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β-catenin pathway by repressing ITCH expression
Gene 710 (2019) 39–47
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Research paper
miR-10b promoted melanoma progression through Wnt/β-catenin pathway by repressing ITCH expression
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Shengqiang Wang , Yi Wu, Yan Xu, Xianjun Tang General Department, Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital, No. 181 Han Yu Road, Shapingba District, Chongqing 400030, China
A R T I C LE I N FO
A B S T R A C T
Keywords: miR-10b Melanoma Proliferation Invasion ITCH Wnt/β-catenin pathway
Dysregulation of microRNAs (miRNAs) have been reported to contribute to malignant progression in melanoma. However, the roles and mechanisms of several miRNAs in melanoma remain poorly understood. In our study, we showed that miR-10b was significantly up-regulated in melanoma tissues and cell lines, and was associated with overall survival of melanoma patients. Inhibition of miR-10b dramatically suppressed melanoma cell proliferation, migration and invasion in vitro and inhibited tumor growth in vivo. Moreover, we defined ITCH as a direct and functional downstream target of miR-10b, and showed that there was an inverse correlation between the expression of ITCH and miR-10b on melanoma tissues. Down-regulation of ITCH partially attenuated the inhibitory effects of miR-10b inhibition on melanoma cell proliferation, migration and invasion. Furthermore， we found that miR-10b exerted its effects on melanoma by regulating the Wnt/β-catenin pathway. Taken together, our results demonstrated that miR-10b was an important epigenetic modifier, promoting melanoma progression through regulating ITCH/Wnt/β-catenin pathway. These results offer a new strategy for epigenetic cancer therapy.
1. Introduction Malignant melanoma originating from melanocytes is an aggressive form of skin cancer with high patient morbidity and mortality, which is a high degree of malignancy and poor prognosis of tumor (Isola et al., 2016; Miller et al., 2016; Siegel et al., 2016). Melanoma is a major health problem in many countries and accounts for approximately 50,000 deaths annually worldwide (Slipicevic and Herlyn, 2012). Global incidence of melanoma continues to increase, in contrast to the stable or declining trends for most cancer types (Siegel et al., 2014). Despite recent advances in targeted therapy and immunotherapies, understanding of melanoma pathogenesis is still limited. Therefore, understanding the molecular pathogenesis of melanoma development is urgent. MicroRNAs (miRNAs) are a class of single-stranded, small noncoding RNAs that play important roles in negatively regulating gene expression either by inducing translational silencing or by causing mRNA degradation (Bartel, 2009). They have been implicated in the oncogenesis and progression of several cancers (Olive et al., 2010; Tili et al., 2013), and involved in cancer-relevant processes, such as
proliferation (F. Ma et al., 2017), invasion (Lu et al., 2017) and apoptosis (Li et al., 2017). Studies have shown that several miRNAs are implicated in melanoma progression. For example, miR-622 was downregulated in melanoma and miR-622 re-expression inhibited clonogenicity, proliferation, and migration in melanoma (Dietrich et al., 2018). While miR-338-5p promotes melanoma cells growth and metastasis via targeting CD82 (Long et al., 2018). miR-10b expression was up-regulated in metastatic CRC tissues and cell lines, and inhibition of miR-10b prevented cancer cell metastasis and growth by inducing cellcycle arrest and apoptosis (Xie et al., 2019); miR-10b promotes invasion by targeting KLF4 in osteosarcoma cells (Wang et al., 2016). Recent studies have reported that up-regulation of serum miR-10b is associated with poor prognosis in patients with melanoma, and suggested miR-10b is a new prognostic microRNA for melanoma and that there could be a place for miRNA analysis in stratifying melanoma for therapy (Bai et al., 2017; Saldanha et al., 2016). However, the function and molecular mechanisms of miR-10b in melanoma pathogenesis has not been fully understood. In our study, we clearly demonstrated that miR-10b functioned as an oncogenic miRNA. We demonstrated that knockdown of miR-10b
Abbreviations: miRNAs, microRNAs; MTT, 3-[4, 5-dimthylthiazol-2yl]-2,5-diphenyltetrazolium bromide ⁎ Corresponding author at: General Department, Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital, Chongqing 400030, China. E-mail address: [email protected] (S. Wang). https://doi.org/10.1016/j.gene.2019.05.043 Received 9 January 2019; Received in revised form 3 May 2019; Accepted 22 May 2019 Available online 23 May 2019 0378-1119/ © 2019 Published by Elsevier B.V.
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Fig. 1. miR-10b was up-regulated in melanoma cell lines and tissues. (A) miR-10b expression was detected in primary melanocytes and five melanoma cell lines by qRT-PCR. (B) qRT-PCR analysis of miR-10b expression in 30 melanoma specimens and 22 non-neoplastic skin tissues. (C) The Kaplan-Meier survival analysis evaluated the prognostic value of miR-10b for melanoma patients. ⁎P < 0.05.
specific primer sequence of GAPDH is as followed: Sense: 5′- GACTCA TGACCACAGTCCATGC-3′ and antisense: 3′- AGAGGCAGGGATGATGT TCTG-5′. The specific primer sequence of ITCH is as followed: Sense: 5′-CTGCCTGTTGCACATCTTGT-3′ and antisense: 5′-TGTCCAATTTT ACAAGATGT-3′.
dramatically inhibited melanoma cell proliferation, migration and invasion in vitro and suppressed tumor growth in vivo through regulating Wnt/β-catenin pathway via repression ITCH expression. Thus, our findings provide valuable clues toward understanding the mechanisms of melanoma pathogenesis and may contribute to the development of novel therapeutic strategies for melanoma in the future.
2.4. Western blot 2. Materials and methods Total proteins were extracted from the frozen tissues and cells and protein concentrations were determined using the BCA protein assay kit (Beyotime Institute of Biotechnology, Jiangsu, China). Protein were electrophoresed in 10% sodium dodecyl sulfate–polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk and incubated with primary antibodies as follows: ITCH (Abcam, Cambridge, UK) and β-actin (Sigma-Aldrich, USA), β-catenin, TCF4, c-Myc and cyclin D1 both purchased from Proteintech (Wuhan, China), followed by incubation with appropriate correlated HRP-conjugated secondary antibody. Protein detection was performed by enhanced chemiluminescence (ECL kit, Santa Cruz Biotechnology).
2.1. Ethics statement and human tissue samples All experimental procedures were approved by the Chongqing University Cancer Hospital. 78 melanoma tissues were collected from patients with melanoma at Chongqing University Cancer Hospital, and 30 non-neoplastic skin specimens from patients with chronic inflammatory skin diseases from 2013 to 2015, which were approved by the Institutional Review Board of Chongqing University Cancer Hospital (SYXK-2017-0002). 2.2. Cell culture and transfection Melanoma cell lines (A375, SK-MEL-1, SK-MEL-28, WM451) and human primary melanocytes were obtained from the American Type Culture Collection, and cultured in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS. miR-10b mimic, miR-10b inhibitor, or their negative controls were transfected into A375 and SK-MEL-1 cells by using Lipofectamine 2000 (Invitrogen). ITCH-silencing oligonucleotides (siITCH) or the respective controls (RiboBio, Guangzhou, China) were transfected into the cells using Lipofectamine 2000 (Invitrogen).
