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• W2893427972 abstract "Laryngeal squamous cell carcinoma (LSCC) is a common form of head and neck cancer with poor prognosis. However, the mechanism underlying the pathogenesis of LSCC remains unclear. Here, we demonstrated increased expression of fascin actin-bundling protein 1 (FSCN1) and decreased expression of microRNA-145-5p (miR-145-5p) in a clinical cohort of LSCC. Luciferase assay revealed that miR-145-5p is a negative regulator of FSCN1. Importantly, low miR-145-5p expression was correlated with TNM (tumor, node, metastasis) status and metastasis. Moreover, cases with low miR-145-5p/high FSCN1 expression showed poor prognosis, and these characteristics together served as independent prognostic indicators of survival. Gain- and loss-of-function studies showed that miR-145-5p overexpression or FSCN1 knockdown inhibited LSCC migration, invasion, and growth by suppressing the epithelial-mesenchymal transition along with inducing cell-cycle arrest and apoptosis. Additionally, hypermethylation of the miR-145-5p promoter suggested that repression of miR-145-5p arises through epigenetic inactivation. LSCC tumor growth in vivo could be inhibited by using miR-145-5p agomir or FSCN1 small interfering RNA (siRNA), which highlights the potential for clinical translation. Collectively, our findings indicate that miR-145-5p plays critical roles in inhibiting the progression of LSCC by suppressing FSCN1. Both miR-145-5p and FSCN1 are important potential prognostic markers and therapeutic targets for LSCC. Laryngeal squamous cell carcinoma (LSCC) is a common form of head and neck cancer with poor prognosis. However, the mechanism underlying the pathogenesis of LSCC remains unclear. Here, we demonstrated increased expression of fascin actin-bundling protein 1 (FSCN1) and decreased expression of microRNA-145-5p (miR-145-5p) in a clinical cohort of LSCC. Luciferase assay revealed that miR-145-5p is a negative regulator of FSCN1. Importantly, low miR-145-5p expression was correlated with TNM (tumor, node, metastasis) status and metastasis. Moreover, cases with low miR-145-5p/high FSCN1 expression showed poor prognosis, and these characteristics together served as independent prognostic indicators of survival. Gain- and loss-of-function studies showed that miR-145-5p overexpression or FSCN1 knockdown inhibited LSCC migration, invasion, and growth by suppressing the epithelial-mesenchymal transition along with inducing cell-cycle arrest and apoptosis. Additionally, hypermethylation of the miR-145-5p promoter suggested that repression of miR-145-5p arises through epigenetic inactivation. LSCC tumor growth in vivo could be inhibited by using miR-145-5p agomir or FSCN1 small interfering RNA (siRNA), which highlights the potential for clinical translation. Collectively, our findings indicate that miR-145-5p plays critical roles in inhibiting the progression of LSCC by suppressing FSCN1. Both miR-145-5p and FSCN1 are important potential prognostic markers and therapeutic targets for LSCC. Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide.1Cancer Genome Atlas NetworkComprehensive genomic characterization of head and neck squamous cell carcinomas.Nature. 2015; 517: 576-582Crossref PubMed Scopus (2472) Google Scholar Laryngeal squamous cell carcinoma (LSCC) has the second highest incidence among these cancers and is especially prevalent in the northern areas of China.2Gao W. Zhang C. Feng Y. Chen G. Wen S. Huangfu H. Wang B. Fascin-1, ezrin and paxillin contribute to the malignant progression and are predictors of clinical prognosis in laryngeal squamous cell carcinoma.PLoS ONE. 2012; 7: e50710Crossref PubMed Scopus (32) Google Scholar The limitations of throat anatomy and the richness of submucous lymph readily facilitate local invasion and neck lymphatic metastasis in LSCC and are important risk factors for recurrence and poor prognosis after treatment. Despite therapeutic advances for many cancer types, the outcomes for most patients with advanced LSCC have not improved significantly in the last 30 years.3Liu H.C. Chen G.G. Vlantis A.C. Tong M.C. van Hasselt C.A. Chemotherapy for laryngeal cancer—an apoptotic approach.Curr. Drug Targets. 