Background
Dishevelled-associated activator of morphogenesis 1 (DAAM1) is a member of microfilament-related formins and is involved in cell motility through mediating Wnt signaling pathway [
1‐
3]. In the cytoplasm, DAAM1 exists in an autoinhibited state by intramolecular interaction between its N-terminal GBD and C-terminal DAD domains. When Dishevelled 2 binds to DAAM1, leading to disrupting the interaction between the GBD and DAD that mediates DAAM1 auto-inhibition, DAAM1 will exposure FH1 and FH2 domains to polymerize straight actin filaments [
2,
4]. DAAM1 is essential for Wnt-11/Frizzled-induced activation of RhoA and
Xenopus gastrulation [
2]. DAAM1 directly collaborates to fascin in actin filaments and thus controls the formation of filopodia [
5]. Our previous studies find that active DAAM1 is involved in Wnt5a/Dishevelled 2 and Collagen/Integrin αvβ3 signaling pathways, resulting in the elevation of the migratory and haptotatic ability of breast cancer (BrCa) cells [
3,
6]. However, the genetic mutation status of DAAM1 mRNA and its correlation with pathological characteristics are still unknown in BrCa patients.
Single nucleotide polymorphisms (SNPs) located in untranslated region (UTR) have been reported to be associated with dysregulation of genes expression. A recent global transcriptional network study identifies mutations at somatic expression quantitative trait locus (eQTL) located 5′-UTR of
DAAM1, directly regulating the expression of
DAAM1 mRNA [
7]. Nevertheless, noncoding mutations in the 3′-UTR of
DAAM1 has not been reported. An increasing evidence revealed that functional 3′-UTR SNPs are participated in the progression of multiple cancers [
8‐
11]. Most 3′-UTR of mRNAs function as the target sequences of microRNAs (miRNAs) by base pairing, thereby leading to the degradation of mRNAs and decrease of their translation [
12,
13]. SNPs in the miRNA binding sites in the 3′-UTRs of target genes represent their differential binding affinities for corresponding miRNAs [
14‐
16].
Here, we demonstrate that DAAM1 is highly expressed in lymphnode metastatic BrCa tissues. We also elucidate the functional role of SNP rs79036859 in the miR-208a-5p binding site of DAAM1 3′-UTR and its involvement in BrCa metastasis. Overall these data identify miR-208a-5p/DAAM1 axis as a potential therapeutic target in limiting BrCa metastasis and reducing death from this disease.
Methods
157 BrCa patients were recruited by the First Affiliated Hospital with Nanjing Medical University and Affiliated Cancer Hospital to Nanjing Medical University (NJMU) from 2015 to 2018. All cases had been diagnosed with BrCa by a pathologist on the basis of hematoxylin–eosin (HE) staining. Relevant clinicopathological characteristics record for each case were collected by review of patients’ medical files. Ethical approval for the study was obtained from the Clinical Research Ethics Committee, NJMU. Pathologic staging was determined by a pathologist based on AJCC Cancer Staging Manual 8th classification criteria. All the participants voluntarily joined this study with informed contents.
Immunohistochemistry (IHC)
A total of 46 BrCa sections were deparaffinised at 55 °C for 30 min. The sections were then washed with xylene for three 5-min. The sections were rehydrated by successive washes in 100, 90 and 70% graded ethanol. Hydrogen peroxidase (0.3%, ZSGB-Bio, Beijing, China) was used to block endogenous peroxidase activity for 20 min. The primary anti-DAAM1 (1:100 dilution, Cat. 14876-1-AP, ProteinTech, Wuhan, China) antibody and DAB and hematoxylin counterstain (ZSGB-Bio) were used to visualize its expression. The percentage of positively stained cells was scored as 0–4: 0 (< 5%), 1 (6–25%), 2 (26–50%), 3 (51–75%) and 4 (> 75%). The staining intensity was scored as 0–3: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong). The immunoreactivity score (IRS) equals to the percentages of positive cells multiplied with staining intensity. Immunostained sections were scanned by a microscope (Olympus Corporation, Tokyo, Japan).
