferative, migratory and invasive abilities of HeLa and SiHa cells. Western blot was performed to measure the expression levels of proteins related to epithelial–mesenchymal transition (EMT) and proteins in the Wnt signaling pathway. In vivo tumorigenesis assay was conducted to evaluate the effect of MAGE-A3 on tumor growth. Results: MAGE-A3 expression was significantly up-regulated in CC cell lines (HeLa, SiHa and C33A) compared with that in End1/E6E7 cell line. Knockdown of MAGE-A3 could significantly suppress migration, invasion and proliferation in HeLa cells; whereas, overexpression of MAGE-A3 in SiHa cells presented the opposite results. Moreover, knockdown of MAGE-A3 presented a suppressive effect on the activation of EMT and Wnt signaling pathway in HeLa cells, whilst up-regulation of MAGE-A3 exhibited the opponent outcomes in SiHa cells. Through in vivo tumorigenesis assay, we further verified that MAGE-A3 acted as a facilitator in tumor growth. Conclusions: MAGE-A3 is overexpressed in CC cells and possibly facilitates the viability and motility of CC cells via modulating EMT and Wnt signaling. This study implied that MAGE-A3 might be a potential therapeutic target as well as a prognosis predictor for patients with CC.
1. Introduction Gynecologic malignancies, containing cervical, vulvar, vaginal and endometrial cancers, pose a major threat to the health of women worldwide [1,2]. Cervical cancer (CC), one of the most common cancer around the world, is considered as one of the main causes of death in feminine [3] and most of the mortality occurs in the less developed countries [4]. Human papillomavirus (HPV) persistent infection is a major risk factor for CC occurrence and progression [5]. Although progress has been made in early diagnosis and effective vaccines during the past years [6], CC is often diagnosed at a later stage especially in developing countries. Currently, few strategies are available for ⁎
effective treatment of advanced CC [7]. Moreover, there is a great chance of recurrence after receiving therapies [8]. Therefore, it is urgently needed to identify and carry out novel as well as effective predictors, diagnostics and treatments for this disease. As a member of melanoma-associated antigen-A (MAGE-A) family, MAGE-A3 is commonly expressed in germline cells and weakly expressed in somatic cells [9]. It has been demonstrated that cancer-testis antigens contribute to cell survival and transformation to neoplastic phenotypes [10]. However, little is known about the biological functions of MAGE-A3 so far. Previous studies showed that the expression of MAGE-A3 is up-regulated in bladder cancer [11], non-small cell lung cancer [12], colorectal cancer [13], melanoma [14] and gastric cancer
Corresponding author at: Department of Obstetrics and Gynecology, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510282, China. E-mail address: [email protected] (Y. He).
https://doi.org/10.1016/j.biopha.2019.109710 Received 17 June 2019; Received in revised form 23 November 2019; Accepted 25 November 2019 0753-3322/ © 2019 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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quantification was calculated using the 2–ΔΔCT method.
[9,15] tissues, suggesting a vital role of it in the development and metastasis of tumors. Recently, Xie et al. have reported that MAGE-A3 facilitates cell viability and drug resistance in gastric cancer-derived cells [9]. In addition, the up-regulation of MAGE-A3 in CC tissues has been illustrated in our previous study and higher MAGE-A3 expression is found to be correlated with poor prognosis of patients with CC [16]. However, the detailed biological function of MAGE-A3 in CC remains to be clarified. The epithelial to mesenchymal transition (EMT) takes a major part during the development and metastasis of CC [17]. Primary CC with an EMT phenotype presented enhanced tumor development, invasion and metastasis [18]. The Wnt signaling acted critical roles in cell growth, migration and differentiation, and its abnormal activation is implicated in the progression of several cancers including CC [19]. Moreover, Wnt signaling is a signal transduction pathway which could induce EMT [20]. Identifying molecules that are implicated in the induction of EMT and in the activation of Wnt signaling pathway in CC may assist in finding effective therapeutic targets. Our present study illustrates for the first time that MAGE-A3 is overexpressed in CC and plays a facilitating role in the oncogenic proliferation and migration of CC cells, which is possibly achieved by regulating EMT and Wnt signaling pathway. These data implied that MAGE-A3 is a potential predictor and therapeutic target for CC.
2.4. Cell counting kit-8 (CCK8) assay HeLa cells with silenced MAGE-A3 and SiHa cells with over-expressed MAGE-A3 were seeded into a 96-well plate (1000 cells/well) and cultured at 37 °C with 5 % CO2. Cell proliferation ability was determined at 24 h, 48 h, 72 h and 96 h time points by adding 10 μl of CCK8 chemicals (DOJINDO, Kumamoto, Japan) and incubation for 1.5 h at 37 °C. Afterwards, the optical density (OD) was detected at 450 nm using a Model 680 microplate reader (Bio-Rad, Hercules, CA, USA). Graph Pad Prism 5 (GraphPad Software Inc, CA, USA) was used to draw the proliferation curves. 2.5. Colony formation assay Cell suspensions of HeLa cells with silenced MAGE-A3 and SiHa cells with over-expressed MAGE-A3 were prepared. Then cells were plated into 12-well plates (500 cells/well) containing DMEM medium added with 10 % FBS. After being cultured for 2 weeks at 37 °C with 5 % CO2, the cells were fixed with 4 % paraformaldehyde and stained by using 0.1 % crystal violet dye. The numbers of formatted colonies were counted under a light microscopy with 2×magnification.
2. Methods
2.6. Wound healing assay
2.1. Cell lines and cell culture
Cells were seeded on a 6-well plate and grown to confluence. Afterwards, the cells were starved for 24 h and a horizontal wound was made in the confluence using 200-μl pipette tips. Next, the wounds were captured under an optical microscope (Olympus, Japan) at 100× magnification after 0 h and 24 h scratching and the widths of the wounds were measured.
CC cell lines (Hela, SiHa and C33A) and endocervical epithelial cell line End1/E6E7 were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA) containing 10 % fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Sigma, St. Louis, MO) at 37 °C with 5 % CO2.
2.7. Transwell invasion assay Transwell invasion assays were carried out to study the invasive ability of cells with silenced or overexpressed MAGE-A3. About 1 × 105 cells were added to the upper chamber which was pre-coated with matrigel (BD Biosciences, Franklin Lakes, NJ, USA). After incubation at 37 °C for 24 h, we removed the cells on the upper chamber using cotton swabs. Afterwards, cells on the lower surface were fixed with 4 % paraformaldehyde and stained by 0.1 % crystal violet stain. The invaded cells were photographed and counted under a light microscope at 200× magnification.
2.2. Transfection MAGE-A3 si RNA1 (si-MAGE-A3 1; 5′-UCCUAUGAGGACUCCA GCA-3′) and MAGE-A3 si RNA2 (si-MAGE-A3 2; 5′-AGCUUCCAGUUC CUUGCAG-3′) were synthesized by GENEWIZ Co., Ltd. (Suzhou, China) and used for the depletion of MAGE-A3. Scrambled siRNA (si-con) was used as the negative control. The plasmid pcDNA3.1-MAGE-A3 was constructed and used to overexpress MAGE-A3, with pcDNA3.1 vector used as the control. The siRNA was transfected into HeLa cells and the overexpression plasmid was transfected into SiHa cells utilizing Lipofectamine 2000 (Invitrogen, Shanghai, China) following the instructions. Afterwards, the cells were kept at 37 °C for 6 h followed by replacing the medium with fresh medium. After 48 h cultivation, cells were used for the next experiments.