2.5. Cell proliferation
2.3. qRT-PCR
Cells transfected with miR-10b inhibitor or NC inhibitor were seeded in a 6-well plate at a density of 5 × 105 cells/well. After starvation for 24 h in 1% FBS medium, the cells were removed using 200 μL tips while the remaining cells continued to grow in a monolayer. Cells were then washed and cultured in DMEM with 10% FBS for 24 h, the cell growth was observed using a Nikon TS100 microscope. The invasive ability of cells was determined using the transwell assay. The transwell membrane of the upper chamber coated with Matrigel (BD Biosciences Discovery Labware, Woburn, MA, USA) was used. The chambers were rehydrated in incubator with serum-free medium for 2 h. The top chambers were subsequently hydrated with 200 μL of cell suspension (containing 1 × 105 cells). The bottom chambers were added medium containing 10% FBS. After 24 h, the cells on the lower surface of the membrane were fixed, stained, washed and counted under a microscope (Chang et al., 2014).
A375 and SK-MEL-1 cells were seeded at 2000 cells per well in 96well plates after transfection. MTT (3-[4, 5-dimthylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay was used to measure cell viability, and the absorbance was determined using a spectrophotometric plate reader at 450 nm (Bio-Rad 680, USA). 2.6. Cell migration and invasion
Total RNA was extracted from cell lines and frozen tissues using Trizol reagent (Invitrogen). cDNA synthesis was performed using PrimeScript reverse transcriptase reagent kit (Takara, Dalian, China) according to the manufacturer's instructions. qRT-PCR analyses were performed using SYBR Green master mix Universal RT for miR-10b and FastStart Universal SYBR Green Master kit (Roche Life Sciences, Switzerland) for ITCH. U6 or GAPDH was measured as an internal control for miRNA or mRNA. Relative changes in gene and miRNA expression were determined using the 2−ΔΔCt method. The primer sequence of U6 is as followed: Sense: 5′-CTTCGGCAG CACATATACT-3′ and antisense: 5′-AAAATATGGAACGCTTCACG-3′. The specific primer sequence of miR-10b is as followed: Sense: 5′-TACCCTGTAGAA CCGA ATTTGTG-3′ and antisense: 5′-CAGTGCGTGTCGTGGAGT-3′. The 40
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Fig. 2. Knockdown of miR-10b inhibited melanoma cell proliferation, migration and invasion. (A) Relative expression of miR-10b in A375 and SK-MEL-1 cells transfected with miR-10b inhibitor or NC inhibitor was detected. (B) Cell viability was determined by measuring MTT absorbance at the indicated times after transfection. (C) Representative images and quantification of the wound healing assay of A375 and SK-MEL-1 cells transfected with miR-10b inhibitor or NC inhibitor. (D) Representative images and quantification of the Transwell invasion assay of A375 and SK-MEL-1 cells transfected with miR-10b inhibitor or NC inhibitor. ⁎P < 0.05.
2.7. In vivo tumor growth model
shortest and longest tumor diameter, respectively). Mice were sacrificed after 25 days. All tumors were excised, weighed, harvested, fixed, and embedded. All animals received humane care per the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” issued by the National Institutes of Health.
A375 cells (5 × 106 cells/mice) transfected with antagomiR-10b or antagomiR-NC cells were subcutaneously injected into the nude mice. Tumor size was determined every 5 days by measuring tumor diameter using Vernier calipers, and tumor weight was measured in grams after dissection. Tumor volume (mm3) = d2 × D/2 (d and D represent the 41
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Fig. 3. Knockdown of miR-10b inhibited tumor growth in vivo. (A) Images of the excised xenograft tumors. A375 cells transfected with antagomiR-10b or antagomiRNC were subcutaneously injected into the flank of nude mice. (B) The tumor growth curves of two groups in nude mice. Tumor growth was monitored for up to 25 days. (C) Tumor weight was calculated. (D) The expression of miR-10b was measures in tumor tissues using qRT-PCR. (E) Cell proliferation marker Ki67 protein level was measured by immunohistochemical staining. ⁎P < 0.05.
IL, USA) was used in this study.
2.8. Luciferase reporter assay Oligonucleotides containing the WT or mutant target site of miR10b in the 3′ UTR of ITCH was ligated into the pGL3-basic vector (Promega, Madison, WI). HEK293T cells were co-transfected with the ITCH vector or control, miR-10b mimic or NC mimic with Lipofectamine 2000 (Invitrogen). Topflash luciferase reporter plasmid containing TCF wild-type copies of the TCF/LEF binding sites and Fopflash luciferase reporter plasmid containing mutated binding sites were purchased from Upstate Biotechnology (Lake Placid, NY, USA), and transfected into miR-10b inhibitor-cells. After 48 h, the DualLuciferase Reporter Assay System (Promega) was used to detect the luciferase activity.
2.10. Statistical analysis Data are presented as mean ± SD, and all statistical analyses were performed using Prism 5.0 software (GraphPad, San Diego, CA, USA). Each experiment was repeated at least three times, and differences between groups were compared using Student's t-test or ANOVA. Pearson's correlation was used for analyzing the correlation between miR-10b and ITCH levels. p < 0.05 were defined as statistically significant.
2.9. Immunohistochemical staining
3. Results
The cell proliferation marker ki67 protein level in the sections from the paraffin-embedded subcutaneous tumor tissue was detected using immunohistochemical staining (Li et al., 2009). The degree of immunostaining was reviewed and scored independently by two observers based on the proportion of positively stained tumor cells and intensity of staining. Tumor cell proportion was scored as follows: 0 (no positive tumor cells), 1 (< 10% positive tumor cells), 2 (10–35% positive tumor cells), 3 (35–70% positive tumor cells), and 4 (> 70% positive tumor cells). Staining intensity was graded according to the following criteria: 0 (no staining), 1 (weak staining = light yellow), 2 (moderate staining = yellow brown), and 3 (strong staining = brown). Staining index was calculated as the product of staining intensity score and the proportion of positive tumor cells. ki67 antibody (Proteintech, Chicago,
3.1. Up-regulation of miR-10b in melanoma cell lines and tissues To validate the importance of miR-10b in melanoma, the expression of miR-10b was analyzed in melanoma cell lines and tissues. As shown in Fig. 1A, miR-10b was significantly higher in five human melanoma cells than primary melanocytes. Additionally, miR-10b expression was evaluated in 30 primary melanoma tissues and 22 non-neoplastic skin specimens, and qRT-PCR results showed that miR-10b expression was up-regulated in melanoma tissues (Fig. 1B). Moreover, we revealed that the high miR-10b expression was significantly associated with shorter overall in melanoma patients (Fig. 1C). These results indicated that miR-10b was up-regulated in melanoma and could do duty for a potential prognostic biomarker in melanoma patients. 42
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Fig. 4. miR-10b inhibited ITCH expression by targeting its 3′UTR. (A) Schematic representation of the predicted binding sites for miR-10b in the 3′UTR of ITCH and the site mutagenesis design for the reporter assay. Complementarities between miR-10b and the target sites are shown. (B) Relative luciferase activity of ITCH 3′ UTR in the HEK293 cells co-transfected with the indicated reporters and miR-10b mimic or NC mimic. (C) ITCH mRNA level was measured by qRT-PCR analysis in A375 and SK-MEL-1 cells transfected with miR-10b mimic or NC mimic. (D) ITCH protein level was measured by western blot assay in A375 and SK-MEL-1 cells transfected with miR-10b mimic or NC mimic. (E) qRT-PCR analysis of ITCH expression in 30 melanoma specimens and 22 non-neoplastic skin tissues. (F) Inverse relationship between miR-10b and ITCH expression in melanoma tissues. ⁎P < 0.05.