Selection of SNPs and TaqMan genotyping
A next-generation sequencing of metastatic BrCa tissues (data not published) revealed some SNPs, including rs79036859 and rs45476291, locating in the 3′-UTR of
DAAM1. After a review of dbSNP database (
https://www.ncbi.nlm.nih.gov/snp) and PolymiRTS Database 3.0 (
http://compbio.uthsc.edu/miRSNP), we selected the candidate SNP (rs79036859) suggested as a transcriptional regulation factor for the 3′-UTR of
DAAM1. SNP genotyping was conducted by allelic discrimination using the TaqMan SNP Genotyping Assays according to the manufacturer’s instructions (Applied Biosystems). Specific primers (TATCTCCTGAAAGAGATAAGA, GTTTTTCCAACAACTCCAGT) and FAM/VIC-labeled TaqMan probes (FAM-labeled CAAACAAACAAAAAA
AGCTTGCAAAATATTTT, VIC-labeled CAAACAAACAAAAAA
GGCTTGCAAAATATTTT) were designed and supplied by Synbio Technologies (Soochow, China). The PCR conditions were as follows: initiation at 98 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 30 s and annealing/extension at 60 °C for 60 s. PCR application was undergoing in a StepOnePlus Real-Time PCR System (Applied Biosystems).
Cell culture and transfection
MCF-10A, MDA-MB-231, MCF-7, and COS-7 cell lines were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). MCF-7, MDA-MB-231, and COS-7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, high glucose) (Hyclone, Thermo Scientific, Waltham, MA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Hyclone) at 37 °C with 5% CO2. MCF-10A cells were cultured in DMEM/F12 media supplemented with 5% (v/v) horse serum, 20 ng/mL human EGF, 10 μg/mL insulin, 0.5 μg/mL hydrocortisone, penicillin, streptomycin and 100 ng/mL cholera toxin (Sigma-Aldrich, St. Louis, MO).
For subsequent assays, cells were transfected with miR-208a-5p mimic, miR-208a-5p inhibitor (an anti-sense of miR-208a-5p), or miRNA mimic control, which is synthesized in RiboBio Co., Ltd. (Guangzhou, China), using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.
Western blotting analysis and pulldown assays
Cells were placed in 35-mm dishes (6 × 105 cells/dish) and transfected with synthesized miR-208a-5p mimic, miR-208a-5p inhibitor (anti-sense of miR-208a-5p), or miRNA mimic control. 72 h after transfection, all cells were harvested the proteins with lysis buffer. SDS–polyacrylamide gel electrophoresis and Western blotting analysis were performed as standard protocols. The primary antibodies for DAAM1 (1:1000 dilution, Cat. 14876-1-AP, ProteinTech), RhoA (1:1000 dilution, Cat. 10749-1-AP, ProteinTec), β-actin (1:5000 dilution, Cat. 60008-1-Ig, ProteinTec) were used. DAAM1 protein levels were normalized to β-actin for each sample.
To detect the active level of DAAM1, we employed GST-RhoA beads as bait. The activate level of DAAM1 was detected by the Pulldown assays and subsequently immunoblotted with anti-DAAM1 antibody (Cat. 14876-1-AP, ProteinTech) [
6]. SDS-PAGE and Western blotting were performed using the above methods.
Dual-luciferase activity assay
The 3′-UTR of DAAM1 which contained the putative target site of miR-208a-5p was synthesized by Integrated Biotech Solutions Co., Ltd (Shanghai, China) and ligated into pGL3 construct (Promega, Madison, WI). The constructs pGL3-DAAM1-3′-UTR-WT or pGL3-DAAM1-3′-UTR-mutant (200 ng) and pRL-TK (80 ng, Promega) were co-transfected with 60 pmol miR-208a-5p mimic or miRNA mimic control by Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, Dual-Luciferase Reporter Assay System (Promega) was performed to measure luciferase activity.
Boyden chamber assays
Migratory ability of cancer cells was tested in a modified Boyden chamber (Cat. 3422, Costar, Corning, NY). The detail protocol was described as previously [
6].
Immunofluorescence and actin cytoskeleton staining
Cells were placed on glass cover-slides and subjected to actin cytoskeleton staining. The detail protocol was described as previously [
17]. The images were captured by a laser scanning confocal microscope (Zeiss LSM710, Oberkochen, Germany).
Quantitative real-time PCR
MiRNA of BrCa cells and tissues were extracted by using mirVana™ miRNA isolation kit (Ambion, Austin, TX). The primers for miRNA reverse transcription were synthesized in RiboBio Co., Ltd. (Guangzhou, China) called Bulge-Loop™ miRNA qRT-PCR Primer Set as previously described [
17]. Primers used for
DAAM1 amplification were
GAPDH: 5′-TGAACGGGAAGCTCACTGG-3′ (sense) and 5′-TCCACCACCCTGTTGCTGTA-3′ (antisense);
DAAM1: 5′-AAATTGAAACGGAATCGCAAAC-3′ (sense) and 5′-GCAAGGCAGTGTAATGAAACG-3′ (antisense). SYBR Green (SYBR
® Premix Ex Taq™ II, TaKaRa, Dalian, China) was used to label the amplified genes. The 2
−ΔΔCt method was used for miR-208a-5p or
DAAM1 expression analysis.