2.8. Western blot Western blot was performed according to the steps described previously [21] with some minor changes. Briefly, total protein was obtained by lysing cells in RIPA lysates (Beyotime, Jiangsu, China) and separated by 12 % polyacrylamide gel electrophoresis. Afterwards, the proteins on the gel were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA) and blocked by 5 % nonfat milk. Then the membranes were incubated at 4 °C with the primary anti-bodies overnight and incubated with the secondary antibodies at room temperature for 1 h. The signals were detected using an Enhanced chemi-luminescence (ECL) kit (Perkin-Elmer Life Science, Fremont, CA, USA). A gel imaging system (Kodak, 4000R PRO, Rochester, NY, USA) was used to conduct the band density analysis. GAPDH was used as an internal reference. The extraction of nuclei and cytosol proteins in HeLa and SiHa cells was conducted utilizing a Nuclear and Cytoplasmic Protein Extraction Kit (KeyGEN Biotech, Jiangsu, China) following the supplier’s instructions. Actin was utilized as a reference for cytoplasmic protein, while Lamin B was regarded as a reference for nuclear protein. The primary antibodies used include: MAGE-A3 (#25800, CST), β-
2.3. Real-time quantitative reverse transcription PCR (qRT-PCR) Total RNA was extracted from the CC cell lines or End1/E6E7 cell line using RNAiso Plus (TaKaRa Biotechnology, Dalian, China) in accordance with the instruction provided by the manufacturer. Afterwards, the extracted RNA was reverse-transcribed and amplified using a HiFiScript cDNA Synthesis Kit (CwBio, Beijing, China). Next, the mNRA expression of MAGE-A3 was analyzed quantitatively using Applied Biosystems 7300 Sequence Detection System (Applied Biosystems). The following primers were used: MAGE-A3 F: 5′−CCCCTGCATGAGTGGGTTTT-3′, MAGE-A3 R: 5′-TGAAGAATGGGCCTCACGTC-3′; GAPDH F: 5′-GGAGCGAGATCCCTCCAAAAT-3′, GAPDH R: 5′-GGCTGTTGTCATACTTCTCATGG-3′. GAPDH was used as an internal reference. The reaction conditions of qPCR were: 95 °C for 30 s, then for 40 cycles of 5 s at 95 °C, 34 s at 60 °C. Relative 2
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investigate the influence of MAGE-A3 on cell motility. We identified that down-regulation of MAGE-A3 in HeLa cells remarkably decreased the migration distance (Fig. 3A) and the number of invading cells compared with the si-con group (Fig. 3B and C, p < 0.01). On the contrary, over-expression of MAGE-A3 in SiHa cells dramatically increased the migration distance (Fig. 3D) and the number of invading cells compared to the control (vector) group (Fig. 3E and F, p < 0.01). In summary, these results demonstrated that MAGE-A3 might be a promoter in the migration and invasion of CC cells in vitro.
actin (#4967, CST), GAPDH (#2118, CST), E-cadherin (#3195, CST), N-cadherin (#4061, CST), Vimentin (#5741, CST), β-catenin (#9562, CST), Cyclin D1(#2922, CST), C-Myc (#9402, CST) and Lamin B (ab16048; Abcam). 2.9. In vivo tumorigenesis assay Female athymic BALB/c nu/nu mice (five-week-old) were purchased from Vital River Laboratories (Beijing, China). si-MAGE-A3 or si-con transfected HeLa cells (5 × 106) and pcDNA 3.1 or pcDNA 3.1MAGE-A3 transfected SiHa cells (5 × 106) were injected sub-cutaneously into the right flank of mice. Tumors were measured using a caliper every 7 days. The formula used for tumor volume calculation was: tumor volume = 1/2(width × width × length). Twenty eight days after tumor cell injection, the mice were sacrificed to take out the tumors. All animal studies were approved by the local Ethics Committee.
3.2. MAGE-A3 promotes EMT in HeLa and SiHa cells To study whether MAGE-A3 facilitated the motility of HeLa and SiHa cells by inducing EMT, we examined the expression levels of EMTrelated proteins by Western blot. The results showed that the protein expression levels of N-cadherin and Vimentin were declined, whilst the level of E-cadherin was increased dramatically after silencing MAGE-A3 in HeLa cells (Fig. 4A and B, p < 0.01). On the contrast, when overexpressing MAGE-A3 in SiHa cells, the protein expression levels of Ncadherin and Vimentin were increased, meanwhile the level of E-cadherin was declined significantly (Fig. 4C and D, p < 0.01). These data hinted that MAGE-A3 possibly affected the migration and invasion of CC cells via regulation of EMT.
2.10. Statistical analysis Statistical analyses were performed using SPSS 15.0 software. Student’s t-test and One-way analysis of variance were used to assess the significance of differences between groups as appropriate. All the data were presented as mean ± standard deviation (SD). It was considered as statistically significant when p < 0.05. 3. Results
3.3. MAGE-A3 promotes the proliferation of CC cells
3.1. MAGE-A3 is up-regulated and performs a promoting role in the migration and invasion of CC cells
Afterwards, we performed CCK8 and colony formation assays to examine the influence of MAGE-A3 on the proliferative ability of HeLa and SiHa cells. The results of CCK8 assays indicated that depletion of MAGE-A3 by si-MAGE-A3 significantly reduced the OD value of HeLa cells at 72 h and 96 h compared to the si-con group (Fig. 5A, p < 0.01). Similarly, fewer colonies were formed in the si-MAGE-A3 group than that in the si-con group (Fig. 5B and C, p < 0.01). On the other hand, the OD value of SiHa cells with overexpressed MAGE-A3 showed an obviously increase compared with the vector group (Fig. 5D, p < 0.01). Furthermore, overexpression of MAGE-A3 in SiHa cells remarkably increased the colony numbers compared to the vector group (Fig. 5E and F, p < 0.01). Collectively, these results implied the promoting effect of MAGE-A3 on the proliferative capability of CC cells.
We analyzed the mRNA expression of MAGE-A3 in End1/E6E7, HeLa, SiHa and C33A cell lines by qRT-PCR. Our results demonstrated that the expression of MAGE-A3 is obviously up-regulated in HeLa, SiHa and C33A cells compared to End1/E6E7 cells (Fig. 1), which further confirmed the over-expression of this gene in CC tissues that observed in our previous study [16]. We then developed HeLa cell line with silenced MAGE-A3 (Fig. 2A–C) and SiHa cell line with over-expressed MAGE-A3 (Fig. 2D–F) to probe the biological functions of MAGE-A3. SiMAGE-A3 1 was elected for conducting the next loss-of- function assays since it presented a higher knockdown efficiency than si-MAGE-A3 2. Wound healing and transwell invasion assays were implemented to
3.4. MAGE-A3 modulates Wnt signaling pathway in HeLa and SiHa cells To better understand the underlying mechanism of MAGE-A3mediated CC cell growth and migration, we then detected the expression changes of proteins in Wnt signaling pathway. The results of western blot showed that down-regulation of MAGE-A3 remarkably decreased the expression of nuclear β-catenin and reduced the levels of C-Myc and CyclinD1 in HeLa cells (p < 0.01, Fig. 6A and B). On the contrast, overexpression of MAGE-A3 obviously up-regulated the expression of nuclear β-catenin, as well as enhanced the expression of CMyc and CyclinD1 in SiHa cells (p < 0.01, Fig. 6C and D). These results suggested that MAGE-A3 could promote the activation of Wnt signaling pathway in CC.