10b was significantly decreased in the antagomiR-10b group compared with antagomiR-NC group (Fig. 3D). Moreover, immunhistochemical staining of ki67 revealed that knockdown of miR-10b inhibited tumor cell proliferation (Fig. 3E). All data demonstrated that miR-10b inhibitor suppressed tumor growth in vivo, suggesting that miR-10b functions to positively regulate tumor growth in melanoma.
3.2. Knockdown of miR-10b inhibited melanoma cell proliferation, migration and invasion in vitro To investigate the biological function of miR-10b in melanoma, A375 and SK-MEL-1 cells were transiently transfected with miR-10b inhibitor or NC inhibitor (Fig. 2A), and then a series of biological experiments were performed in vitro. The MTT assay indicated that cell viability of cells transfected with miR-10b inhibitor was obviously reduced compared to cells transfected with NC inhibitor (Fig. 2B). The wound healing assay showed that the migratory ability of melanoma cells transfected with miR-10b inhibitor was lower than that of cells transfected with NC inhibitor (Fig. 2C). In addition, the Transwell invasion assay revealed that the invasive ability of cells transfected with miR-10b inhibitor was significantly suppressed compared to cells transfected with NC inhibitor (Fig. 2D). These results revealed that down-regulation of miR-10b significantly inhibited melanoma progression in vitro.
3.4. ITCH was a target gene of miR-10b in melanoma cells In order to explore the molecular mechanism through which miR10b promotes progression of melanoma, the bioinformatics databases (TargetScan and miRanda) were adopted to identify functionally relevant targets of miR-10b. ITCH was found to be the target gene of miR10b, which contains putative target sequence (Fig. 4A). We performed a luciferase reporter assay to test whether miR-10b directly targets ITCH. Two luciferase reporters were constructed, including a reporter plasmid containing the wide-type 3′UTR of ITCH (ITCH-3′UTR-WT) and a reporter construct of mutated the predicted miR-10b binding site (ITCH3′UTR-MUT), and these luciferase reporters were transfected with miR10b mimic or NC mimic. Co-transfection of miR-10b mimic and ITCH3′UTR-WT strongly decreased the luciferase activity, whereas cotransfection of NC mimic and ITCH-3′UTR-WT did not change the luciferase activity. In contrast, co-transfection of miR-10b mimic and ITCH-3′UTR-MUT or NC mimic and ITCH-3′UTR-MUT both did not change the luciferase activity (Fig. 4B). Furthermore, qRT-PCR and western blot assays were used to determine the role of the direct interaction between miR-10b and ITCH. As shown in Fig. 4C and D, A375 and SK-MEL-1 cells transfected with miR-10b mimic could suppress mRNA and protein levels of ITCH. In addition, we showed that ITCH
3.3. Knockdown of miR-10b inhibited tumor growth in vivo To further determine the above findings, an in vivo xenograft tumor model of melanoma was established. A375 cells transfected with antagomiR-10b or antagomiR-negative control (antagomiR-NC) were subcutaneously injected into the flank of nude mice. As shown in Fig. 3A and B, the tumors derived from miR-10b-downregulating A375 melanoma cells grew significantly slower than those derived from antagomiR-NC group. The average tumor weight was lower in the antagomiR-10b group than in antagomiR-NC group (Fig. 3C). We also measured miR-10b expression in the cancer tissues and found that miR43
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Fig. 5. ITCH abrogates the effects of miR-10b on melanoma cell proliferation, migration and invasion. A375 and SK-MEL-1 cells were co-transfected with miR-10b inhibitor and siITCH or siNC. (A) Cell proliferation was measured in A375 and SK-MEL-1 cells co-transfected with miR-10b inhibitor and siITCH or siNC by MTT assay at the indicated time points. (B) Cell migration and (C) cell invasion was measured by wound healing assay and transwell invasion assay in A375 and SK-MEL-1 cells co-transfected with miR10b inhibitor and siITCH or siNC. ⁎P < 0.05.
TOPflash which was used to evaluate β-catenin-mediated transcriptional activation and FOPflash constructs which contains mutated TCF binding sites, and is used as a negative control. The results showed that miR-10b inhibitor inhibited TOP luciferase reporter activity, whereas had no effect on FOP luciferase reporter activity, revealing a significant down-regulation of β-catenin signaling in response to miR-10b inhibitor in A375 and SK-MEL-1 cells (Fig. 6B). Furthermore, the expression of several Wnt target genes was examined to determine miR-10b regulated Wnt/β-catenin pathway in melanoma. We found that knockdown of miR-10b suppressed TCF4, c-Myc and cyclin D1 levels in melanoma cells (Fig. 6C). In contrast, knockdown of ITCH expression enhanced the expressions of β-catenin, cyclin D1, and c-Myc, which were opposite to the effects of miR-10b inhibitor (Fig. 6D–F). We further revealed that ITCH inhibitor reversed the effect of miR-10b inhibitor on TCF/LEF activity in cells (Fig. 6G). These findings demonstrated that miR-10b positively regulated Wnt/β-catenin signaling through repressing ITCH expression in melanoma.
was down-regulated in melanoma tissues compared with non-neoplastic skin specimens, and there was an inverse relationship between miR-10b and ITCH levels in melanoma tissues by Pearson's correlation analysis (Fig. 4E and F). Thus, miR-10b can regulate ITCH by binding its 3′UTR directly in melanoma. 3.5. Down-regulation of ITCH alleviates inhibitory effects of miR-10b inhibitor on melanoma cell proliferation, migration and invasion To evaluate whether ITCH is involved in the process of miR-10bregulated melanoma cell proliferation, migration and invasion, A375 and SK-MEL-1 cells were co-transfected with miR-10b inhibitor and siITCH or transfected with miR-10b inhibitor and siRNA control (siNC). MTT assay results showed that the cell viability of melanoma cells cotransfected with miR-10b inhibitor and siITCH were significantly higher than that transfected with miR-10b inhibitor and siNC (Fig. 5A). Moreover, the migratory and invasive abilities of A375 and SK-MEL-1 cells co-transfected with miR-10b inhibitor and siITCH were obviously enhanced compared with cells transfected with miR-10b inhibitor and siNC (Fig. 5B and C). These results indicated that deletion of miR-10b significantly inhibited cell proliferation, migration and invasion of melanoma cells, though these effects can be reversed by inhibition of ITCH, suggesting that ITCH is a functional target of miR-10b.