2008; 9: 878-886Crossref PubMed Scopus (12) Google Scholar, 4Jenckel F. Knecht R. State of the art in the treatment of laryngeal cancer.Anticancer Res. 2013; 33: 4701-4710PubMed Google Scholar Consequently, understanding the molecular mechanisms of the proliferation, invasion, and metastasis of LSCC is essential for its diagnosis, treatment, and prognosis. An actin-filament bundling oncogene, fascin actin-bundling protein 1 (FSCN1), has been studied in human cancers.5Lin C. Zhang S. Wang Y. Wang Y. Nice E. Guo C. Zhang E. Yu L. Li M. Liu C. et al.Functional role of a novel long noncoding RNA TTN-AS1 in esophageal squamous cell carcinoma progression and metastasis.Clin. Cancer Res. 2018; 24: 486-498Crossref PubMed Scopus (110) Google Scholar, 6Zhang Y. Lu Y. Zhang C. Huang D. Wu W. Zhang Y. Shen J. Cai Y. Chen W. Yao W. FSCN-1 increases doxorubicin resistance in hepatocellular carcinoma through promotion of epithelial-mesenchymal transition.Int. J. Oncol. 2018; (Published online March 20, 2018)https://doi.org/10.3892/ijo.2018.4327Crossref Scopus (28) Google Scholar, 7Wang C.Q. Li Y. Huang B.F. Zhao Y.M. Yuan H. Guo D. Su C.M. Hu G.N. Wang Q. Long T. et al.EGFR conjunct FSCN1 as a novel therapeutic strategy in triple-negative breast cancer.Sci. Rep. 2017; 7: 15654Crossref PubMed Scopus (31) Google Scholar We previously reported that FSCN1 was frequently upregulated in LSCC tissues as compared with adjacent normal margin (ANM) tissue. Moreover, upregulation of FSCN1 was associated with poor prognosis of LSCC.2Gao W. Zhang C. Feng Y. Chen G. Wen S. Huangfu H. Wang B. Fascin-1, ezrin and paxillin contribute to the malignant progression and are predictors of clinical prognosis in laryngeal squamous cell carcinoma.PLoS ONE. 2012; 7: e50710Crossref PubMed Scopus (32) Google Scholar Therefore, understanding the tumor-specific regulation of FSCN1 is crucial and has potential clinical significance. MicroRNAs (miRNAs) regulate the expression of protein-coding genes at the post-transcription level, and almost 60% of human genes are thought to be regulated by miRNAs.8Kozomara A. Griffiths-Jones S. miRBase: annotating high confidence microRNAs using deep sequencing data.Nucleic Acids Res. 2014; 42: D68-D73Crossref PubMed Scopus (3810) Google Scholar Dysregulated miRNAs are associated with the malignant progression of LSCC. The functions of miRNAs in LSCC have been revealed. The expression of miR-155 is upregulated in LSCC, and miR-155 overexpression promotes the proliferation and invasion of LSCC by targeting suppressor of cytokine signaling 1 and signal transducer and activator of transcription 3.9Zhao X.D. Zhang W. Liang H.J. Ji W.Y. Overexpression of miR -155 promotes proliferation and invasion of human laryngeal squamous cell carcinoma via targeting SOCS1 and STAT3.PLoS ONE. 2013; 8: e56395Crossref PubMed Scopus (99) Google Scholar miR-27a targets polo-like kinase 2 to promote proliferation and suppresses apoptosis in LSCC cells.10Tian Y. Fu S. Qiu G.B. Xu Z.M. Liu N. Zhang X.W. Chen S. Wang Y. Sun K.L. Fu W.N. MicroRNA-27a promotes proliferation and suppresses apoptosis by targeting PLK2 in laryngeal carcinoma.BMC Cancer. 