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. 2018 May 4;293(18):6693-6706.
doi: 10.1074/jbc.RA118.001689. Epub 2018 Mar 16.

Wnt7a activates canonical Wnt signaling, promotes bladder cancer cell invasion, and is suppressed by miR-370-3p

Xiaojing Huang  1 , Hongwen Zhu  2 , Zemin Gao  1 , Junzun Li  1 , Junlong Zhuang  3 , Yu Dong  2   4 , Bing Shen  5 , Meiqian Li  1 , Hu Zhou  2 , Hongqian Guo  6 , Ruimin Huang  7 , Jun Yan  8   9
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"VSports注册入口" Wnt7a activates canonical Wnt signaling, promotes bladder cancer cell invasion, and is suppressed by miR-370-3p

V体育平台登录 - Xiaojing Huang et al. J Biol Chem. .

Abstract

Once urinary bladder cancer (UBC) develops into muscle-invasive bladder cancer, its mortality rate increases dramatically. However, the molecular mechanisms of UBC invasion and metastasis remain largely unknown. Herein, using 5637 UBC cells, we generated two sublines with low (5637 NMI) and high (5637 HMI) invasive capabilities. Mass spectrum analyses revealed that the Wnt family protein Wnt7a is more highly expressed in 5637 HMI cells than in 5637 NMI cells VSports手机版. We also found that increased Wnt7a expression is associated with UBC metastasis and predicted worse clinical outcome in UBC patients. Wnt7a depletion in 5637 HMI and T24 cells reduced UBC cell invasion and decreased levels of active β-catenin and its downstream target genes involved in the epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM) degradation. Consistently, treating 5637 NMI and J82 cells with recombinant Wnt7a induced cell invasion, EMT, and expression of ECM degradation-associated genes. Moreover, TOP/FOPflash luciferase assays indicated that Wnt7a activated canonical β-catenin signaling in UBC cells, and increased Wnt7a expression was associated with nuclear β-catenin in UBC samples. Wnt7a ablation suppressed matrix metalloproteinase 10 (MMP10) expression, and Wnt7a overexpression increased MMP10 promoter activity through two TCF/LEF promoter sites, confirming that Wnt7a-mediated MMP10 activation is mediated by the canonical Wnt/β-catenin pathway. Of note, the microRNA miR-370-3p directly repressed Wnt7a expression and thereby suppressed UBC cell invasion, which was partially restored by Wnt7a overexpression. Our results have identified an miR-370-3p/Wnt7a axis that controls UBC invasion through canonical Wnt/β-catenin signaling, which may offer prognostic and therapeutic opportunities. .

Keywords: MMP10; Wnt signaling; Wnt7a; bladder cancer; cancer biology; invasion; mass spectrometry (MS); metalloprotease; miR-370-3p; microRNA (miRNA) V体育安卓版. .