4. Discussion Emerging evidences have reported that miRNAs play an important role in melanoma development by serving as potential biomarkers and therapeutic targets (Wu et al., 2017). In our study, we showed that miR10b was up-regulated in melanoma tissues and cell lines. Functional studies revealed that knockdown of miR-10b expression suppressed melanoma cell proliferation, migration and invasion in vitro and inhibited tumor growth in vivo. Mechanistic investigations identified that miR-10b negatively regulated ITCH gene expression by directly targeting the 3′ UTR of ITCH, and there was an inverse correlation between the expression of ITCH and miR-10b. Furthermore，inhibition of ITCH significantly attenuated the suppressive effects of miR-10b inhibitor on melanoma and miR-10b exhibited its function through Wnt/ β-catenin pathway. Taken together, all data suggested that miR-10b play an important role in the melanoma progression.
3.6. Down-regulation of miR-10b inhibited Wnt/β-catenin signaling through ITCH regulation It has been confirmed that ITCH negatively regulates canonical Wnt/β-catenin signaling, thus we explored whether miR-10b exerted its effects through ITCH regulation of Wnt/β-catenin signaling. Analysis of nuclear and cytosolic extracts of A375 and SK-MEL-1 cells found that miR-10b inhibitor reduced β-catenin levels in the nuclei of melanoma cells concomitant with an increase in β-catenin levels in the cytosolic fraction (Fig. 6A). In addition, melanoma cells were transfected with 44
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Fig. 6. Downregulation of miR-10b inhibited Wnt/β-catenin signaling through ITCH regulation. (A) Nuclear and cytosolic obtained from A375 and SK-MEL-1 cells were detected using western blot. (B) Cells were transfected with TOPflash or FOPflash and subjected to dual-luciferase assays. (C) Expression of TCF4, c-Myc and cyclin D1 were measured in A375 and SK-MEL-1 cells. (D) ITCH level was measured in A375 and SK-MEL-1 cells transfected with siITCH or negative control. (E) Nuclear and cytosolic obtained from melanoma cells were detected using western blot. (F) Expression of TCF4, c-Myc and cyclin D1 were measured in siITCH transfected cells. (G) TCF/LEF activities were determined in ITCH siRNA transfected cells using TOP/FOPflash assay. ⁎P < 0.05.
expression was remarkably decreased in melanoma tissues and cell lines. The results revealed that miR-10b was up-regulated in melanoma. Dysregulation of miR-10b have been demonstrated that it exerted pivotal biological and pathological functions, including tumor cell proliferation, migration and invasion (Chen et al., 2016; Ouyang et al., 2014). We then explored the biological function of miR-10b in melanoma. Down-regulation of miR-10b inhibited melanoma cell
Several studies have reported that the dysregulation of specific miRNAs has been implicated in melanoma development and progression. Recent studies have reported that miR-10b expression was increased in serum of melanoma patients, and was associated with poor prognosis in patients with melanoma (Bai et al., 2017). However, the function and the molecular mechanisms of miR-10b in melanoma pathogenesis remain unclear. Here, we first determined that miR-10b 45
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References
proliferation, migration and invasion in vitro and suppressed xenograft tumor growth in vivo. These results are consistent with observations in breast cancer (Knirsh et al., 2016), glioblastoma (C. Ma et al., 2017) and hepatocellular carcinoma (Zhu et al., 2016). All results revealed that miR-10b may contribute to melanoma development and progression. It has been reported that miRNAs exert their function by inducing target mRNA cleavage or translational repression through binding to its 3′ UTR. Several genes have been identified as downstream targets of miR-10b, such as CMTM5 (Guan et al., 2018), KLF4 (Zhang et al., 2016) and PTEN (Bahena-Ocampo et al., 2016). In our study, we identified that ITCH was a target of miR-10b in melanoma. ITCH, a member of E3 ubiquitin ligases, which plays a critical role in the regulation of cell growth and apoptosis (Ho et al., 2011; Salah et al., 2011). Xia et al. have reported that miR-411 regulated ITCH expression and promoted cell proliferation in hepatocellular carcinoma cells (Xia et al., 2015) and Li et al. have concluded that circular RNA ITCH has an antitumor role by controlling miRNA activity, which increases the concentration of ITCH, and results in suppression of the canonical Wnt/β-catenin pathway in esophageal squamous cell carcinoma (Li et al., 2015). Here, we indicated that miR-10b overexpression dramatically reduced ITCH expression at mRNA and protein level. Moreover, knockdown of ITCH attenuated the suppressive effects of miR-10b inhibitor on cell proliferation, migration and invasion. These finding indications that there may be additional miRNAs that can affect tumor cell phenotypes via regulation of ITCH expression. For melanoma, we first demonstrated that miR-10b was a new modulatory regulator of ITCH expression in melanoma cells, which provides at least one mechanism for the upregulation of miR-10b expression in melanoma proliferation, invasion and migration. It has been proposed that a single miRNA can target several genes and multiple miRNAs can target a single gene, which consist of a network regulatory mechanism in cancer development. Furthermore, change in tissue-specific miRNA could cause a series of phenotypic changes via target key genes. All results suggested that miR10b involved in melanoma progression partly through negatively regulating ITCH expression. Number of studies has shown that the Wnt/β-catenin pathway is crucial for the development and progression of melanoma (Brown et al., 2017; Fan et al., 2017). It has been confirmed that ITCH negatively regulates canonical Wnt signaling by targeting dishevelled protein (Wei et al., 2012). Circular RNA ITCH may have an inhibitory effect on ESCC, which increase ITCH expression and suppressing the Wnt/β-catenin pathway (Li et al., 2015). Therefore, we determined whether Wnt/βcatenin pathway was involved in miR-10b-ITCH-mediated cell proliferation and invasion. Our results revealed that miR-10b inhibitor decreased β-catenin levels in the nuclei of melanoma cells and an increase in β-catenin levels in the cytosolic fraction. In addition, we showed that miR-10b inhibitor caused a significant down-regulation of β-catenin pathway in A375 and SK-MEL-1 cells. In contrast, knockdown of ITCH expression enhanced the expressions of β-catenin, cyclin D1, and c-Myc. We further revealed that ITCH inhibitor reversed the effect of miR-10b inhibitor on TCF/LEF activity in cells. Together, these findings demonstrated that miR-10b positively regulated Wnt/β-catenin signaling through down-regulating ITCH in melanoma. In summary, our study demonstrates that miR-10b is up-regulated in melanoma, and miR-10b inhibitor can inhibit melanoma cell proliferation, migration and invasion in vitro and suppress tumor growth in vivo by regulating ITCH/Wnt/β-catenin pathway. The newly identified miR-10b-ITCH pathway provides a novel insight into the molecular mechanisms regulating melanoma progression, and may provide novel therapeutic targets for the treatment of melanoma.