2014; 14: 678Crossref PubMed Scopus (62) Google Scholar Conversely, miR-1 overexpression suppresses the migration and invasion of Hep-2 LSCC cells.11Wang F. Song G. Liu M. Li X. Tang H. miRNA-1 targets fibronectin1 and suppresses the migration and invasion of the HEp2 laryngeal squamous carcinoma cell line.FEBS Lett. 2011; 585: 3263-3269Crossref PubMed Scopus (52) Google Scholar Given the critical oncogenic role of FSCN1 in LSCC, we sought to test whether FSCN1 expression is regulated by specific miRNAs, with the hypothesis that the regulatory miRNAs could also be crucial for LSCC pathogenesis. We found that miR-145-5p is a negative regulator of FSCN1 expression in LSCC, and the expression of miR-145-5p was significantly lower in LSCC with poor prognosis. miR-145-5p and FSCN1 levels affected the phenotypes of LSCC cells, including migration, invasion, colony formation, proliferation, cell-cycle, apoptosis, and tumorigenesis both in vitro and in vivo. Moreover, miR-145-5p overexpression or FSCN1 knockdown inhibited the epithelial-to-mesenchymal transitions (EMT) and impaired cytoskeleton organization. Furthermore, miR-145-5p downregulation was attributed to hypermethylation of its promoter. Our microarray data showed that FSCN1 was upregulated in LSCC tissues as compared with ANM tissue (Figure 1A), and we previously reported that differentially high expression of FSCN1 was associated with poor prognosis in LSCC.2Gao W. Zhang C. Feng Y. Chen G. Wen S. Huangfu H. Wang B. Fascin-1, ezrin and paxillin contribute to the malignant progression and are predictors of clinical prognosis in laryngeal squamous cell carcinoma.PLoS ONE. 2012; 7: e50710Crossref PubMed Scopus (32) Google Scholar Furthermore, a recent genome-wide analysis of cancer transcriptomes highlighted that FSCN1 expression was prognostic in HNSCC.12Uhlen M. Zhang C. Lee S. Sjöstedt E. Fagerberg L. Bidkhori G. Benfeitas R. Arif M. Liu Z. Edfors F. et al.A pathology atlas of the human cancer transcriptome.Science. 2017; 357 (eaan2507)Crossref PubMed Scopus (1720) Google Scholar Interrogation of The Cancer Genome Atlas (TCGA) data showed that FSCN1 was not frequently amplified in LSCC (Figure S1A), and we hypothesized that other regulatory mechanisms were involved, particularly that altered expression of miRNAs and affected FSCN1 expression in HNSCC. To address this concept, we turned to our prior analysis of miRNA expression profiles in LSCC determined by genome-wide miRNA microarray assay.13Gao W. Zhang C. Ma T. Wen S. Fu R. Zhao D. Wu Y. Wang B. Potential biomarkers and their regulatory relationships in laryngeal squamous cell carcinoma with lymph node metastasis revealed by integrating mRNA, microRNA and long non-coding RNA profiles.Int. J. Clin. Exp. Pathol. 2016; 9: 5103-5116Google Scholar, 14Zhang C. Gao W. Wen S. Wu Y. Fu R. Zhao D. Chen X. Wang B. Potential key molecular correlations in laryngeal squamous cell carcinoma revealed by integrated analysis of mRNA, miRNA and lncRNA microarray profiles.Neoplasma. 2016; 63: 888-900Crossref PubMed Scopus (16) Google Scholar To determine potential miRNA regulators of FSCN1, we performed bioinformatics analysis with miRwalk-2.15Dweep H. Gretz N. miRWalk2.0: a comprehensive atlas of microRNA-target interactions.Nat. Methods. 2015; 12: 697Crossref PubMed Scopus (970) Google Scholar The miRNAs hit by miRwalk-2 were intersected with miRNAs downregulated in LSCC microarray data (Figure 1B; Tables S1 and S2). Notably, miR-145-5p was the only miRNA that was derived in common with the two approaches (Figure 1C). Therefore, we focused on miR-145-5p as a putative regulator of FSCN1 in LSCC. Next, we compared miR-145-5p and FSCN1 RNA expression across 40 paired samples of fresh LSCC and ANM tissue. For all cases, miR-145-5p was significantly downregulated in LSCC tissue as compared with ANM tissue, and FSCN1 was differentially upregulated (Figures 1D and 1E). As compared with LSCC cases with low FSCN1 level, LSCC cases with high FSCN1 level showed low miR-145-5p level (Figure 1F), with Pearson correlation analyses revealing a clear inverse relation between miR-145-5p expression and FSCN1 mRNA level (r = −0.737; p < 0.001) (Figure 1G). In support, in situ hybridization experiments illustrated that LSCC lesions with high FSCN1 level showed low miR-145-5p level (Figure 1H). In contrast, miR-145-5p expression was readily observed in surrounding cells and ANM tissues (Figure 