Background
Breast cancer is one of the most common types of cancer diagnosed among women world-wide and the second leading cause of cancer death among women in the US [
1]. In China, the burden of female breast cancer is getting increasingly severe, especially in urban areas of China [
2]. Despite the great advancement in the understanding, surgical and meditations; breast cancer remains to be a major cause of death worldwide [
3]. Consequently, further investigation of novel oncogenes involved in the pathogenesis of breast cancer is needed and necessary.
TMED3, a contraction of Transmembrane Emp24 Protein Transport Domain Containing 3, a member of TMED family thought of being implicated in the vesicular trafficking of proteins and innate immune signaling [
4]. Till now, the ten members of TMED family have been discovered [
5]; many TMED proteins [
6‐
9] have been investigated and reported except for TMED3 which has been comparatively seldom described in the context of cancer, with the exception of the only three relevant studies performed regarding TMED3 in cancers, including prostate cancer [
10], colon cancer [
11] and liver cancer [
12]. Evidence from the three studies of TMED3 suggest the role of TMED3 involved in malignancies seems to be different even somewhat controversial. Considering that no evidence has been available concerning TMED3 in breast cancer, herein we attempted to explore both of the clinicopathological and biological roles of TMED3 in breast cancer. We firstly defined the oncogenic role of TMED3 in breast.
Methods
Tissue microarray preparation
Formalin-fixed, paraffin-embedded blocks of breast cancer and its paired normal control tissues, totaling 224, were retrieved from the archives at the Department of pathology, The First Affiliated Hospital of Anhui Medical University. All these blocks we retrieved were outsourced to prepare the tissue microarray by Shanghai Outdo Biotech Co. Ltd. (Shanghai, China). Another additional independent 37 cases of fresh frozen breast cancer tissues were collected in The Department of Breast Surgery, The First Affiliated Hospital of Anhui Medical University. All cancer tissues were staged and graded following the World Health Organization classification and grading system (2016 version). None of the patients had received any treatment before mastectomy. Both the cancer and its adjacent normal controls were histopathologically confirmed by experienced pathologist (Zhengsheng Wu M.D) who was totally blind to our study from the outset. Informed consent was obtained from each participant involved, and the study got approval from the Medical Ethics Committee of the First Affiliated Hospital of Anhui Medical University.
Cell culture and siRNA transfection
The human breast cancer cell lines MDA-MB-231, MDA-MB-468 and MCF-7 as well as the normal human breast mammary epithelial cell lines MCF-10A were commercially from American Type Culture Collection (ATCC, Manassas, VA, USA). The transformed but not malignant human mammary epithelial cell line HBL-100 (HBT-124, ATCC, USA) has been routinely stored in liquid nitrogen in our cell laboratory in The First Affiliated Hospital of Anhui Medical University. The MCF10A cells were cultured in a DMEM/F12 medium containing EGF (20 ng/mL), hydrocortisone (0.5 mg/mL), cholera toxin (100 ng/mL) (Sigma-Aldrich; St. Louis, MO, USA), insulin (10 μg/mL), and 1× penicillin–streptomycin (Gibco; Carlsbad, CA, USA). HBL-100 cell line was cultured in MEM media supplemented with 10% fetal calf serum, 0.2% sodium bicarbonate, 10 mM HEPES, 1% non-essential amino acids, 2 mM
l-glutamine and 6 ng/mL insulin (Life Technologies, Grand Island, NY USA). The breast cancer cells were cultured in DMEM (HyClone; Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). All cells were maintained at 37 °C in a humidified 5% CO
2 atmosphere incubator. The specific siRNA to TMED3 was designed and synthesized by GenePharm Company (GenePharma, Shanghai, China), the sequences were tabulated in Additional file
1: Table S1. The lenti-viral based shRNA for TMED or re-expression of miR-188-3p vectors were outsourced to GeneChem Company (GeneChem, Shanghai, China) to construct. Cells were seeded in six-well plates at the concentration of 4 × 10
5 cells/well 24 h before the transfection with TMED3-siRNA and its control using Lipofectamine™ 2000 (Invitrogen, Life Technologies Inc, USA) following the manufacturer’s instructions.
Quantitative real-time PCR (qRT-PCR)
Total RNA were isolated using the Trizol (Invitrogen™, Life Technologies Inc, USA) according to the manufacture’s protocol. RNA from each sample was transcribed into cDNA using Revert Aid First Strand cDNA Synthesis Kit (Catalog number: K1621, Thermo Fisher Scientific, MA, USA) according to the manufacturer’s instructions. qRT-PCR was conducted using Fast Start Universal SYBR Green Master (Rox) (Roche, Germany). TMED3 PCR detection was performed using SYBR Green method (Catalog number: 4309155, Thermo Fisher Scientific, MA, USA). The PCR cycle consisted of 95 °C for 3 min, 95 °C for 30 s, 62 °C for 30 s (β-actin was 55 °C for 30 s), 72 °C for 30 s. Expression of TMED3 was normalized to the β-actin to control the variability in expression levels. The relative expression levels were calculated using standard curve method. The sequence of primers was listed in Additional file
1: Table S1.