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Declaration of Competing Interest The authors declare no conflict of interest. 46
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Gene journal homepage: www.elsevier.com/locate/gene
Research paper
miR-10b promoted melanoma progression through Wnt/β-catenin pathway by repressing ITCH expression
T
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Shengqiang Wang , Yi Wu, Yan Xu, Xianjun Tang General Department, Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital, No. 181 Han Yu Road, Shapingba District, Chongqing 400030, China
A R T I C LE I N FO
A B S T R A C T
Keywords: miR-10b Melanoma Proliferation Invasion ITCH Wnt/β-catenin pathway
Dysregulation of microRNAs (miRNAs) have been reported to contribute to malignant progression in melanoma. However, the roles and mechanisms of several miRNAs in melanoma remain poorly understood. In our study, we showed that miR-10b was significantly up-regulated in melanoma tissues and cell lines, and was associated with overall survival of melanoma patients. Inhibition of miR-10b dramatically suppressed melanoma cell proliferation, migration and invasion in vitro and inhibited tumor growth in vivo. Moreover, we defined ITCH as a direct and functional downstream target of miR-10b, and showed that there was an inverse correlation between the expression of ITCH and miR-10b on melanoma tissues. Down-regulation of ITCH partially attenuated the inhibitory effects of miR-10b inhibition on melanoma cell proliferation, migration and invasion. Furthermore， we found that miR-10b exerted its effects on melanoma by regulating the Wnt/β-catenin pathway. Taken together, our results demonstrated that miR-10b was an important epigenetic modifier, promoting melanoma progression through regulating ITCH/Wnt/β-catenin pathway. These results offer a new strategy for epigenetic cancer therapy.
1. Introduction Malignant melanoma originating from melanocytes is an aggressive form of skin cancer with high patient morbidity and mortality, which is a high degree of malignancy and poor prognosis of tumor (Isola et al., 2016; Miller et al., 2016; Siegel et al., 2016). Melanoma is a major health problem in many countries and accounts for approximately 50,000 deaths annually worldwide (Slipicevic and Herlyn, 2012). Global incidence of melanoma continues to increase, in contrast to the stable or declining trends for most cancer types (Siegel et al., 2014). Despite recent advances in targeted therapy and immunotherapies, understanding of melanoma pathogenesis is still limited. Therefore, understanding the molecular pathogenesis of melanoma development is urgent. MicroRNAs (miRNAs) are a class of single-stranded, small noncoding RNAs that play important roles in negatively regulating gene expression either by inducing translational silencing or by causing mRNA degradation (Bartel, 2009). They have been implicated in the oncogenesis and progression of several cancers (Olive et al., 2010; Tili et al., 2013), and involved in cancer-relevant processes, such as
proliferation (F. Ma et al., 2017), invasion (Lu et al., 2017) and apoptosis (Li et al., 2017). Studies have shown that several miRNAs are implicated in melanoma progression. For example, miR-622 was downregulated in melanoma and miR-622 re-expression inhibited clonogenicity, proliferation, and migration in melanoma (Dietrich et al., 2018). While miR-338-5p promotes melanoma cells growth and metastasis via targeting CD82 (Long et al., 2018). miR-10b expression was up-regulated in metastatic CRC tissues and cell lines, and inhibition of miR-10b prevented cancer cell metastasis and growth by inducing cellcycle arrest and apoptosis (Xie et al., 2019); miR-10b promotes invasion by targeting KLF4 in osteosarcoma cells (Wang et al., 2016). Recent studies have reported that up-regulation of serum miR-10b is associated with poor prognosis in patients with melanoma, and suggested miR-10b is a new prognostic microRNA for melanoma and that there could be a place for miRNA analysis in stratifying melanoma for therapy (Bai et al., 2017; Saldanha et al., 2016). However, the function and molecular mechanisms of miR-10b in melanoma pathogenesis has not been fully understood. In our study, we clearly demonstrated that miR-10b functioned as an oncogenic miRNA. We demonstrated that knockdown of miR-10b
Abbreviations: miRNAs, microRNAs; MTT, 3-[4, 5-dimthylthiazol-2yl]-2,5-diphenyltetrazolium bromide ⁎ Corresponding author at: General Department, Chongqing University Cancer Hospital & Chongqing Cancer Institute & Chongqing Cancer Hospital, Chongqing 400030, China. E-mail address: [email protected] (S. Wang). https://doi.org/10.1016/j.gene.2019.05.043 Received 9 January 2019; Received in revised form 3 May 2019; Accepted 22 May 2019 Available online 23 May 2019 0378-1119/ © 2019 Published by Elsevier B.V.
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Fig. 1. miR-10b was up-regulated in melanoma cell lines and tissues. (A) miR-10b expression was detected in primary melanocytes and five melanoma cell lines by qRT-PCR. (B) qRT-PCR analysis of miR-10b expression in 30 melanoma specimens and 22 non-neoplastic skin tissues. (C) The Kaplan-Meier survival analysis evaluated the prognostic value of miR-10b for melanoma patients. ⁎P < 0.05.
specific primer sequence of GAPDH is as followed: Sense: 5′- GACTCA TGACCACAGTCCATGC-3′ and antisense: 3′- AGAGGCAGGGATGATGT TCTG-5′. The specific primer sequence of ITCH is as followed: Sense: 5′-CTGCCTGTTGCACATCTTGT-3′ and antisense: 5′-TGTCCAATTTT ACAAGATGT-3′.
dramatically inhibited melanoma cell proliferation, migration and invasion in vitro and suppressed tumor growth in vivo through regulating Wnt/β-catenin pathway via repression ITCH expression. Thus, our findings provide valuable clues toward understanding the mechanisms of melanoma pathogenesis and may contribute to the development of novel therapeutic strategies for melanoma in the future.
2.4. Western blot 2. Materials and methods Total proteins were extracted from the frozen tissues and cells and protein concentrations were determined using the BCA protein assay kit (Beyotime Institute of Biotechnology, Jiangsu, China). Protein were electrophoresed in 10% sodium dodecyl sulfate–polyacrylamide gels and then transferred onto polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% skimmed milk and incubated with primary antibodies as follows: ITCH (Abcam, Cambridge, UK) and β-actin (Sigma-Aldrich, USA), β-catenin, TCF4, c-Myc and cyclin D1 both purchased from Proteintech (Wuhan, China), followed by incubation with appropriate correlated HRP-conjugated secondary antibody. Protein detection was performed by enhanced chemiluminescence (ECL kit, Santa Cruz Biotechnology).
2.1. Ethics statement and human tissue samples All experimental procedures were approved by the Chongqing University Cancer Hospital. 78 melanoma tissues were collected from patients with melanoma at Chongqing University Cancer Hospital, and 30 non-neoplastic skin specimens from patients with chronic inflammatory skin diseases from 2013 to 2015, which were approved by the Institutional Review Board of Chongqing University Cancer Hospital (SYXK-2017-0002). 2.2. Cell culture and transfection Melanoma cell lines (A375, SK-MEL-1, SK-MEL-28, WM451) and human primary melanocytes were obtained from the American Type Culture Collection, and cultured in DMEM (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS. miR-10b mimic, miR-10b inhibitor, or their negative controls were transfected into A375 and SK-MEL-1 cells by using Lipofectamine 2000 (Invitrogen). ITCH-silencing oligonucleotides (siITCH) or the respective controls (RiboBio, Guangzhou, China) were transfected into the cells using Lipofectamine 2000 (Invitrogen).