1H; Figure S1B). To better understand the association between miR-145-5p and FSCN1, we used cell models of LSCC (Hep-2 and TU177) along with normal HOK cells for comparison. Notably, qRT-PCR-based analysis demonstrated relatively low expression of miR-145-5p in Hep-2 and TU177 cells, and high level of FSCN1 as compared with HOK cells showing the reverse pattern (Figure 1I). Moreover, manipulating miR-145-5p levels with a mimic sharply decreased FSCN1 mRNA and protein levels in both Hep-2 and TU177 cells (Figures 1J and 1K). Together these findings indicate that FSCN1 expression in LSCC depended on miR-145-5p level, but the underlying basis of this relation remained undefined. We then sought to determine whether FSCN1 mRNA was directly regulated by miR-145-5p binding. Indeed, bioinformatics analysis identified that the 3′ UTR region of FSCN1 contains four putative seed regions with complementarity to miR-145-5p binding (Figure S1C). To verify that miR-145-5p targets FSCN1 via 3′ UTR-mediated regulation, we conducted experiments with HEK293T cells transfected with FSCN1 3′ UTR luciferase reporter constructs. Notably, co-introduction of a miR-145-5p mimic, but not a control, significantly depleted the reporter activity of wild-type FSCN1 3′ UTR construct binding (Figure 1L). Similar results were obtained when each of the four miR-145-5p binding sites in the FSCN1 3′ UTR construct were individually mutated (Figure 1L). Only mutation of all four miR-145-5p binding sites abrogated the ability of miR-145-5p mimics to inhibit reporter activity (Figure 1L), which suggests that all seed regions participate in FSCN1 regulation. Overall, these findings support a mechanism whereby miR-145-5p targets FSCN1 directly, and downregulation of miR-145-5p results in FSCN1 upregulation in LSCC. To evaluate the clinical and prognostic significance of miR-145-5p expression and FSCN1 protein level in LSCC, we examined their expression in 188 formalin-fixed paraffin-embedded (FFPE) tissue samples (Table S3) by using qRT-PCR and immunohistochemistry. miR-145-5p level was significantly lower in samples from LSCC patients with distant metastases (p = 0.024). On multivariate analysis, the expression of miR-145-5p in LSCC tissue was negatively associated with T staging (p < 0.001), N status (p = 0.018), and clinical stage (p = 0.004; Table 1). Conversely, high FSCN1 protein level in LSCC was positively associated with T staging (p = 0.007), N status (p = 0.005), and clinical stage (p = 0.007; Table S4). Moreover, miR-145-5p expression and FSCN1 protein level were inversely related (p < 0.001; Table 1).Table 1Association between Expression of miR-145-5p/FSCN1 and Clinical Features of LSCC PatientsmiR-145-5p ExpressionFSCN1 Protein ExpressionParametersCases (n)Average Rankp ValueaMann-Whitney U test used for two-group analysis. Kruskal-Wallis H test used for three-group analysis.Average Rankp ValueaMann-Whitney U test used for two-group analysis. Kruskal-Wallis H test used for three-group analysis.Age (Years)≤609995.920.66288.070.038>608992.92101.65SexFemale2187.790.488100.140.540Male16795.3493.79Primary Cancer SiteGlottic101102.410.04381.40<0.001Supraglottic8384.87108.88Subglottic494.50127.00DifferentiationHigh72112.78<0.00168.25<0.001Medium or low11683.16110.79T StagingT1+T2111105.93<0.00187.200.007T3+T47778.02105.03Cervical Lymph Node MetastasisN014299.130.01889.270.005N+4680.20110.65Distant MetastasisM018395.780.02493.610.100M1547.50127.00Clinical StageI+II96104.290.00485.880.007III+IV9284.28103.50Smoke PreoperativelyNo75100.140.18184.390.012Yes11390.76101.21FSCN1 Protein ExpressionLow65119.81<0.001––High12381.13–a Mann-Whitney U test used for two-group analysis. Kruskal-Wallis H test used for three-group analysis. Open table in a new tab To examine FSCN1 expression across HNSCC and normal tissues at the mRNA level, we queried TCGA RNA-sequencing (RNA-seq) data. FSCN1 mRNA level was markedly higher in HNSCC than normal