RhoA GTPase activation assays (G-LISA small GTPase activation assays)
Total protein lysates extracted from BrCa cells were turned to measure RhoA activity by using RhoA GTPase activation assays (Cat. BK121, Cytoskeleton Inc., Denver, CO). RhoA activation was described as previously [
6].
Kaplan–Meier plotter analysis
DAAM1 (Affy ID: 216060_s_at) mRNA expression data and survival information (progression free survival, overall survival, post progression survival and distant metastasis free survival) were collected from Kaplan–Meier plotter (
http://kmplot.com). Then, the prognostic significance of
DAAM1 mRNA was analyzed in BrCa. Kaplan–Meier survival plots were generated with survival curves compared by log-rank test.
Statistical analysis
All statistical analyses were calculated using SPSS 25.0 software (Chicago, IL). Most of the data were analyzed by Student’s t-test or one-way ANOVA followed by Dunnett’s multiple posthoc tests. All data are presented as means ± SDs of five independent experiments if not noted. The associations between DAAM1 mRNA expression levels, genotypes distribution of DAAM1 SNP, along with miR-208a-5p expression status and clinicopathological characteristics were performed using Pearson’s Chi squared test. The correlations between DAAM1 mRNA expression and miR-208a-5p expression or between DAAM1 mRNA level and protein level were assessed by Spearman’s correlation analysis.
Discussion
The first finding in this study is that the high level of DAAM1 expresses in lymphnode metastatic BrCa tissues. These clinicopathological and biochemical characteristics imply DAAM1 is likely to function as a metastatic promoter in BrCa, which is consistent with our previous findings verified on the cellular levels [
3,
6,
17].
Protein is translated from RNA, which is strictly conformed to the genetic central dogma [
19]. The mRNA modification exists extensively in eukaryotic cells and leads to the diversity of protein expression. Zhang et al. has analyzed the big data from the Cancer Genome Atlas (TCGA) and identified novel mutations at eQTL situated in
DAAM1 promotor (− 91 bp), which are able to increase
DAAM1 mRNA expression levels [
7]. In this study, we focus on the polymorphism of 3′-UTR of
DAAM1 gene. Patients with the G (GG/GA) allele genotypes have the prominently higher risk of metastasis in BrCa than AA genotype of SNP rs79036859. These results demonstrate that G genotype of rs79036859 in
DAAM1 3′-UTR enhances the risk of metastasis in BrCa.
The most common post-transcriptional regulation is that miRNAs bind to 3′-UTR of target gene and induce the degradation of whole mRNAs. We find that miR-208a-5p targets to the 3′-UTR of
DAAM1 gene. Wang et al. reported the downregulation of miR-208a/miR-208b significantly suppresses the harmful effect of extracellular vesicles on hypoxia/reoxygenation damage in cardiac myoblasts [
20]. The regulatory feedback loop of miR-208a-SOX2/β-catenin-LIN28-let-7a-DICER1 mediates the renovation of BrCa stem cells [
21]. MiR-208a is capable of promoting gastric cancer progression by suppressing SFRP1 and downregulating MEG3 [
22]. Expression analysis demonstrate that miR-208a-5p is significantly suppressed in lymphnode metastatic tumor samples than that in non-lymphnode metastatic tumor samples, indicating the potential role of miR-208a-5p on anti-tumor metastasis via DAAM1 signaling.
DAAM1, an element of cellular actin cytoskeleton, transduces Wnt/Dishevelled signaling to RhoA and then assemble microfilaments in mammalian cells [
2,
3,
23]. Actin polymerization is the key step of the assemblage of microfilament in the process of cancer metastasis [
24]. The functional and dynamic microfilaments are generally provided a possible route for the degradation of extracellular matrix and available for cancer cells escaping from the primary lesion [
24,
25]. Our previous studies find that active DAAM1 is elevated by the treatment of Wnt5a or type IV collagen and participates in the tumor cell migration and haptotaxis [
3,
6]. Here, we demonstrate that miR-208a-5p overexpression decreases DAAM1 mRNA and protein expression levels, resulting in the decrease of the migratory ability of BrCa cells. We also find that miR-208a-5p downregulates the RhoA activity, disrupts the formation of microfilaments of BrCa cells. We summarize the potential DAAM1 signaling pathways in Fig.
5e. Thus, miR-208a-5p/DAAM1/RhoA axis is the potential therapeutic target in regulating microfilament assemblage and cell migration.
Authors’ contributions
YZ and TY conceived the study and participated in the study design, performance, coordination and manuscript writing. JM, TY, YH, TX, LH, SS, YC, ZW, XJ, and BX carried out the assays and analysis. YZ, TY and JM revised the manuscript. All authors read and approved the final manuscript.
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