3.5. MAGE-A3 promotes tumor growth in vivo To further explore the effect of MAGE-A3 on tumor growth in vivo, we conducted nude mouse tumorigenicity assay. The results showed that the tumor volume of the si- MAGE-A3 group was smaller than that of the si-con group. On the contrary, the volume of the tumors in the pcDNA 3.1-MAGE-A3 group was notably larger than that of the pcDNA 3.1 group (Fig. 7A–C). These results further proved the promoting effect of MAGE-A3 on CC tumor growth in vivo.
Fig. 1. The expression of MAGE-A3 is enhanced in CC cell lines. The mRNA expression levels of MAGE-A3 in CC cells and normal endocervical epithelial cells were examined by qRT-PCR. *p < 0.05, **p < 0.01 vs. End1/E6E7. 3
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Fig. 2. Knockdown or overexpression of MAGE-A3 in HeLa and SiHa cells, correspondingly. (A) The mRNA expression level of MAGE-A3 in HeLa cells transfected with siRNA was assessed by qRT-PCR. (B) The protein expression level of MAGE-A3 in HeLa cells transfected with siRNA was determined by Western blot. (C) Quantification of MAGE-A3 protein expression in (B) using quantity one software. Columns, mean (n = 6); bars, SD. **p < 0.01, vs. MAGE-A3 expression in HeLa cells transfected with scramble siRNA (si-con). (D) The mRNA expression level of MAGE-A3 in SiHa cells transfected with pcDNA3.1 or pcDNA3.1-MAGE-A3 was assessed by qRTPCR. (E) The protein expression level of MAGEA3 in SiHa cells transfected with pcDNA3.1 or pcDNA3.1-MAGE-A3 was evaluated by Western blot. (F) Quantification of MAGE-A3 protein expression in (E) using quantity one software. Columns, mean (n = 6); bars, SD. **p < 0.01, vs. MAGE-A3 expression in SiHa cells transfected with pcDNA3.1 (vector).
Fig. 3. The effect of depletion or overexpression of MAGE-A3 on cell migration and invasion. (A) The migratory ability of HeLa cell was measured by wound healing assays. (B) The invasive ability of HeLa cell was examined by Transwell invasion assay. (C) The numbers of invading HeLa cells. **p < 0.01, vs. si-con groups. (D) The migratory ability of SiHa cell was measured by wound healing assays. (E) The invasive ability of SiHa cell was examined by Transwell invasion assay. (F) The numbers of invading SiHa cells. **p < 0.01, vs. vector groups.
4. Discussion
vaccines [23]. Formerly, MAGE proteins have been found to be correlated with enhanced tumor growth, motility and resistance to chemotherapeutic drugs [9]. Moreover, the interference of MAGE-A with cell apoptosis and proliferation has been demonstrated recently [24,25]. In gastric cancer tissues, the mRNA expression of MAGE-A3 was identified to be enhanced compared with that in the normal control tissues. What’s more, higher MAGE-A3 expression showed significant association with lower overall survival rate in gastric cancer patients [9]. In our previous clinical study, we found that the positive expression ratio of MAGE-A3 in CC tissues was dramatically higher than that in cervical intraepithelial neoplasia (CIN) and normal tissues. Moreover, our analyses indicated that higher MAGE-A3 expression is correlated
The function of MAGE-A3 in cancer occurrence and progression is still not well understood so far. Here, we illustrated that the expression of MAGE-A3 is enhanced in CC cells and overexpression of MAGE-A3 obviously promotes cell motility, proliferation, EMT and enhances the activation of Wnt signaling pathway in CC, indicating that MAGE-A3 acts as a facilitator during the malignant development of CC. In addition to being expressed in testis, placenta and some stages of embryonal development, MAGE-A proteins have also been found in many types of cancers [22]. On the basis of the immunogenicity of MAGE proteins in tumor, they are considered as ideal targets for tumor 4
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Fig. 4. The expression of EMT-related proteins in HeLa cells with depleted MAGE-A3 and SiHa cells with overexpressed MAGEA3. (A) The protein expression levels of Ecadherin, N- cadherin and Vimentin in HeLa cells with depleted MAGE-A3 were determined by Western blot. (B) Quantification of E-cadherin, N- cadherin and Vimentin expression levels in (A). **p < 0.01, vs. si-con groups. (C) The protein expression levels of E-cadherin, Ncadherin and Vimentin in SiHa cells with overexpressed MAGE-A3 were measured using Western blot. (D) Quantification of E-cadherin, N- cadherin and Vimentin expression levels in (C). **p < 0.01, vs. vector groups.
proliferation using CCK8 and colony formation assays as well as in vivo tumorigenesis assay. The results revealed that MAGE-A3 remarkably accelerated CC cell proliferation in vitro and in vivo. The stimulatory role of MAGE A3 in thyroid cancer cell proliferation, migration and invasion has also been proved by Wei Liu et al. [29]. Besides, early study has suggested that loss of MAGE-A3 expression obviously decreased proliferation and colony formation in gastric cancer-derived cells [9]. Taken together, these findings further confirmed the involvement of MAGE-A3 in CC progression. EMT, a process by which polarized epithelial cells gain mesenchymal characteristics, is a vital mechanism underlying increased migration and invasion of tumors [30,31]. It has been illustrated by many studies that EMT promotes the metastasis of cancer [32,33]. The EMT process is characterized by a significant enhancement in the expression of some mesenchymal markers (such as N-catenin and vimentin) and a notable decrease in the levels of epithelial markers including E-cadherin
with poor prognosis of patients with CC [16]. In the present study, the notable overexpression of MAGE-A3 was also observed in CC cell lines. Our results together with the previous results observed in gastric cancer and CC suggested that MAGE-A3 might play a carcinogenic role in cancer progression. In order to further investigate the biological function of MAGE-A3 in CC progression, we developed HeLa cell line with down-regulated MAGE-A3 and SiHa cell line with over-expressed MAGE-A3. The ability of metastasis to distant organs is one of the main characteristic of neoplasms [26] and the occurrence of metastasis in CC patients is a vital event that influence the prognosis and therapy of CC [27]. Using wound healing assays and transwell invasion assays, we uncovered that MAGEA3 dramatically facilitated the invasion and migration of CC cells, suggesting that MAGE-A3 possibly functioned as a forwarder during the metastasis of CC. Infinite proliferation is another important feature of tumors [28]. Hence, we then explored the effect of MAGE-A3 on cell
Fig. 5. The effect of depletion or overexpression of MAGE-A3 on cell proliferation. (A) The proliferative ability of HeLa cell was determined by CCK8 assays. (B) The colony formation ability of HeLa cells was tested by colony formation assay. (C) Quantification of formatted colonies in (B). **p < 0.01, vs. si-con groups. (D) The proliferative ability of SiHa cells was determined by CCK8 assays. (E) The colony formation ability of SiHa cells was measured by colony formation assay. (F) Quantification of formatted colonies in (E) **p < 0.01, vs. vector groups.