tissues (Figure 1M). Notably, Kaplan-Meier analysis of TCGA cohorts revealed that upregulated FSCN1 was significantly associated with poor outcome with HNSCC (Figure 1N). In contrast, HNSCC patients with upregulated miR-145-5p exhibited longer survival time (Figure 1O). Our analysis revealed better overall survival (OS) in LSCC patients with comparatively high than low level of miR-145-5p (106.09 ± 4.12 versus 85.01 ± 5.24 months; Figure 1P) and poorer OS with high than low level of FSCN1 (79.74 ± 4.43 versus 122.83 ± 1.77 months; Figure 1Q). Subdividing patients into four groups based on miR-145-5p expression and FSCN1 protein level provided an opportunity to further delineate outcomes associated with biological subtype. As expected, LSCC patients with low miR-145-5p/high FSCN1 levels showed low OS, whereas those with high miR-145-5p/low FSCN1 levels showed the best OS (76.98 ± 5.59 versus 123.16 ± 2.03; p < 0.001; Figure 1R). Furthermore, on regression analysis, the combination of low miR-145-5p expression and high FSCN1 protein level was an independent prognostic indicator for LSCC survival (relative risk = 12.69; 95% confidence interval [CI] 2.83–56.91; p = 0.001; Table S4). Nevertheless, cases with high miR-145-5p/high FSCN1 levels also partitioned with the poor-performing low miR-145-5p/high FSCN1 level cases (Figure 1R). Indeed, FSCN1 level proved to be an independent predictor of LSCC survival (relative risk = 12.27; 95% CI 3.49–43.19; p = 0.001; Table S5), although in contrast, miR-145-5p expression alone was not significant in this analysis. Collectively, these data support the existence of a high-risk biological subtype of LSCC exhibiting low miR-145-5p/high FSCN1 levels, but further propose that high FSCN1 protein level itself is an independent prognostic indicator for LSCC. Thus, we propose that miR-145-5p and its target FSCN1 are potential biomarkers for the diagnosis and prognosis of LSCC. Next, we sought to better understand the functional impact of miR-145-5p and FSCN1 expression on the growth behavior of LSCC cells. First, overexpression of miR-145-5p by using mimics in Hep-2 and TU177 cell lines with low endogenous miR-145-5p expression substantially reduced growth and colony formation (Figures 2A and 2B ). Measurement of EdU incorporation established reduced cell proliferation rates (Figure 2C), and cell-cycle analyses established that miR-145-5p overexpression increased the proportion of cells in the G0/G1 stage, with concurrent reduction in the S and G2/M stages (Figure 2D). In addition, the proportion of apoptotic cells increased with the introduction of miR-145-5p mimic (Figure 2E). Cells transfected with miR-145-5p showed increased cleavage of caspase-3 and apoptotic bodies, which was consistent with the induction of apoptosis (Figure 2F; Figure S2A). These data indicate that high miR-145-5p expression in LSCC likely inhibits growth by inducing both G0/G1 phase arrest and apoptosis. We further conducted similar functional analyses after manipulating FSCN1 levels in LSCC. Both Hep-2 and TU177 cells were depleted of FSCN1 by using an optimized small interfering RNA (siRNA) protocol (Figure S2B); the reduced FSCN1 protein level was confirmed using immunoblotting (Figure 3A). FSCN1 knockdown inhibited both cell proliferation and colony formation of Hep2 and TU177 cells (Figures 3B–3D), thereby largely phenocopying the results obtained with the miR-145-5p mimic. In accordance with these data, FSCN1 knockdown pushed cells toward the G0/G1 phase (Figure 3E) and increased the proportion of cells undergoing apoptosis (Figure 3F). Moreover, immunoblotting results showed that FSCN1 knockdown increased cleavage of caspase-3 (Figure 3G). Thus, these effects of miR-145-5p and FSCN1 in LSCC in vitro reconcile well with the clinical behavior of LSCC tumors in vivo. In addition to having effects on cell proliferation and survival, the miR-145-5p mimic appeared to affect the