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"V体育官网" Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
Establishment and characterization of 5637 NMI and HMI cells. A, schematic illustration of the establishment of low-invasive (5637 NMI) and high-invasive (5637 HMI) sublines from primary 5637 cell line. B, transwell assay showed the invasive capacities of NMI 5637 and 5637 HMI cells. The upper photographs are representative fields of invaded cells 15 h after seeding. Scale bar, 100 μm. The lower graphs indicate the invaded cell number. C, wound healing assay show the migration capacities of 5637 NMI and 5637 HMI cells. The upper photographs are representative fields of wound closure at 0, 24, and 48 h, respectively. The lower graphs indicate the relative percent of wound closure at 24 and 48 h. Scale bar, 200 μm. The assays were performed in three independent biological replicates. D and E, invasive capacities of 5637 parental cells and derivatives in 3D Matrigel. 5637 parental cells, NMI, and HMI cells were formed spheroid in ultra-low 6-well plates and embedded in Matrigel for culture. The individual sphere in each group was monitored within 12 h and photographed (D), and the invasion area change fold was quantified (E) at the indicated time points. n = 16 for each group. Scale bar, 100 μm. **, p < 0.01; ***, p < 0.001; n.s., not significant.
Figure 2.
Figure 2.
Mass spectrum analysis of differentially expressed proteins in 5637 NMI and HMI cells. A, experimental workflow. 5637 HMI samples were labeled with 126, 127, and 128; 5637 NMI samples were labeled with 129, 130, and 131. The labeled samples were pooled and subjected to fractionation. Each fraction was analyzed on an Orbitrap Elite MS. B, volcano plot illustrating proteins with different abundances in 5637 HMI and NMI samples. It was displayed by −log10 (p value) versus log2 of the relative protein abundance of 5637 HMI to NMI cells. Orange points represent proteins with changes in abundance of greater than ±1.2-fold and p < 0.05. C, protein association network analysis of regulated proteins by STRING. Orange indicates up-regulation, and blue indicates down-regulation. Proteins were represented by log2 of the relative abundance of 5637 HMI to NMI cells. D and E, validation of mass spectrum data in 5637 NMI and HMI cells by qRT-PCR (D) and Western blotting. E, active β-catenin was also examined in 5637 NMI and HMI cells. GAPDH was used as loading control. F, quantification of Western blotting data in E. *, p < 0.05; ***, p < 0.001; n.s., not significant.
Figure 3.
Figure 3.
Wnt7a overexpression is associated with canonical Wnt/β-catenin pathway and predicts clinical outcome. A, qRT-PCR showed Wnt7a mRNA expression level in matched clinical UBCs and corresponding normal tissues (n = 41). B, Western blotting results demonstrated the overexpression of Wnt7a in human UBC samples (n = 20). Wnt7a protein levels by quantitation of density of protein bands from Western blotting in UBCs (C) was relative to the matched normal tissues (n = 20) (B). N, nontumor; T, tumor. D, analysis of Wnt7a mRNA expression level fragments per kilobase million (FPKM) in normal bladder (n = 19), UBC without lymph node metastatic lesion (N0; n = 237), and UBC with lymph node metastatic lesions (≥N1; n = 128), which is derived from TCGA provisional database. E, analysis of Wnt7a mRNA expression level (FPKM) in normal bladder (n = 19), UBC without metastatic lesion (M0; n = 195), and UBC with distal metastatic lesions (M1; n = 11), which is derived from TCGA provisional database. F and G, correlation of Wnt7a mRNA expression with overall survival in TCGA provisional dataset (F) and one TCGA (6) dataset (G). H, representative IHC staining of Wnt7a in normal urothelial cells, and different staining intensities (weak, moderate, and strong) of Wnt7a in UBC samples. I, Kaplan-Meier plot of overall survival of 45 UBC patients, stratified by Wnt7a protein level.
Figure 4.
Figure 4.
Wnt7a deficiency decreased the invasiveness of 5637 HMI and T24 cells. A, expression level of Wnt7a and constitutively active β-catenin in UBC cell lines. B and C, Wnt7a mRNA expression levels in 5637 HMI (B) and T24 cells (C) transfected with control (siCTL) and two Wnt7a siRNAs (siWnt7a-1 and siWnt7a-2) by qRT-PCR. D, Western blotting showed EMT and invasiveness-associated protein expression in 5637 HMI and T24 cells transfected with siCTL, siWnt7a-1, and -2. E, transwell assay showed the invasive capacities of 5637 HMI and T24 cells followed by siCTL and Wnt7a siRNA treatment. The upper photograph are representative fields of staining invaded cells 16 h after seeding. Scale bar, 100 μm. The lower graphs indicated the counting number of invaded cells. The assays were performed in three independent biological replicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Figure 5.