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Fig. 6. The effect of depletion or overexpression of MAGE-A3 on the activation of Wnt signaling pathway. (A) The protein expression levels of β-catenin, CMyc and Cyclin D1 in HeLa cells with silenced MAGE-A3 were determined by Western blot. (B) Quantification of β-catenin, C-Myc and Cyclin D1 expression levels in (A). Columns, mean (n = 6); bars, SD. **p < 0.01, vs. expressions of these proteins in Hela cells in si-con group. (C) The protein expression levels of β-catenin, CMyc and Cyclin D1 in SiHa cells with overexpressed MAGE-A3 were determined by Western blotting. (D) Quantification of β-catenin, C-Myc and Cyclin D1 expression levels in (C). Columns, mean (n = 6); bars, SD. **p < 0.01, vs. expressions of these proteins in SiHa cells in si-con group. Actin was utilized as a reference for cytoplasmic protein, while Lamin B was regarded as a reference for nuclear protein.
of cadherin and then promote EMT [37,38]. In addition, KAP-1 also could regulate the expression of fibroblast-specific protein 1 (FSP1) expression, which takes an important part in EMT [39]. The crosstalk between MAGE-A3 and EMT in CC deserves to be further explored. The Wnt/β-catenin signaling pathway is often abnormally activated in cancer and helps in the initiation and development of CC [40]. This pathway is a critical modulator in regulation of multiple processes including proliferation, differentiation, cell cycle etc. [41]. Furthermore, Wnt signaling pathway is one of the transduction pathways that
[31,34]. Hence, we then explored the effect of MAGE-A3 on the expression of EMT-related markers in CC. Western blot analysis demonstrated that enhanced MAGE-A3 expression contributes to EMT in CC cells. It has been reported that MAGE-A3 could bind to KAP1 (a wellknown transcriptional repressor) [35]. The binding of MAGE-A3 and KAP1 lead to ZNF382 polyubiquitination and degradation. The overexpression of MAGE-A3 decreased the KAP1 bound to ID1 gene (a target of ZNF382) and the expression of ID1 was then up-regulated [36]. The up-regulation of ID1 has been reported to regulate the switch
Fig. 7. The effect of MAGE-A3 on the tumor growth in vivo. (A) The images of tumors that isolated from the mice after injection of CC cells for 28 days. (B) Tumor volumes were measured every 7 days after the injection of HeLa cells. (C) Tumor volumes were measured every 7 days after the injection of SiHa cells. *p < 0.05, **p < 0.01 vs. si-con group or vector group.
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contributes to EMT [20,32]. Researchers have revealed that overexpression of Klotho (a Wnt antagonist) in SiHa cells notably decreased the migrating and invading abilities via repressing EMT and up-regulating E-cadherin [42]. The activation of β-catenin signaling is important for maintaining the EMT-related stem cell-like features of CC cells [43]. Thus, we next examined the expression of proteins in Wnt signaling pathway. Western blot analysis found that overexpression of MAGE-A3 obviously up-regulated the protein levels of nuclear β-catenin and the important downstream genes (C-Myc and CyclinD1), while down-regulation of MAGE-A3 showed the opposite results. Upregulated cytoplasmic and nuclear β-catenin expression has been found in several tumors. However, the mechanism underlying the disorder of β-catenin remains unclear [44]. Our present data suggested that overexpression of MAGE-A3 up-regulated the activity of Wnt/β-catenin signaling pathway and induced EMT thereby promoted CC cell proliferation and motility. The detailed underlying mechanism of how MAGE-A3 talk with Wnt/β-catenin signaling pathway and EMT is worth our further study. Currently, there are few studies concerning the pathways or mechanisms involved in the regulation of MAGE-A3. DNA methylation and histone deacetylation have been illustrated to involve in regulating the expression of MAGE-A genes [45]. Furthermore, it has been revealed that Methyl-CpG binding protein MBD1 can bind to the unmethylated MAGE-A promoters and lead to gene repression [46]. In thyroid cancer, MAGE-A3 is identified as a new target of FGFR2-IIIb signaling, which can take part in epigenetic modifications of MAGE-A3 gene and lead to its downregulation [47]. In esophageal squamous cell carcinoma (ESCC), ciRS-7 functioned as a miR-876-5p sponge to increase the expression of MAGE-A genes thereby promoting the progression of ESCC [48]. However, the pathways and mechanisms implicated in the regulation of MAGE-A3 in CC remains largely unknown and will be explored in our future research. In conclusion, our study provide new evidences that MAGE-A3 is up-regulated in CC and might involve in the progression of CC by regulating EMT and Wnt signaling pathway. These data implied that MAGE-A3 might be a valuable therapeutic target and a prognostic predictor for patients with CC.
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Declaration of Competing Interest None. Acknowledgement This study was supported by the project of Expression and clinical significance of MAGE-A3, MAGE-A9 in cervical lesions (361007). References [1] J.E. Scholey, P.J. Boimel, M. Kirk, L. Lin, Gynecologic Malignancies, (2018). [2] H.H. Cai, M.B. Liu, Y.L. He, Treatment of Early Stage Endometrial Cancer by Transumbilical Laparoendoscopic Single-Site Surgery Versus Traditional Laparoscopic Surgery: A Comparison Study, Medicine 95 (2016) e3211. [3] J. Ferlay, H.R. Shin, F. Bray, D. Forman, C. Mathers, et al., Cancer incidence and mortality worldwide: IARC CancerBase No, Int. J. Cancer Suppl. 136 (2010) E359–E386. [4] N. Thapa, M. Maharjan, M.A. Petrini, R. Shah, S. Shah, et al., Knowledge, attitude, practice and barriers of cervical cancer screening among women living in midwestern rural, Nepal, J. Gynecol. Oncol. (2018) 29. [5] D.M. Gershenson, W.P. Mcguire, M. Gore, M.A. Quinn, G. Thomas, Gynecologic Cancer: Controversies in Management, (2004). [6] R. Chhabra, Cervical cancer stem cells: opportunities and challenges, J. Cancer Res. Clin. Oncol. 141 (2015) 1889–1897. [7] R. Chhabra, Cervical cancer stem cells: opportunities and challenges, J. Cancer Res. Clin. Oncol. 141 (2015) 1889–1897. [8] R. Chhabra, let-7i-5p, miR-181a-2-3p and EGF/PI3K/SOX2 axis coordinate to maintain cancer stem cell population in cervical cancer, Sci. Rep. 8 (2018) 7840. [9] C. Xie, V.V. Subhash, A. Datta, N. Liem, S.H. Tan, et al., Melanoma associated antigen (MAGE)-A3 promotes cell proliferation and chemotherapeutic drug resistance in gastric cancer, Cell Oncol (Dordr). 39 (2016) 175–186. [10] A.J. Simpson, O.L. Caballero, A. Jungbluth, Y.T. Chen, L.J. Old, Cancer/testis antigens, gametogenesis and cancer, Nat. Rev. Cancer 5 (2005) 615–625.