adhesive properties of LSCC cells (Figure S2C). Given the well-known relation between cell adhesion and motility migration, we further investigated this phenomenon. Indeed, overexpression of miR-145-5p with a mimic significantly inhibited cell migration and invasion in Transwell assays as compared with controls (Figures 4A and 4B ). Consistent with the mechanistic relation uncovered between miR-145-5p and FSCN1, on repeating these assays with FSCN1 siRNA, Hep-2 and TU177 cells showed decreased migration and invasion (Figures 4C and 4D). Hence our findings propose that FSCN1 is an oncogene in LSCC that promotes cell proliferation and is also involved in promoting migration and invasion. A corollary to these findings was the phenotype of LSCC cells observed by scanning electron microscopy. Control cells showed the typical morphology of cultured cancer cells with prominent lamellipodial and filopodial structures, whereas LSCC cells treated with miR-145-5p mimic or si-FSCN1 showed very few lamellipodia and filopodia, for a relatively rounded cell morphology (Figure 4E). Furthermore, impaired F-actin polymerization and filopodium formation were observed after miR-145-5p restoration or FSCN1 knockdown (Figure 4F). Hence, the miR-145-5p/FSCN1 mechanism may be involved in controlling the EMT in LSCC, generally known as a critical determinant of tumor cell metastasis.16Pang B. Wu N. Guan R. Pang L. Li X. Li S. Tang L. Guo Y. Chen J. Sun D. et al.Overexpression of RCC2 enhances cell motility and promotes tumor metastasis in lung adenocarcinoma by inducing epithelial-mesenchymal transition.Clin. Cancer Res. 2017; 23: 5598-5610Crossref PubMed Scopus (44) Google Scholar To determine whether the regulation of FSCN1 expression via miR-145-5p and consequent modulation of migration and invasion of LSCC depended on the EMT, we analyzed EMT marker expression. Immunoblotting of Hep-2 and TU177 LSCC cells treated with miR-145-5p mimic or with FSCN1 siRNA revealed increased expression of E-cadherin level along with reduced levels of Vimentin, Snail, N-cadherin, and matrix metalloproteinase 2 (MMP2) and matrix metalloproteinase 9 (MMP9) (Figures 4G and 4H). Hence, miR-145-5p inhibited the EMT, and its target, FSCN1, promoted the EMT in LSCC. A final question remained as to why downregulation of miR-145-5p occurs in the malignant transition of LSCC cells. To understand this, we analyzed the DNA methylation of the miR-145-5p proximal promoter in LSCC cell lines by using pyrosequencing. As compared with normal control HOK cells, in Hep-2 and TU177 LSCC cells, the CpG sites 2–7 were highly methylated (Figures 5A and 5B ), which suggests that promoter hypermethylation underpinned the loss of miR-145-5p expression in LSCC. In support, miR-145-5p expression was significantly increased in Hep-2 and TU177 cells treated with the DNA methyltransferase inhibitor 5-Aza-deoxycytidine (5-Aza) (Figure 5C). In addition, we tested the effect of the histone deacetylase inhibitor trichostatin A (TSA) and chemopreventive agent genistein on miR-145-5p expression. TSA and genistein alone inhibited miR-145-5p expression, whereas their combination increased miR-145-5p expression. Cells treated with 5-Aza combined with TSA and/or genistein showed increased miR-145-5p expression (Figure 5C). Endogenous FSCN1 protein expression exhibited corresponding changes in 5-Aza-treated Hep-2 and TU177 cells (Figure 5D). Notably, genistein treatment reduced both miR-145-5p level and FSCN1 protein level (Figure 5D), which might be due to its different regulatory pathways for miR-145-5p and FSCN1. Moreover, by MassARRAY methylation analysis, we determined the DNA methylation level of miR-145-5p promoter in clinical LSCC tissues separated by laser capture microdissection (LCM) (Figure S3). The methylation level of the miR-145-5p promoter was significantly higher in LSCC tissue than paired ANM tissue (Figure 5E), and the methylation level of the miR-145-5p