Immunohistochemistry (IHC)
The tissue microarray was incubated at 60 °C overnight and then subjected to de-paraffinization and dehydration in gradient ethanol. The activity of endogenous peroxide was blocked in 3% H2O2 for 15 min. The antigen was retrieved in 0.01 M citrate buffer heated in a microwave oven at 98 °C for 15 min and then was cooled at room temperature for 15 min. After washing in phosphate-buffered saline (PBS), the tissue microarray was incubated with normal serum to block nonspecific staining for 30 min. Polyclonal rabbit anti-TMED3 (1:150 dilution; Ab151056, Abcam, MA, Cambridge, USA) was incubated with the tissue section overnight at 4 °C in a humidified chamber. After washing with PBS, the tissue section was treated with biotinylated anti-mouse secondary antibody, followed by further incubation with streptavidin–horseradish peroxidase complex and staining using the diaminobenzidine kit (Beijing Zhongshan Golden Bridge Biotechnology Co, Ltd). IHC staining was assessed in a series of randomly selected four fields at 400× magnification.
Immunoscoring of TMED3
Four fields per section were randomly selected, and the immunostaining of TMED3 was scored by two separate clinical pathologists. The immunostaining of TMED3 was classified into the following four categories: 0, negative (no positive cells); 1, weak positive (between 20% and 40% positive cells per mm2); 2, moderate positive (between 40% and 60% positive cells per mm2); 3, strong positive (> 60% per mm2). For statistical analysis, these categories were categorized into two groups, low (0–1) and high (2–3) expression.
Flow cytometry analysis
Cells were harvested with tyrosine after transfection for 48 h, fixed with cold 70% ethanol at 4 °C overnight, followed by adding RNase A enzyme (0.05 mg/mL) and staining with propidium iodide (PI, 0.05 mg/mL), cell cycle distribution was analyzed on a FACS Calibur system (3550UV, BioRad, USA). For apoptosis detection, cells were stained by the Annexin V-FITC kit (Invitrogen™, Life Technologies Inc, USA) following the accompanying instruction. Viable cells were not stained with Annexin V. The necrotic cells were Annexin V and PI double positive, whereas apoptotic cells were Annexin V positive and PI negative.
Mitomycin C treatment
Cells were trypsinized (0.25% trypsin–EDTA; Invitrogen™, Life Technologies Inc, USA) for 2 min at 37 °C, washed with SMC medium, and then incubated as a single-cell suspension in 40 μg/mL Mitomycin-C (catalog number: M4287; Sigma-Aldrich Chemical Company, St. Louis, MO, USA) in DMEM medium for 30 min at 37 °C. After two additional washes in PBS, cells were assessed for viability, proliferation, or migratory ability. For the wound healing assay, Mitomycin-C treatment was performed after DMEM plating and when cells were 100% confluent; cells were incubated in 40 μg/mL mitomycin-C in DMEM medium for 30 min at 37 °C and then washed twice in PBS before assay.
MTT
Cells were placed in a 96-well plate at a density of 4 × 103/well and were incubated overnight. At 0, 24, 48, 72, and 96 h, 20 μL (5 g/L) of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) reagent was added to the designated wells. After a 4 h incubation, the MTT formazan precipitate was dissolved in dimethylsulphoxide (DMSO) (150 μL/well, Sigma-Aldrich, St. Louis, MO, USA) in a shaker before reading the absorbance at 490 nm using a 96-well plate reader (Bio-Rad, Winooski, VT USA).
Wound healing assay
Cells were seeded in 6-well plates. After overnight incubation, the cells were preceded by treatment with Mitomycin C (40 μg/mL) for 30 min; followed by scrape with a 10 µL pipette tip to generate a straight-line cell-free scratch. Migrating cells were photographed after 24 h. Each well was washed with PBS to remove the remaining unattached cells before imaging. Cell motility was quantified by measuring the distance between the migrating cell boundaries.