Figure 5.
Wnt7a treatment induced invasive ability of 5637 NMI and J82. A, transwell assay showed the invasive capacities of 5637 NMI and J82 following treatment with recombinant Wnt7a protein (rWnt7a; 100 ng/ml) or vehicle (Veh) for 24 h. Photographs are representative fields of invaded cells 16 h after seeding. Scale bar, 100 μm. The graphs indicate the counting number of invaded cells in 5637 NMI and J82 cells. The assays were performed in three independent biological replicates. **, p < 0.01; ***, p < 0.001. B, Western blotting showed EMT and invasiveness associated protein expression in vehicle and rWnt7a (100 ng/ml) for 24 h in 5637 NMI and J82 cells.
Figure 6.
Figure 6.
Wnt7a overexpression promoted UBC cell metastasis in vivo. A, ectopic expression of Wnt7a in J82 cells activated β-catenin pathway and induced MMP1 and MMP10, detected by Western blotting. B, photograph of lung tissues from vehicle control (pLuci) and experimental group (pWnt7a). Arrows indicated the metastatic lesions. C, histological analysis of lung metastatic lesions from pLuci and pWnt7a groups. Scale bar, 1 mm. Below are two enlarged fields from the sections above. Scale bar, 50 μm. D, quantification of lung metastatic nodules in pLuci (n = 4) and pWnt7a (n = 4) groups. *, p < 0.05.
Figure 7.
Figure 7.
Wnt7a/β-catenin regulated MMP10 expression in UBC cells. A and B, TOP/FOPflash reporter assay to measure WNT/β-catenin signaling in 5637 NMI (A) and J82 (B) cells with indicated treatment. The treatment with 10 mm LiCl and transfection of (S33Y) β-catenin plasmid served as positive controls, respectively. C, IHC staining showed Wnt7a and β-catenin expression in clinical UBC samples (n = 25). The representative photos of Wnt7a and β-catenin staining data in two cases of UBC samples are presented. Scale bar, 20 μm. The correlation between Wnt7a and β-catenin expression in UBC samples are shown below. D, qRT-PCR assay showed MMP10 and Wnt7a mRNA expression levels in Wnt7a knockdown 5637 HMI cells. E, schematic illustration of the promoter of human MMP10 gene. It showed wildtype (WT) contained two consensus TCF/LEF-binding sites, whereas mutants 1, 2, and 1/2 had indicated the mutations in TCF4-1 or/and TCF4-2 sites. F, luciferase assay showed relative luciferase activities of WT, mutant 1, mutant 2, and mutant 1/2 MMP10 promoters within the rWnt7a protein (100 ng/ml) treatment in 5637 NMI cells for 24 h. All the luciferase activity was calculated in triplicate and repeated three times. **, p < 0.01; ***, p < 0.001; n.s., not significant. G, relative expression level of MMP10 in a cohort of human UBCs and adjacent normal bladder tissues (n = 41). H, association of Wnt7a and MMP10 expression levels in a cohort of human UBC samples (n = 41).
Figure 8.
Figure 8.
miR-370-3p reduced Wnt7a to regulate UBC cell invasion. A, qRT-PCR showed the expression levels of two miRNAs: miR-370-3p and miR-195-5p in 5637 NMI and HMI cells. B, Western blotting showed protein expression of Wnt7a, active β-catenin, total β-catenin, MMP1, and MMP10 in 5637 HMI cells transfected with mimic miR-370-3p and control mimic. C, quantification of Western blotting is shown. D, schematic representation of the putative miR-370-3p-binding site in WT Wnt7a 3′UTR (WT). Wnt7a (WT) and Wnt7a (mutant) indicated the psiCHECK2 plasmid containing the Wnt7a 3′UTR WT sequence and mutated sequence. HMI 5637 cells were co-transfected with reporter vector (WT or mutant Wnt7a 3′UTR) and mimic miR-370-3p or control (CTL) as indicated. The relative luciferase activity of WT and mutant Wnt7a 3′UTR was measured 24 h after transfection. E, transwell assay showed invasive capacities of 5637 HMI cells with indicated treatment. The representative fields of invaded cells 16 h after seeding are shown. Scale bar, 100 μm. The graphs indicated the counting number of invaded cells in 5637 HMI cells with the indicated treatment. All assays were performed in three independent biological replicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant. F, relative expression level of miR-370-3p in a cohort of human UBC samples (n = 41). G, negative association of Wnt7a protein level and miR-370-3p level in the same human UBC samples (n = 20). H, hypothetical working model of the miR-370-3p/Wnt7a axis activating canonical Wnt/β-catenin pathway and inducing matrix metalloproteinases (MMPs) to promote UBC cell invasion.
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