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mechanisms, and diseases, Dev. Cell 17 (2009) 9–26. [42] B. Chang, J. Kim, D. Jeong, Y. Jeong, S. Jeon, et al., Klotho inhibits the capacity of cell migration and invasion in cervical cancer, Oncol. Rep. 28 (2012) 1022–1028. [43] J. Li, B.P. Zhou, Activation of β-catenin and Akt pathways by Twist are critical for the maintenance of EMT associated cancer stem cell-like characters, BMC Cancer 11 (2011) 49. [44] C.O.N. Leung, D. Wen, Y. Tian-Min, H.Y.S. Ngan, T. Sai Wah, et al., miR-135a leads to cervical cancer cell transformation through regulation of β-catenin via a SIAH1dependent ubiquitin proteosomal pathway, Carcinogenesis 35 (2014) 1931–1940. [45] F. Wischnewski, K. Pantel, H. Schwarzenbach, Promoter demethylation and histone acetylation mediate gene expression of MAGE-A1, -A2, -A3, and -A12 in human
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Aberrantly enhanced melanoma-associated antigen (MAGE)-A3 expression facilitates cervical cancer cell proliferation and metastasis via actuating Wnt signaling pathway
T
Xinping Gaoa, Guobin Chenb, Huihua Caic, Xuefeng Wanga, Kaijing Songa, Limin Liub, Tianmei Qiua, Yuanli Hea,* a b c
Department of Obstetrics and Gynecology, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China Department of Obstetrics and Gynecology, Shenzhen Maternity and Child Healthcare Hospital, Shenzhen, Guangdong, China Department of Obstetrics and Gynecology, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong, China
A R T I C LE I N FO
A B S T R A C T
Keywords: MAGE-A3 Knockdown Overexpression Proliferation Migration EMT
Background: The over-expression of melanoma-associated antigen (MAGE)-A3 in cervical cancer (CC) has been observed in our previous study, suggesting that it possibly take a vital role during the development and metastasis of CC. The present study aimed to investigate the biological function of MAGE-A3 in the progression of CC and explore how it executes its roles. Methods: The mRNA expression of MAGE-A3 in End1/E6E7 and CC cell lines (HeLa, SiHa and C33A) was measured by real-time quantitative reverse transcription PCR (qRT-PCR). Loss- and gain-of-function methods were used to assess the effect of MAGE-A3 on the proliferative, migratory and invasive abilities of HeLa and SiHa cells. Western blot was performed to measure the expression levels of proteins related to epithelial–mesenchymal transition (EMT) and proteins in the Wnt signaling pathway. In vivo tumorigenesis assay was conducted to evaluate the effect of MAGE-A3 on tumor growth. Results: MAGE-A3 expression was significantly up-regulated in CC cell lines (HeLa, SiHa and C33A) compared with that in End1/E6E7 cell line. Knockdown of MAGE-A3 could significantly suppress migration, invasion and proliferation in HeLa cells; whereas, overexpression of MAGE-A3 in SiHa cells presented the opposite results. Moreover, knockdown of MAGE-A3 presented a suppressive effect on the activation of EMT and Wnt signaling pathway in HeLa cells, whilst up-regulation of MAGE-A3 exhibited the opponent outcomes in SiHa cells. Through in vivo tumorigenesis assay, we further verified that MAGE-A3 acted as a facilitator in tumor growth. Conclusions: MAGE-A3 is overexpressed in CC cells and possibly facilitates the viability and motility of CC cells via modulating EMT and Wnt signaling. This study implied that MAGE-A3 might be a potential therapeutic target as well as a prognosis predictor for patients with CC.
1. Introduction Gynecologic malignancies, containing cervical, vulvar, vaginal and endometrial cancers, pose a major threat to the health of women worldwide [1,2]. Cervical cancer (CC), one of the most common cancer around the world, is considered as one of the main causes of death in feminine [3] and most of the mortality occurs in the less developed countries [4]. Human papillomavirus (HPV) persistent infection is a major risk factor for CC occurrence and progression [5]. Although progress has been made in early diagnosis and effective vaccines during the past years [6], CC is often diagnosed at a later stage especially in developing countries. Currently, few strategies are available for ⁎
effective treatment of advanced CC [7]. Moreover, there is a great chance of recurrence after receiving therapies [8]. Therefore, it is urgently needed to identify and carry out novel as well as effective predictors, diagnostics and treatments for this disease. As a member of melanoma-associated antigen-A (MAGE-A) family, MAGE-A3 is commonly expressed in germline cells and weakly expressed in somatic cells [9]. It has been demonstrated that cancer-testis antigens contribute to cell survival and transformation to neoplastic phenotypes [10]. However, little is known about the biological functions of MAGE-A3 so far. Previous studies showed that the expression of MAGE-A3 is up-regulated in bladder cancer [11], non-small cell lung cancer [12], colorectal cancer [13], melanoma [14] and gastric cancer
Corresponding author at: Department of Obstetrics and Gynecology, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong 510282, China. E-mail address: [email protected] (Y. He).
https://doi.org/10.1016/j.biopha.2019.109710 Received 17 June 2019; Received in revised form 23 November 2019; Accepted 25 November 2019 0753-3322/ © 2019 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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quantification was calculated using the 2–ΔΔCT method.
[9,15] tissues, suggesting a vital role of it in the development and metastasis of tumors. Recently, Xie et al. have reported that MAGE-A3 facilitates cell viability and drug resistance in gastric cancer-derived cells [9]. In addition, the up-regulation of MAGE-A3 in CC tissues has been illustrated in our previous study and higher MAGE-A3 expression is found to be correlated with poor prognosis of patients with CC [16]. However, the detailed biological function of MAGE-A3 in CC remains to be clarified. The epithelial to mesenchymal transition (EMT) takes a major part during the development and metastasis of CC [17]. Primary CC with an EMT phenotype presented enhanced tumor development, invasion and metastasis [18]. The Wnt signaling acted critical roles in cell growth, migration and differentiation, and its abnormal activation is implicated in the progression of several cancers including CC [19]. Moreover, Wnt signaling is a signal transduction pathway which could induce EMT [20]. Identifying molecules that are implicated in the induction of EMT and in the activation of Wnt signaling pathway in CC may assist in finding effective therapeutic targets. Our present study illustrates for the first time that MAGE-A3 is overexpressed in CC and plays a facilitating role in the oncogenic proliferation and migration of CC cells, which is possibly achieved by regulating EMT and Wnt signaling pathway. These data implied that MAGE-A3 is a potential predictor and therapeutic target for CC.
2.4. Cell counting kit-8 (CCK8) assay HeLa cells with silenced MAGE-A3 and SiHa cells with over-expressed MAGE-A3 were seeded into a 96-well plate (1000 cells/well) and cultured at 37 °C with 5 % CO2. Cell proliferation ability was determined at 24 h, 48 h, 72 h and 96 h time points by adding 10 μl of CCK8 chemicals (DOJINDO, Kumamoto, Japan) and incubation for 1.5 h at 37 °C. Afterwards, the optical density (OD) was detected at 450 nm using a Model 680 microplate reader (Bio-Rad, Hercules, CA, USA). Graph Pad Prism 5 (GraphPad Software Inc, CA, USA) was used to draw the proliferation curves. 2.5. Colony formation assay Cell suspensions of HeLa cells with silenced MAGE-A3 and SiHa cells with over-expressed MAGE-A3 were prepared. Then cells were plated into 12-well plates (500 cells/well) containing DMEM medium added with 10 % FBS. After being cultured for 2 weeks at 37 °C with 5 % CO2, the cells were fixed with 4 % paraformaldehyde and stained by using 0.1 % crystal violet dye. The numbers of formatted colonies were counted under a light microscopy with 2×magnification.