2.5. Cell proliferation
2.3. qRT-PCR
Cells transfected with miR-10b inhibitor or NC inhibitor were seeded in a 6-well plate at a density of 5 × 105 cells/well. After starvation for 24 h in 1% FBS medium, the cells were removed using 200 μL tips while the remaining cells continued to grow in a monolayer. Cells were then washed and cultured in DMEM with 10% FBS for 24 h, the cell growth was observed using a Nikon TS100 microscope. The invasive ability of cells was determined using the transwell assay. The transwell membrane of the upper chamber coated with Matrigel (BD Biosciences Discovery Labware, Woburn, MA, USA) was used. The chambers were rehydrated in incubator with serum-free medium for 2 h. The top chambers were subsequently hydrated with 200 μL of cell suspension (containing 1 × 105 cells). The bottom chambers were added medium containing 10% FBS. After 24 h, the cells on the lower surface of the membrane were fixed, stained, washed and counted under a microscope (Chang et al., 2014).
A375 and SK-MEL-1 cells were seeded at 2000 cells per well in 96well plates after transfection. MTT (3-[4, 5-dimthylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay was used to measure cell viability, and the absorbance was determined using a spectrophotometric plate reader at 450 nm (Bio-Rad 680, USA). 2.6. Cell migration and invasion
Total RNA was extracted from cell lines and frozen tissues using Trizol reagent (Invitrogen). cDNA synthesis was performed using PrimeScript reverse transcriptase reagent kit (Takara, Dalian, China) according to the manufacturer's instructions. qRT-PCR analyses were performed using SYBR Green master mix Universal RT for miR-10b and FastStart Universal SYBR Green Master kit (Roche Life Sciences, Switzerland) for ITCH. U6 or GAPDH was measured as an internal control for miRNA or mRNA. Relative changes in gene and miRNA expression were determined using the 2−ΔΔCt method. The primer sequence of U6 is as followed: Sense: 5′-CTTCGGCAG CACATATACT-3′ and antisense: 5′-AAAATATGGAACGCTTCACG-3′. The specific primer sequence of miR-10b is as followed: Sense: 5′-TACCCTGTAGAA CCGA ATTTGTG-3′ and antisense: 5′-CAGTGCGTGTCGTGGAGT-3′. The 40
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Fig. 2. Knockdown of miR-10b inhibited melanoma cell proliferation, migration and invasion. (A) Relative expression of miR-10b in A375 and SK-MEL-1 cells transfected with miR-10b inhibitor or NC inhibitor was detected. (B) Cell viability was determined by measuring MTT absorbance at the indicated times after transfection. (C) Representative images and quantification of the wound healing assay of A375 and SK-MEL-1 cells transfected with miR-10b inhibitor or NC inhibitor. (D) Representative images and quantification of the Transwell invasion assay of A375 and SK-MEL-1 cells transfected with miR-10b inhibitor or NC inhibitor. ⁎P < 0.05.
2.7. In vivo tumor growth model
shortest and longest tumor diameter, respectively). Mice were sacrificed after 25 days. All tumors were excised, weighed, harvested, fixed, and embedded. All animals received humane care per the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” issued by the National Institutes of Health.
A375 cells (5 × 106 cells/mice) transfected with antagomiR-10b or antagomiR-NC cells were subcutaneously injected into the nude mice. Tumor size was determined every 5 days by measuring tumor diameter using Vernier calipers, and tumor weight was measured in grams after dissection. Tumor volume (mm3) = d2 × D/2 (d and D represent the 41
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Fig. 3. Knockdown of miR-10b inhibited tumor growth in vivo. (A) Images of the excised xenograft tumors. A375 cells transfected with antagomiR-10b or antagomiRNC were subcutaneously injected into the flank of nude mice. (B) The tumor growth curves of two groups in nude mice. Tumor growth was monitored for up to 25 days. (C) Tumor weight was calculated. (D) The expression of miR-10b was measures in tumor tissues using qRT-PCR. (E) Cell proliferation marker Ki67 protein level was measured by immunohistochemical staining. ⁎P < 0.05.
IL, USA) was used in this study.
2.8. Luciferase reporter assay Oligonucleotides containing the WT or mutant target site of miR10b in the 3′ UTR of ITCH was ligated into the pGL3-basic vector (Promega, Madison, WI). HEK293T cells were co-transfected with the ITCH vector or control, miR-10b mimic or NC mimic with Lipofectamine 2000 (Invitrogen). Topflash luciferase reporter plasmid containing TCF wild-type copies of the TCF/LEF binding sites and Fopflash luciferase reporter plasmid containing mutated binding sites were purchased from Upstate Biotechnology (Lake Placid, NY, USA), and transfected into miR-10b inhibitor-cells. After 48 h, the DualLuciferase Reporter Assay System (Promega) was used to detect the luciferase activity.
2.10. Statistical analysis Data are presented as mean ± SD, and all statistical analyses were performed using Prism 5.0 software (GraphPad, San Diego, CA, USA). Each experiment was repeated at least three times, and differences between groups were compared using Student's t-test or ANOVA. Pearson's correlation was used for analyzing the correlation between miR-10b and ITCH levels. p < 0.05 were defined as statistically significant.
2.9. Immunohistochemical staining
3. Results
The cell proliferation marker ki67 protein level in the sections from the paraffin-embedded subcutaneous tumor tissue was detected using immunohistochemical staining (Li et al., 2009). The degree of immunostaining was reviewed and scored independently by two observers based on the proportion of positively stained tumor cells and intensity of staining. Tumor cell proportion was scored as follows: 0 (no positive tumor cells), 1 (< 10% positive tumor cells), 2 (10–35% positive tumor cells), 3 (35–70% positive tumor cells), and 4 (> 70% positive tumor cells). Staining intensity was graded according to the following criteria: 0 (no staining), 1 (weak staining = light yellow), 2 (moderate staining = yellow brown), and 3 (strong staining = brown). Staining index was calculated as the product of staining intensity score and the proportion of positive tumor cells. ki67 antibody (Proteintech, Chicago,
3.1. Up-regulation of miR-10b in melanoma cell lines and tissues To validate the importance of miR-10b in melanoma, the expression of miR-10b was analyzed in melanoma cell lines and tissues. As shown in Fig. 1A, miR-10b was significantly higher in five human melanoma cells than primary melanocytes. Additionally, miR-10b expression was evaluated in 30 primary melanoma tissues and 22 non-neoplastic skin specimens, and qRT-PCR results showed that miR-10b expression was up-regulated in melanoma tissues (Fig. 1B). Moreover, we revealed that the high miR-10b expression was significantly associated with shorter overall in melanoma patients (Fig. 1C). These results indicated that miR-10b was up-regulated in melanoma and could do duty for a potential prognostic biomarker in melanoma patients. 42
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Fig. 4. miR-10b inhibited ITCH expression by targeting its 3′UTR. (A) Schematic representation of the predicted binding sites for miR-10b in the 3′UTR of ITCH and the site mutagenesis design for the reporter assay. Complementarities between miR-10b and the target sites are shown. (B) Relative luciferase activity of ITCH 3′ UTR in the HEK293 cells co-transfected with the indicated reporters and miR-10b mimic or NC mimic. (C) ITCH mRNA level was measured by qRT-PCR analysis in A375 and SK-MEL-1 cells transfected with miR-10b mimic or NC mimic. (D) ITCH protein level was measured by western blot assay in A375 and SK-MEL-1 cells transfected with miR-10b mimic or NC mimic. (E) qRT-PCR analysis of ITCH expression in 30 melanoma specimens and 22 non-neoplastic skin tissues. (F) Inverse relationship between miR-10b and ITCH expression in melanoma tissues. ⁎P < 0.05.