promoter was inversely associated with the differentiation degree of LSCC (Figure 5E). Together these findings propose that promoter hypermethylation underpins the low expression of miR-145-5p observed in LSCC. To establish the proof of principle that modulating miR-145-5p or FSCN1 expression in LSCC could be an effective therapy, we used a xenograft model. Subcutaneous (s.c.) tumors of Hep-2 or TU177 LSCC cells were established in opposing flanks of nude mice and allowed to grow to 0.5 mm3. Thereafter, miR-145-5p agomir or control (negative control [NC]) agomir was intra-tumorally injected, and tumor growth was monitored over 4 weeks. Compared with controls, injection of miR-145-5p agomir significantly decreased tumor growth rates and tumor size (Figure 6A). Consistent with previous experiments, injecting tumors with FSCN1-targeting oligos also decreased the growth rate and tumor weight of Hep-2 and TU177 tumors (Figure 6B). Moreover, histopathological examination of tissues from miR-145-5p or FSCN1 siRNA-injected tumors showed a higher abundance of shrunken and fragmentized nuclei in sections as compared with control tissues (Figure 6C), which suggests increased rates of apoptosis. This finding was confirmed on TUNEL assay, whereby miR-145-5p overexpression or FSCN1 knockdown increased cell apoptosis as compared with controls (Figure 6D). Further analysis of xenografted LSCC tissues by immunohistochemical staining confirmed that the injection of both miR-145-5p agomir and FSCN1 siRNA decreased FSCN1 protein level (Figure 6E). Moreover, the expression of cyclin-D1 and the proliferation marker Ki67 was decreased in tumors with miR-145-5p agomir and FSCN1 siRNA injection (Figure 6E). Furthermore, as compared with controls, tumors with miR-145-5p agomir or FSCN1 siRNA injection showed upregulated E-cadherin and downregulated MMP2 and MMP9 (Figure 6F). Thus, we established the proof of principle that modulating miR-145-5p expression in vivo could inhibit LSCC tumor growth ostensibly via the same mechanisms revealed in in vitro experiments. The prognostic value of FSCN1 expression has previously emerged across a number of cancer types, with its upregulation known to promote migration and invasion.17Ma Y. Machesky L.M. Fascin1 in carcinomas: its regulation and prognostic value.Int. J. Cancer. 2015; 137: 2534-2544Crossref PubMed Scopus (27) Google Scholar, 18El-Balat A. Arsenic R. Sänger N. Karn T. Becker S. Holtrich U. Engels K. Fascin-1 expression as stratification marker in borderline epithelial tumours of the ovary.J. Clin. Pathol. 2016; 69: 142-148Crossref PubMed Scopus (7) Google Scholar Our previous study involving LSCC found FSCN1 protein overexpression associated with clinical features and poor prognosis.2Gao W. Zhang C. Feng Y. Chen G. Wen S. Huangfu H. Wang B. Fascin-1, ezrin and paxillin contribute to the malignant progression and are predictors of clinical prognosis in laryngeal squamous cell carcinoma.PLoS ONE. 2012; 7: e50710Crossref PubMed Scopus (32) Google Scholar However, the regulatory mechanisms of FSCN1 in LSCC remain undefined. In the present study, we found that miR-145-5p functioned as a negative regulator of FSCN1 in LSCC tissue and cells, with decreased miR-145-5p level and accompanying increased FSCN1 level acting as critical determinants of poor prognosis. miR-145-5p and FSCN1 levels may have potential for predicting outcomes with LSCC. Indeed, their applicability as novel biomarkers to identify LSCC patients with distinct clinical features is underscored by our findings relating miR-145-5p expression and FSCN1 protein level with tumor, node, metastasis (TNM) and clinical staging. Moreover, we demonstrated that miR-145-5p has an important regulatory role in LSCC, inhibiting LSCC cell proliferation along with the accompanying cell-cycle arrest and apoptosis. Nevertheless, we also found evidence of a high-risk LSCC group displaying elevated miR-145-5p level