2. Methods
2.6. Wound healing assay
2.1. Cell lines and cell culture
Cells were seeded on a 6-well plate and grown to confluence. Afterwards, the cells were starved for 24 h and a horizontal wound was made in the confluence using 200-μl pipette tips. Next, the wounds were captured under an optical microscope (Olympus, Japan) at 100× magnification after 0 h and 24 h scratching and the widths of the wounds were measured.
CC cell lines (Hela, SiHa and C33A) and endocervical epithelial cell line End1/E6E7 were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA) containing 10 % fetal bovine serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Sigma, St. Louis, MO) at 37 °C with 5 % CO2.
2.7. Transwell invasion assay Transwell invasion assays were carried out to study the invasive ability of cells with silenced or overexpressed MAGE-A3. About 1 × 105 cells were added to the upper chamber which was pre-coated with matrigel (BD Biosciences, Franklin Lakes, NJ, USA). After incubation at 37 °C for 24 h, we removed the cells on the upper chamber using cotton swabs. Afterwards, cells on the lower surface were fixed with 4 % paraformaldehyde and stained by 0.1 % crystal violet stain. The invaded cells were photographed and counted under a light microscope at 200× magnification.
2.2. Transfection MAGE-A3 si RNA1 (si-MAGE-A3 1; 5′-UCCUAUGAGGACUCCA GCA-3′) and MAGE-A3 si RNA2 (si-MAGE-A3 2; 5′-AGCUUCCAGUUC CUUGCAG-3′) were synthesized by GENEWIZ Co., Ltd. (Suzhou, China) and used for the depletion of MAGE-A3. Scrambled siRNA (si-con) was used as the negative control. The plasmid pcDNA3.1-MAGE-A3 was constructed and used to overexpress MAGE-A3, with pcDNA3.1 vector used as the control. The siRNA was transfected into HeLa cells and the overexpression plasmid was transfected into SiHa cells utilizing Lipofectamine 2000 (Invitrogen, Shanghai, China) following the instructions. Afterwards, the cells were kept at 37 °C for 6 h followed by replacing the medium with fresh medium. After 48 h cultivation, cells were used for the next experiments.
2.8. Western blot Western blot was performed according to the steps described previously [21] with some minor changes. Briefly, total protein was obtained by lysing cells in RIPA lysates (Beyotime, Jiangsu, China) and separated by 12 % polyacrylamide gel electrophoresis. Afterwards, the proteins on the gel were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA) and blocked by 5 % nonfat milk. Then the membranes were incubated at 4 °C with the primary anti-bodies overnight and incubated with the secondary antibodies at room temperature for 1 h. The signals were detected using an Enhanced chemi-luminescence (ECL) kit (Perkin-Elmer Life Science, Fremont, CA, USA). A gel imaging system (Kodak, 4000R PRO, Rochester, NY, USA) was used to conduct the band density analysis. GAPDH was used as an internal reference. The extraction of nuclei and cytosol proteins in HeLa and SiHa cells was conducted utilizing a Nuclear and Cytoplasmic Protein Extraction Kit (KeyGEN Biotech, Jiangsu, China) following the supplier’s instructions. Actin was utilized as a reference for cytoplasmic protein, while Lamin B was regarded as a reference for nuclear protein. The primary antibodies used include: MAGE-A3 (#25800, CST), β-
2.3. Real-time quantitative reverse transcription PCR (qRT-PCR) Total RNA was extracted from the CC cell lines or End1/E6E7 cell line using RNAiso Plus (TaKaRa Biotechnology, Dalian, China) in accordance with the instruction provided by the manufacturer. Afterwards, the extracted RNA was reverse-transcribed and amplified using a HiFiScript cDNA Synthesis Kit (CwBio, Beijing, China). Next, the mNRA expression of MAGE-A3 was analyzed quantitatively using Applied Biosystems 7300 Sequence Detection System (Applied Biosystems). The following primers were used: MAGE-A3 F: 5′−CCCCTGCATGAGTGGGTTTT-3′, MAGE-A3 R: 5′-TGAAGAATGGGCCTCACGTC-3′; GAPDH F: 5′-GGAGCGAGATCCCTCCAAAAT-3′, GAPDH R: 5′-GGCTGTTGTCATACTTCTCATGG-3′. GAPDH was used as an internal reference. The reaction conditions of qPCR were: 95 °C for 30 s, then for 40 cycles of 5 s at 95 °C, 34 s at 60 °C. Relative 2
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investigate the influence of MAGE-A3 on cell motility. We identified that down-regulation of MAGE-A3 in HeLa cells remarkably decreased the migration distance (Fig. 3A) and the number of invading cells compared with the si-con group (Fig. 3B and C, p < 0.01). On the contrary, over-expression of MAGE-A3 in SiHa cells dramatically increased the migration distance (Fig. 3D) and the number of invading cells compared to the control (vector) group (Fig. 3E and F, p < 0.01). In summary, these results demonstrated that MAGE-A3 might be a promoter in the migration and invasion of CC cells in vitro.
actin (#4967, CST), GAPDH (#2118, CST), E-cadherin (#3195, CST), N-cadherin (#4061, CST), Vimentin (#5741, CST), β-catenin (#9562, CST), Cyclin D1(#2922, CST), C-Myc (#9402, CST) and Lamin B (ab16048; Abcam). 2.9. In vivo tumorigenesis assay Female athymic BALB/c nu/nu mice (five-week-old) were purchased from Vital River Laboratories (Beijing, China). si-MAGE-A3 or si-con transfected HeLa cells (5 × 106) and pcDNA 3.1 or pcDNA 3.1MAGE-A3 transfected SiHa cells (5 × 106) were injected sub-cutaneously into the right flank of mice. Tumors were measured using a caliper every 7 days. The formula used for tumor volume calculation was: tumor volume = 1/2(width × width × length). Twenty eight days after tumor cell injection, the mice were sacrificed to take out the tumors. All animal studies were approved by the local Ethics Committee.
3.2. MAGE-A3 promotes EMT in HeLa and SiHa cells To study whether MAGE-A3 facilitated the motility of HeLa and SiHa cells by inducing EMT, we examined the expression levels of EMTrelated proteins by Western blot. The results showed that the protein expression levels of N-cadherin and Vimentin were declined, whilst the level of E-cadherin was increased dramatically after silencing MAGE-A3 in HeLa cells (Fig. 4A and B, p < 0.01). On the contrast, when overexpressing MAGE-A3 in SiHa cells, the protein expression levels of Ncadherin and Vimentin were increased, meanwhile the level of E-cadherin was declined significantly (Fig. 4C and D, p < 0.01). These data hinted that MAGE-A3 possibly affected the migration and invasion of CC cells via regulation of EMT.