10b was significantly decreased in the antagomiR-10b group compared with antagomiR-NC group (Fig. 3D). Moreover, immunhistochemical staining of ki67 revealed that knockdown of miR-10b inhibited tumor cell proliferation (Fig. 3E). All data demonstrated that miR-10b inhibitor suppressed tumor growth in vivo, suggesting that miR-10b functions to positively regulate tumor growth in melanoma.
3.2. Knockdown of miR-10b inhibited melanoma cell proliferation, migration and invasion in vitro To investigate the biological function of miR-10b in melanoma, A375 and SK-MEL-1 cells were transiently transfected with miR-10b inhibitor or NC inhibitor (Fig. 2A), and then a series of biological experiments were performed in vitro. The MTT assay indicated that cell viability of cells transfected with miR-10b inhibitor was obviously reduced compared to cells transfected with NC inhibitor (Fig. 2B). The wound healing assay showed that the migratory ability of melanoma cells transfected with miR-10b inhibitor was lower than that of cells transfected with NC inhibitor (Fig. 2C). In addition, the Transwell invasion assay revealed that the invasive ability of cells transfected with miR-10b inhibitor was significantly suppressed compared to cells transfected with NC inhibitor (Fig. 2D). These results revealed that down-regulation of miR-10b significantly inhibited melanoma progression in vitro.
3.4. ITCH was a target gene of miR-10b in melanoma cells In order to explore the molecular mechanism through which miR10b promotes progression of melanoma, the bioinformatics databases (TargetScan and miRanda) were adopted to identify functionally relevant targets of miR-10b. ITCH was found to be the target gene of miR10b, which contains putative target sequence (Fig. 4A). We performed a luciferase reporter assay to test whether miR-10b directly targets ITCH. Two luciferase reporters were constructed, including a reporter plasmid containing the wide-type 3′UTR of ITCH (ITCH-3′UTR-WT) and a reporter construct of mutated the predicted miR-10b binding site (ITCH3′UTR-MUT), and these luciferase reporters were transfected with miR10b mimic or NC mimic. Co-transfection of miR-10b mimic and ITCH3′UTR-WT strongly decreased the luciferase activity, whereas cotransfection of NC mimic and ITCH-3′UTR-WT did not change the luciferase activity. In contrast, co-transfection of miR-10b mimic and ITCH-3′UTR-MUT or NC mimic and ITCH-3′UTR-MUT both did not change the luciferase activity (Fig. 4B). Furthermore, qRT-PCR and western blot assays were used to determine the role of the direct interaction between miR-10b and ITCH. As shown in Fig. 4C and D, A375 and SK-MEL-1 cells transfected with miR-10b mimic could suppress mRNA and protein levels of ITCH. In addition, we showed that ITCH
3.3. Knockdown of miR-10b inhibited tumor growth in vivo To further determine the above findings, an in vivo xenograft tumor model of melanoma was established. A375 cells transfected with antagomiR-10b or antagomiR-negative control (antagomiR-NC) were subcutaneously injected into the flank of nude mice. As shown in Fig. 3A and B, the tumors derived from miR-10b-downregulating A375 melanoma cells grew significantly slower than those derived from antagomiR-NC group. The average tumor weight was lower in the antagomiR-10b group than in antagomiR-NC group (Fig. 3C). We also measured miR-10b expression in the cancer tissues and found that miR43
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Fig. 5. ITCH abrogates the effects of miR-10b on melanoma cell proliferation, migration and invasion. A375 and SK-MEL-1 cells were co-transfected with miR-10b inhibitor and siITCH or siNC. (A) Cell proliferation was measured in A375 and SK-MEL-1 cells co-transfected with miR-10b inhibitor and siITCH or siNC by MTT assay at the indicated time points. (B) Cell migration and (C) cell invasion was measured by wound healing assay and transwell invasion assay in A375 and SK-MEL-1 cells co-transfected with miR10b inhibitor and siITCH or siNC. ⁎P < 0.05.
TOPflash which was used to evaluate β-catenin-mediated transcriptional activation and FOPflash constructs which contains mutated TCF binding sites, and is used as a negative control. The results showed that miR-10b inhibitor inhibited TOP luciferase reporter activity, whereas had no effect on FOP luciferase reporter activity, revealing a significant down-regulation of β-catenin signaling in response to miR-10b inhibitor in A375 and SK-MEL-1 cells (Fig. 6B). Furthermore, the expression of several Wnt target genes was examined to determine miR-10b regulated Wnt/β-catenin pathway in melanoma. We found that knockdown of miR-10b suppressed TCF4, c-Myc and cyclin D1 levels in melanoma cells (Fig. 6C). In contrast, knockdown of ITCH expression enhanced the expressions of β-catenin, cyclin D1, and c-Myc, which were opposite to the effects of miR-10b inhibitor (Fig. 6D–F). We further revealed that ITCH inhibitor reversed the effect of miR-10b inhibitor on TCF/LEF activity in cells (Fig. 6G). These findings demonstrated that miR-10b positively regulated Wnt/β-catenin signaling through repressing ITCH expression in melanoma.
was down-regulated in melanoma tissues compared with non-neoplastic skin specimens, and there was an inverse relationship between miR-10b and ITCH levels in melanoma tissues by Pearson's correlation analysis (Fig. 4E and F). Thus, miR-10b can regulate ITCH by binding its 3′UTR directly in melanoma. 3.5. Down-regulation of ITCH alleviates inhibitory effects of miR-10b inhibitor on melanoma cell proliferation, migration and invasion To evaluate whether ITCH is involved in the process of miR-10bregulated melanoma cell proliferation, migration and invasion, A375 and SK-MEL-1 cells were co-transfected with miR-10b inhibitor and siITCH or transfected with miR-10b inhibitor and siRNA control (siNC). MTT assay results showed that the cell viability of melanoma cells cotransfected with miR-10b inhibitor and siITCH were significantly higher than that transfected with miR-10b inhibitor and siNC (Fig. 5A). Moreover, the migratory and invasive abilities of A375 and SK-MEL-1 cells co-transfected with miR-10b inhibitor and siITCH were obviously enhanced compared with cells transfected with miR-10b inhibitor and siNC (Fig. 5B and C). These results indicated that deletion of miR-10b significantly inhibited cell proliferation, migration and invasion of melanoma cells, though these effects can be reversed by inhibition of ITCH, suggesting that ITCH is a functional target of miR-10b.