Transwell assay
Transwell assay was performed to assay the invasion and migration of breast cancer cells using a 24-well Transwell chamber with a polycarbonate membrane with a pore size of 8 μm (Corning, NY, USA). The membrane was coated with or without 60 μL of a 1:5 mixture of Matrigel (BD Sciences, San Jose, CA, USA) and serum-free DMEM medium to form a matrix barrier. After the Matrigel was allowed to solidify at 37 °C for 2 h, breast cancer cells (1 × 105 cells/mL) were pretreated with Mitomycin C (40 μg/mL) in 200 µL of serum-free DMEM medium were added to the upper compartment of the chamber; The lower chamber was filled with 0.6 ml of medium supplemented with 10% FBS as a chemo-attractant. After incubation at 37 °C for 24 h, cells were then rinsed with PBS and fixed in 100% methanol. The cells remaining at the top of the polycarbonate membrane were removed using a moist cotton-tipped swab. The cells that migrated through pores to the lower surface were stained with crystal violet and hematoxylin and distilled water several times, and the cell numbers in four random fields at a magnification of 200× were counted and averaged.
Clonogenesis assay
Clonogenic growth of breast cancer cells were assessed by incubating 500 cells transfected with lentiviral-based shTMED3 or re-expression of miR-188-3p vectors that were preceded by previously cell sorting for GFP expression using Flow Cytometry. Clonogenic cultures were performed in 12-well plate in 1 ml in DMEM (HyClone; Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco). Colonies of more than 50 cells were automatically counted using Image J software (NIH, Bethesda, MA, USA) after 10 days.
miRNA target prediction and luciferase reporter assay
The potential targets of miR-188-3p were predicted using miRanda (
www.microrna.org) and TargetScan (
www.targetscan.org). TMED3 was found to be a putative target of miR-188-3p. The 3′-UTR of TMED3 containing the complementary sequences of miR-188-3p or mutation sequences was amplified and subcloned into the pMirTarget plasmid (Origene, Rockville, MD, USA), which were named as pMirTarget-TMED3-3′-UTR wild-type (WT) and pMirTarget-TMED3-3′-UTR mutant (MUT), respectively. Cells were inoculated into 24-well plates 1 day before transfection. Cells were co-transfected with miR-188-3p mimics or miR-NC, and pMirTarget-TMED3-3′-UTR WT or MUT, using Lipofectamine 2000
®. Following 48 h of incubation at 37 °C, the luciferase activity was determined using a Dual-Luciferase Reporter Assay system (Promega Corporation, Madison, WI, USA). Renilla luciferase activity was chosen for normalization.
Western blot
Cells were lysed in RIPA lysis buffer. The protein concentrations were determined using the BCA protein quantitation assay (Bioteke, Beijing, China). Equal amounts of protein (50 μg) were separated on 12% SDS-PAGE gels and were transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The membranes were blocked for 1 h using blocking buffer and then were probed overnight with an anti- TMED3 Rabbit mAb (1:800; ab173112, Abcam, MA, Cambridge, USA), and anti-GAPDH antibody (1:1000; #5174; Clonality: D16H11; Cell Signaling Technology) at 4 °C. The membrane was washed three times for 5 min each time with washing buffer and was incubated with secondary antibody anti-rabbit IgG, Fluorescence-linked antibody (catalog number: 926-32211; LI-COR Biosciences, Lincoln, Nebraska, USA) dilution at 1:10,000, incubation for 1.5 h avoid light at 37 °C. Proteins were detected using LI-COR Odyssey Imaging system (LI-COR Biosciences, Lincoln, Nebraska, USA).
Xenograft nude mice model
All animal procedures were carried out following the procedures described in the Laboratory Animal Maintenance Manual of The First Affiliated Hospital of Anhui Medical University (Approval No. AHMU-F1-111208). Six-week-old male nude mice (BALB/cSlcn/n) were purchased from Vitalriver Inc. (Vitalriver, Beijing, China) and maintained under specific pathogen-free conditions. For subcutaneous implantation, tumors were established by injecting MCF-7 cells expressing either control or pcDNA6-miR-188-3p at the density of 1 × 107 cells subcutaneously into the left flank of each mouse. Mice were divided into six groups and tumor lesions were harvested 1 month later.
Statistical analysis
The data were expressed as the mean ± standard error of mean (SEM). Statistical analysis was carried out using SPSS 17.0 software (SPSS, Chicago, IL, USA). Independent sample T-test and one-way ANOVA were used for statistical analyses between groups of continuous variables that followed the normal distribution. Cross-table analysis was conducted for categorical variables. Kaplan–Meier survival analysis was performed for overall prognostic analyses. A value of p < 0.05 was taken as statistically significant.
Discussion
In our present investigation, to the best of our knowledge, we firstly showed that compared to normal controls, TMED3 was markedly up-regulated in breast cancer; that elevated TMED3 significantly clinicopathologically correlated with ER, PR, HER-2 status, lymph nodes metastases and inferior overall prognosis; and that in vitro cell lines, TMED3 was shown to promote the proliferation, migration and invasion of breast cancer cells. Moreover, miR-188-3p was identified to negatively modulate the TMED3. All the data we collectively obtained are strongly suggestive of its oncogenic role in breast cancer. Our findings are important in that we firstly defined the oncogenic role of TMED3 in breast cancer.