with high FSCN1 protein level, likely indicating that non-miR-145-5p mechanism(s) are also responsible for high FSCN1 protein level in some cases. We initiated our study in response to the clinical impact of FSCN1 expression in LSCC. After identifying miR-145-5p by a bioinformatics approach, we sought to learn more about its role and mechanisms, particularly involving LSCC. We now provide definitive evidence that miR-145-5p robustly targets FSCN1 via a multivalent mechanism involving all four binding sites identified within the 3′ UTR of FSCN1 mRNA. However, although we demonstrated that inhibiting FSCN1 expression in LSCC largely reproduced the experimental findings observed when miR-145-5p expression was restored, we cannot exclude the involvement of other important targets. Indeed, a previous study suggested that miR-145-5p acts in LSCC cells to inhibit stem cell markers SRY-box 2, POU class 5 homeobox 1 (also known as OCT4), Kruppel-like factor 4, and ATP binding cassette subfamily G member 2,19Karatas O.F. Suer I. Yuceturk B. Yilmaz M. Hajiyev Y. Creighton C.J. Ittmann M. Ozen M. The role of miR-145 in stem cell characteristics of human laryngeal squamous cell carcinoma Hep-2 cells.Tumour Biol. 2016; 37: 4183-4192Crossref PubMed Scopus (31) Google Scholar although the mechanisms need to be further investigated. More generally, these findings expand on the growing evidence of the importance of the miR-145 locus in cancer progression.20Lei C. Du F. Sun L. Li T. Li T. Min Y. Nie A. Wang X. Geng L. Lu Y. et al.miR-143 and miR-145 inhibit gastric cancer cell migration and metastasis by suppressing MYO6.Cell Death Dis. 2017; 8: e3101Crossref PubMed Scopus (103) Google Scholar, 21Larne O. Hagman Z. Lilja H. Bjartell A. Edsjö A. Ceder Y. miR-145 suppress the androgen receptor in prostate cancer cells and correlates to prostate cancer prognosis.Carcinogenesis. 2015; 36: 858-866Crossref PubMed Scopus (55) Google Scholar, 22Cioce M. Ganci F. Canu V. Sacconi A. Mori F. Canino C. Korita E. Casini B. Alessandrini G. Cambria A. et al.Protumorigenic effects of mir-145 loss in malignant pleural mesothelioma.Oncogene. 2014; 33: 5319-5331Crossref PubMed Scopus (56) Google Scholar, 23Xing A.Y. Wang Y.W. Su Z.X. Shi D.B. Wang B. Gao P. Caten" @default.
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• W2893427972 title "Promoter Methylation-Regulated miR-145-5p Inhibits Laryngeal Squamous Cell Carcinoma Progression by Targeting FSCN1" @default.
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• W2893427972 cites W1963501822 @default.
• W2893427972 cites W1972888632 @default.
• W2893427972 cites W1986241711 @default.
• W2893427972 cites W1996600340 @default.
• W2893427972 cites W2003074738 @default.
• W2893427972 cites W2032050847 @default.
• W2893427972 cites W2036344491 @default.
• W2893427972 cites W2045126262 @default.
• W2893427972 cites W2049852036 @default.
• W2893427972 cites W2055489631 @default.
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• W2893427972 cites W2077327925 @default.
• W2893427972 cites W2096384051 @default.
• W2893427972 cites W2098362718 @default.
• W2893427972 cites W2104348316 @default.
• W2893427972 cites W2112979115 @default.
• W2893427972 cites W2115797469 @default.
• W2893427972 cites W2123527373 @default.
• W2893427972 cites W2137734450 @default.
• W2893427972 cites W2143561574 @default.
• W2893427972 cites W2159586009 @default.
• W2893427972 cites W2177211001 @default.
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• W2893427972 cites W2189153404 @default.
• W2893427972 cites W2470233900 @default.
• W2893427972 cites W2510597175 @default.
• W2893427972 cites W2512973801 @default.
• W2893427972 cites W2521453400 @default.
• W2893427972 cites W2527069674 @default.
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• W2893427972 cites W2767330490 @default.
• W2893427972 cites W2767754396 @default.
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