2.10. Statistical analysis Statistical analyses were performed using SPSS 15.0 software. Student’s t-test and One-way analysis of variance were used to assess the significance of differences between groups as appropriate. All the data were presented as mean ± standard deviation (SD). It was considered as statistically significant when p < 0.05. 3. Results
3.3. MAGE-A3 promotes the proliferation of CC cells
3.1. MAGE-A3 is up-regulated and performs a promoting role in the migration and invasion of CC cells
Afterwards, we performed CCK8 and colony formation assays to examine the influence of MAGE-A3 on the proliferative ability of HeLa and SiHa cells. The results of CCK8 assays indicated that depletion of MAGE-A3 by si-MAGE-A3 significantly reduced the OD value of HeLa cells at 72 h and 96 h compared to the si-con group (Fig. 5A, p < 0.01). Similarly, fewer colonies were formed in the si-MAGE-A3 group than that in the si-con group (Fig. 5B and C, p < 0.01). On the other hand, the OD value of SiHa cells with overexpressed MAGE-A3 showed an obviously increase compared with the vector group (Fig. 5D, p < 0.01). Furthermore, overexpression of MAGE-A3 in SiHa cells remarkably increased the colony numbers compared to the vector group (Fig. 5E and F, p < 0.01). Collectively, these results implied the promoting effect of MAGE-A3 on the proliferative capability of CC cells.
We analyzed the mRNA expression of MAGE-A3 in End1/E6E7, HeLa, SiHa and C33A cell lines by qRT-PCR. Our results demonstrated that the expression of MAGE-A3 is obviously up-regulated in HeLa, SiHa and C33A cells compared to End1/E6E7 cells (Fig. 1), which further confirmed the over-expression of this gene in CC tissues that observed in our previous study [16]. We then developed HeLa cell line with silenced MAGE-A3 (Fig. 2A–C) and SiHa cell line with over-expressed MAGE-A3 (Fig. 2D–F) to probe the biological functions of MAGE-A3. SiMAGE-A3 1 was elected for conducting the next loss-of- function assays since it presented a higher knockdown efficiency than si-MAGE-A3 2. Wound healing and transwell invasion assays were implemented to
3.4. MAGE-A3 modulates Wnt signaling pathway in HeLa and SiHa cells To better understand the underlying mechanism of MAGE-A3mediated CC cell growth and migration, we then detected the expression changes of proteins in Wnt signaling pathway. The results of western blot showed that down-regulation of MAGE-A3 remarkably decreased the expression of nuclear β-catenin and reduced the levels of C-Myc and CyclinD1 in HeLa cells (p < 0.01, Fig. 6A and B). On the contrast, overexpression of MAGE-A3 obviously up-regulated the expression of nuclear β-catenin, as well as enhanced the expression of CMyc and CyclinD1 in SiHa cells (p < 0.01, Fig. 6C and D). These results suggested that MAGE-A3 could promote the activation of Wnt signaling pathway in CC.
3.5. MAGE-A3 promotes tumor growth in vivo To further explore the effect of MAGE-A3 on tumor growth in vivo, we conducted nude mouse tumorigenicity assay. The results showed that the tumor volume of the si- MAGE-A3 group was smaller than that of the si-con group. On the contrary, the volume of the tumors in the pcDNA 3.1-MAGE-A3 group was notably larger than that of the pcDNA 3.1 group (Fig. 7A–C). These results further proved the promoting effect of MAGE-A3 on CC tumor growth in vivo.
Fig. 1. The expression of MAGE-A3 is enhanced in CC cell lines. The mRNA expression levels of MAGE-A3 in CC cells and normal endocervical epithelial cells were examined by qRT-PCR. *p < 0.05, **p < 0.01 vs. End1/E6E7. 3
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Fig. 2. Knockdown or overexpression of MAGE-A3 in HeLa and SiHa cells, correspondingly. (A) The mRNA expression level of MAGE-A3 in HeLa cells transfected with siRNA was assessed by qRT-PCR. (B) The protein expression level of MAGE-A3 in HeLa cells transfected with siRNA was determined by Western blot. (C) Quantification of MAGE-A3 protein expression in (B) using quantity one software. Columns, mean (n = 6); bars, SD. **p < 0.01, vs. MAGE-A3 expression in HeLa cells transfected with scramble siRNA (si-con). (D) The mRNA expression level of MAGE-A3 in SiHa cells transfected with pcDNA3.1 or pcDNA3.1-MAGE-A3 was assessed by qRTPCR. (E) The protein expression level of MAGEA3 in SiHa cells transfected with pcDNA3.1 or pcDNA3.1-MAGE-A3 was evaluated by Western blot. (F) Quantification of MAGE-A3 protein expression in (E) using quantity one software. Columns, mean (n = 6); bars, SD. **p < 0.01, vs. MAGE-A3 expression in SiHa cells transfected with pcDNA3.1 (vector).
Fig. 3. The effect of depletion or overexpression of MAGE-A3 on cell migration and invasion. (A) The migratory ability of HeLa cell was measured by wound healing assays. (B) The invasive ability of HeLa cell was examined by Transwell invasion assay. (C) The numbers of invading HeLa cells. **p < 0.01, vs. si-con groups. (D) The migratory ability of SiHa cell was measured by wound healing assays. (E) The invasive ability of SiHa cell was examined by Transwell invasion assay. (F) The numbers of invading SiHa cells. **p < 0.01, vs. vector groups.
4. Discussion
vaccines [23]. Formerly, MAGE proteins have been found to be correlated with enhanced tumor growth, motility and resistance to chemotherapeutic drugs [9]. Moreover, the interference of MAGE-A with cell apoptosis and proliferation has been demonstrated recently [24,25]. In gastric cancer tissues, the mRNA expression of MAGE-A3 was identified to be enhanced compared with that in the normal control tissues. What’s more, higher MAGE-A3 expression showed significant association with lower overall survival rate in gastric cancer patients [9]. In our previous clinical study, we found that the positive expression ratio of MAGE-A3 in CC tissues was dramatically higher than that in cervical intraepithelial neoplasia (CIN) and normal tissues. Moreover, our analyses indicated that higher MAGE-A3 expression is correlated
The function of MAGE-A3 in cancer occurrence and progression is still not well understood so far. Here, we illustrated that the expression of MAGE-A3 is enhanced in CC cells and overexpression of MAGE-A3 obviously promotes cell motility, proliferation, EMT and enhances the activation of Wnt signaling pathway in CC, indicating that MAGE-A3 acts as a facilitator during the malignant development of CC. In addition to being expressed in testis, placenta and some stages of embryonal development, MAGE-A proteins have also been found in many types of cancers [22]. On the basis of the immunogenicity of MAGE proteins in tumor, they are considered as ideal targets for tumor 4
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Fig. 4. The expression of EMT-related proteins in HeLa cells with depleted MAGE-A3 and SiHa cells with overexpressed MAGEA3. (A) The protein expression levels of Ecadherin, N- cadherin and Vimentin in HeLa cells with depleted MAGE-A3 were determined by Western blot. (B) Quantification of E-cadherin, N- cadherin and Vimentin expression levels in (A). **p < 0.01, vs. si-con groups. (C) The protein expression levels of E-cadherin, Ncadherin and Vimentin in SiHa cells with overexpressed MAGE-A3 were measured using Western blot. (D) Quantification of E-cadherin, N- cadherin and Vimentin expression levels in (C). **p < 0.01, vs. vector groups.