TMED3, also aliased C15orf22 or P26, a contraction of Transmembrane Emp24 Protein Transport Domain Containing 3, whose potential role has been proposed to be implicated in vesicular protein trafficking [
13], mainly in the early secretory pathway. Besides, its physiopathological role remains unknown owing to little study has been emerged regarding TMED3 in the tumor setting, with the exception of three relevant studies performed in prostate cancer [
10], colon cancer [
11] and liver cancer [
12] in chronological order. Analysis of the three previous studies [
10‐
12] is readily indicative of its contradictory roles involved in the malignant behaviors of cancer. Original study of TMED3 on tumor came from prostate cancer by Vainio et al. [
10] reporting that TMED3 whose mRNA was shown to be highly expressed in prostate cancer and high mRNA of TMED3 significantly correlated with AR and ERG oncogene expression, indicating the oncogenic trait of TMED3 that could be used as potential drug target in prostate cancer. Subsequent study of TMED3 carried out in colon cancer by Duquet et al. [
11], however, seems to conflict with it. Duquet et al. [
11] employed subcutaneous xenograft model demonstrating that TMED3 turns out to be able to suppress the metastasis of colon cancer through positive modulation of WNT-TCF pathway, strongly suggesting the tumor-suppressing role in colon cancer. In stark contrast with the observation by Duquet et al., Zheng et al. reported that up-regulated TMED remarkably correlated with aggressive malignant behaviors and poor prognosis in patients with hepatocellular carcinoma and TMED3 was shown to promote cell migration and invasion through IL-11/STAT3 signaling pathway [
12]. In our present study, in terms of expression level, our observation made in breast cancer tissues were fully consistent with what’s been earlier reported in hepatocellular carcinoma [
12] and prostate cancer [
10] where TMED3 was found to be significantly up-regulated in cancer tissues compared with its normal controls. In study of TMED3 on colon cancer by Duquet et al. [
11], in spite of lots of mechanistic data from mice model and cell culture system, the authors failed to analyze the expression patter of TMED3 on clinical tissue level. Moreover, when it comes to correlation with unfavorable overall prognosis and aggressiveness of cancer, our data was totally supported by the study of TMED3 in hepatocellular carcinoma by Zheng et al. [
12]. These discrepancies, whatever in vivo or in vitro, between our study and what has been previous reported regarding TMED3 could be explained by the tissue-specific expression of TMED3, which would be tempting to speculate that TMED3 would be higher in secretory gland tissues compared with other glands, including breast, salivary, pancreas, prostate, gallbladder and thyroid glands etc., mainly based on its proposed biological roles that involved in protein trafficking and secretion. The speculation certainly needs to be confirmed in the following.
Mechanistically, the limited evidence available suggests that TMED3 worked through positive modulation of WNT-TCF [
11] and IL-11/STAT3 [
12] signaling pathways in colon and hepatocellular carcinoma, respectively. Different from the two studies mentioned, we attempted to search out the potential miRNA regulating the TMED3 with the help of miRNA microarray. Five significantly differential miRNAs were eventually screened out. Following the literature review and analysis, miR-188-3p was picked out and identified as negative regulator of TMED3 in breast cancer, among the candidate miRNA available. Although several studies were performed concerning miR-188 in cancers that defined miR-188 as tumor suppressor, miR-188-3p has been little described in cancers, not to mention in breast cancer except from only one recent study performed in colorectal cancer [
14]. it has to be mentioned that miR-188-5p, another paralog of miR-188-3p from the same miR-188, was chorally reported to be tumor suppressing in cancers, including gastric [
15], prostatic [
16] and hepatocellular [
17] carcinoma. Yet, in colorectal cancer, miR-188-3p was turned out to be significantly elevated in cancer tissues relative to normal controls and re-expression of miR-188-3p was therefore shown to promote migration and invasion of colorectal cancer cells in vitro, indicating its oncogenic property in colorectal cancer. By contrast, in our study, miR-188-3p was found to be markedly down-regulated in breast cancer compared with its normal control. In vitro functional analysis of miR-188-3p supported the tumor suppressing role in breast cancer cells, taking the form of suppression of proliferation, migration and invasion in MCF-7 cells. As further verification, Pearson correlation analysis on tissue level corroborated the luciferase reporter assay performed in vitro breast cancer cell lines, confirming the negative modulation of miR-188-3p on TMED3.
Authors’ contributions
Conception and design: BZW; acquisition, analysis and interpretation of data: JP and JZ; tissue specimens collected: XWY and ZRW; pathological consultant: ZSW. Drafting the article or revising it critically for important intellectual content: all authors. Agreement to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved: BZW. All authors read and approved the final manuscript.