4. Discussion Emerging evidences have reported that miRNAs play an important role in melanoma development by serving as potential biomarkers and therapeutic targets (Wu et al., 2017). In our study, we showed that miR10b was up-regulated in melanoma tissues and cell lines. Functional studies revealed that knockdown of miR-10b expression suppressed melanoma cell proliferation, migration and invasion in vitro and inhibited tumor growth in vivo. Mechanistic investigations identified that miR-10b negatively regulated ITCH gene expression by directly targeting the 3′ UTR of ITCH, and there was an inverse correlation between the expression of ITCH and miR-10b. Furthermore，inhibition of ITCH significantly attenuated the suppressive effects of miR-10b inhibitor on melanoma and miR-10b exhibited its function through Wnt/ β-catenin pathway. Taken together, all data suggested that miR-10b play an important role in the melanoma progression.
3.6. Down-regulation of miR-10b inhibited Wnt/β-catenin signaling through ITCH regulation It has been confirmed that ITCH negatively regulates canonical Wnt/β-catenin signaling, thus we explored whether miR-10b exerted its effects through ITCH regulation of Wnt/β-catenin signaling. Analysis of nuclear and cytosolic extracts of A375 and SK-MEL-1 cells found that miR-10b inhibitor reduced β-catenin levels in the nuclei of melanoma cells concomitant with an increase in β-catenin levels in the cytosolic fraction (Fig. 6A). In addition, melanoma cells were transfected with 44
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Fig. 6. Downregulation of miR-10b inhibited Wnt/β-catenin signaling through ITCH regulation. (A) Nuclear and cytosolic obtained from A375 and SK-MEL-1 cells were detected using western blot. (B) Cells were transfected with TOPflash or FOPflash and subjected to dual-luciferase assays. (C) Expression of TCF4, c-Myc and cyclin D1 were measured in A375 and SK-MEL-1 cells. (D) ITCH level was measured in A375 and SK-MEL-1 cells transfected with siITCH or negative control. (E) Nuclear and cytosolic obtained from melanoma cells were detected using western blot. (F) Expression of TCF4, c-Myc and cyclin D1 were measured in siITCH transfected cells. (G) TCF/LEF activities were determined in ITCH siRNA transfected cells using TOP/FOPflash assay. ⁎P < 0.05.
expression was remarkably decreased in melanoma tissues and cell lines. The results revealed that miR-10b was up-regulated in melanoma. Dysregulation of miR-10b have been demonstrated that it exerted pivotal biological and pathological functions, including tumor cell proliferation, migration and invasion (Chen et al., 2016; Ouyang et al., 2014). We then explored the biological function of miR-10b in melanoma. Down-regulation of miR-10b inhibited melanoma cell
Several studies have reported that the dysregulation of specific miRNAs has been implicated in melanoma development and progression. Recent studies have reported that miR-10b expression was increased in serum of melanoma patients, and was associated with poor prognosis in patients with melanoma (Bai et al., 2017). However, the function and the molecular mechanisms of miR-10b in melanoma pathogenesis remain unclear. Here, we first determined that miR-10b 45
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References
proliferation, migration and invasion in vitro and suppressed xenograft tumor growth in vivo. These results are consistent with observations in breast cancer (Knirsh et al., 2016), glioblastoma (C. Ma et al., 2017) and hepatocellular carcinoma (Zhu et al., 2016). All results revealed that miR-10b may contribute to melanoma development and progression. It has been reported that miRNAs exert their function by inducing target mRNA cleavage or translational repression through binding to its 3′ UTR. Several genes have been identified as downstream targets of miR-10b, such as CMTM5 (Guan et al., 2018), KLF4 (Zhang et al., 2016) and PTEN (Bahena-Ocampo et al., 2016). In our study, we identified that ITCH was a target of miR-10b in melanoma. ITCH, a member of E3 ubiquitin ligases, which plays a critical role in the regulation of cell growth and apoptosis (Ho et al., 2011; Salah et al., 2011). Xia et al. have reported that miR-411 regulated ITCH expression and promoted cell proliferation in hepatocellular carcinoma cells (Xia et al., 2015) and Li et al. have concluded that circular RNA ITCH has an antitumor role by controlling miRNA activity, which increases the concentration of ITCH, and results in suppression of the canonical Wnt/β-catenin pathway in esophageal squamous cell carcinoma (Li et al., 2015). Here, we indicated that miR-10b overexpression dramatically reduced ITCH expression at mRNA and protein level. Moreover, knockdown of ITCH attenuated the suppressive effects of miR-10b inhibitor on cell proliferation, migration and invasion. These finding indications that there may be additional miRNAs that can affect tumor cell phenotypes via regulation of ITCH expression. For melanoma, we first demonstrated that miR-10b was a new modulatory regulator of ITCH expression in melanoma cells, which provides at least one mechanism for the upregulation of miR-10b expression in melanoma proliferation, invasion and migration. It has been proposed that a single miRNA can target several genes and multiple miRNAs can target a single gene, which consist of a network regulatory mechanism in cancer development. Furthermore, change in tissue-specific miRNA could cause a series of phenotypic changes via target key genes. All results suggested that miR10b involved in melanoma progression partly through negatively regulating ITCH expression. Number of studies has shown that the Wnt/β-catenin pathway is crucial for the development and progression of melanoma (Brown et al., 2017; Fan et al., 2017). It has been confirmed that ITCH negatively regulates canonical Wnt signaling by targeting dishevelled protein (Wei et al., 2012). Circular RNA ITCH may have an inhibitory effect on ESCC, which increase ITCH expression and suppressing the Wnt/β-catenin pathway (Li et al., 2015). Therefore, we determined whether Wnt/βcatenin pathway was involved in miR-10b-ITCH-mediated cell proliferation and invasion. Our results revealed that miR-10b inhibitor decreased β-catenin levels in the nuclei of melanoma cells and an increase in β-catenin levels in the cytosolic fraction. In addition, we showed that miR-10b inhibitor caused a significant down-regulation of β-catenin pathway in A375 and SK-MEL-1 cells. In contrast, knockdown of ITCH expression enhanced the expressions of β-catenin, cyclin D1, and c-Myc. We further revealed that ITCH inhibitor reversed the effect of miR-10b inhibitor on TCF/LEF activity in cells. Together, these findings demonstrated that miR-10b positively regulated Wnt/β-catenin signaling through down-regulating ITCH in melanoma. In summary, our study demonstrates that miR-10b is up-regulated in melanoma, and miR-10b inhibitor can inhibit melanoma cell proliferation, migration and invasion in vitro and suppress tumor growth in vivo by regulating ITCH/Wnt/β-catenin pathway. The newly identified miR-10b-ITCH pathway provides a novel insight into the molecular mechanisms regulating melanoma progression, and may provide novel therapeutic targets for the treatment of melanoma.
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