proliferation using CCK8 and colony formation assays as well as in vivo tumorigenesis assay. The results revealed that MAGE-A3 remarkably accelerated CC cell proliferation in vitro and in vivo. The stimulatory role of MAGE A3 in thyroid cancer cell proliferation, migration and invasion has also been proved by Wei Liu et al. [29]. Besides, early study has suggested that loss of MAGE-A3 expression obviously decreased proliferation and colony formation in gastric cancer-derived cells [9]. Taken together, these findings further confirmed the involvement of MAGE-A3 in CC progression. EMT, a process by which polarized epithelial cells gain mesenchymal characteristics, is a vital mechanism underlying increased migration and invasion of tumors [30,31]. It has been illustrated by many studies that EMT promotes the metastasis of cancer [32,33]. The EMT process is characterized by a significant enhancement in the expression of some mesenchymal markers (such as N-catenin and vimentin) and a notable decrease in the levels of epithelial markers including E-cadherin
with poor prognosis of patients with CC [16]. In the present study, the notable overexpression of MAGE-A3 was also observed in CC cell lines. Our results together with the previous results observed in gastric cancer and CC suggested that MAGE-A3 might play a carcinogenic role in cancer progression. In order to further investigate the biological function of MAGE-A3 in CC progression, we developed HeLa cell line with down-regulated MAGE-A3 and SiHa cell line with over-expressed MAGE-A3. The ability of metastasis to distant organs is one of the main characteristic of neoplasms [26] and the occurrence of metastasis in CC patients is a vital event that influence the prognosis and therapy of CC [27]. Using wound healing assays and transwell invasion assays, we uncovered that MAGEA3 dramatically facilitated the invasion and migration of CC cells, suggesting that MAGE-A3 possibly functioned as a forwarder during the metastasis of CC. Infinite proliferation is another important feature of tumors [28]. Hence, we then explored the effect of MAGE-A3 on cell
Fig. 5. The effect of depletion or overexpression of MAGE-A3 on cell proliferation. (A) The proliferative ability of HeLa cell was determined by CCK8 assays. (B) The colony formation ability of HeLa cells was tested by colony formation assay. (C) Quantification of formatted colonies in (B). **p < 0.01, vs. si-con groups. (D) The proliferative ability of SiHa cells was determined by CCK8 assays. (E) The colony formation ability of SiHa cells was measured by colony formation assay. (F) Quantification of formatted colonies in (E) **p < 0.01, vs. vector groups.
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Fig. 6. The effect of depletion or overexpression of MAGE-A3 on the activation of Wnt signaling pathway. (A) The protein expression levels of β-catenin, CMyc and Cyclin D1 in HeLa cells with silenced MAGE-A3 were determined by Western blot. (B) Quantification of β-catenin, C-Myc and Cyclin D1 expression levels in (A). Columns, mean (n = 6); bars, SD. **p < 0.01, vs. expressions of these proteins in Hela cells in si-con group. (C) The protein expression levels of β-catenin, CMyc and Cyclin D1 in SiHa cells with overexpressed MAGE-A3 were determined by Western blotting. (D) Quantification of β-catenin, C-Myc and Cyclin D1 expression levels in (C). Columns, mean (n = 6); bars, SD. **p < 0.01, vs. expressions of these proteins in SiHa cells in si-con group. Actin was utilized as a reference for cytoplasmic protein, while Lamin B was regarded as a reference for nuclear protein.
of cadherin and then promote EMT [37,38]. In addition, KAP-1 also could regulate the expression of fibroblast-specific protein 1 (FSP1) expression, which takes an important part in EMT [39]. The crosstalk between MAGE-A3 and EMT in CC deserves to be further explored. The Wnt/β-catenin signaling pathway is often abnormally activated in cancer and helps in the initiation and development of CC [40]. This pathway is a critical modulator in regulation of multiple processes including proliferation, differentiation, cell cycle etc. [41]. Furthermore, Wnt signaling pathway is one of the transduction pathways that
[31,34]. Hence, we then explored the effect of MAGE-A3 on the expression of EMT-related markers in CC. Western blot analysis demonstrated that enhanced MAGE-A3 expression contributes to EMT in CC cells. It has been reported that MAGE-A3 could bind to KAP1 (a wellknown transcriptional repressor) [35]. The binding of MAGE-A3 and KAP1 lead to ZNF382 polyubiquitination and degradation. The overexpression of MAGE-A3 decreased the KAP1 bound to ID1 gene (a target of ZNF382) and the expression of ID1 was then up-regulated [36]. The up-regulation of ID1 has been reported to regulate the switch
Fig. 7. The effect of MAGE-A3 on the tumor growth in vivo. (A) The images of tumors that isolated from the mice after injection of CC cells for 28 days. (B) Tumor volumes were measured every 7 days after the injection of HeLa cells. (C) Tumor volumes were measured every 7 days after the injection of SiHa cells. *p < 0.05, **p < 0.01 vs. si-con group or vector group.
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contributes to EMT [20,32]. Researchers have revealed that overexpression of Klotho (a Wnt antagonist) in SiHa cells notably decreased the migrating and invading abilities via repressing EMT and up-regulating E-cadherin [42]. The activation of β-catenin signaling is important for maintaining the EMT-related stem cell-like features of CC cells [43]. Thus, we next examined the expression of proteins in Wnt signaling pathway. Western blot analysis found that overexpression of MAGE-A3 obviously up-regulated the protein levels of nuclear β-catenin and the important downstream genes (C-Myc and CyclinD1), while down-regulation of MAGE-A3 showed the opposite results. Upregulated cytoplasmic and nuclear β-catenin expression has been found in several tumors. However, the mechanism underlying the disorder of β-catenin remains unclear [44]. Our present data suggested that overexpression of MAGE-A3 up-regulated the activity of Wnt/β-catenin signaling pathway and induced EMT thereby promoted CC cell proliferation and motility. The detailed underlying mechanism of how MAGE-A3 talk with Wnt/β-catenin signaling pathway and EMT is worth our further study. Currently, there are few studies concerning the pathways or mechanisms involved in the regulation of MAGE-A3. DNA methylation and histone deacetylation have been illustrated to involve in regulating the expression of MAGE-A genes [45]. Furthermore, it has been revealed that Methyl-CpG binding protein MBD1 can bind to the unmethylated MAGE-A promoters and lead to gene repression [46]. In thyroid cancer, MAGE-A3 is identified as a new target of FGFR2-IIIb signaling, which can take part in epigenetic modifications of MAGE-A3 gene and lead to its downregulation [47]. In esophageal squamous cell carcinoma (ESCC), ciRS-7 functioned as a miR-876-5p sponge to increase the expression of MAGE-A genes thereby promoting the progression of ESCC [48]. However, the pathways and mechanisms implicated in the regulation of MAGE-A3 in CC remains largely unknown and will be explored in our future research. In conclusion, our study provide new evidences that MAGE-A3 is up-regulated in CC and might involve in the progression of CC by regulating EMT and Wnt signaling pathway. These data implied that MAGE-A3 might be a valuable therapeutic target and a prognostic predictor for patients with CC.
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