MiR-145 Inhibits Metastasis by Targeting Fascin Actin-Bundling Protein 1 in Nasopharyngeal Carcinoma

March MiR-145 Inhibits Metastasis by Targeting Fascin Actin-Bundling Protein 1 in Nasopharyngeal Carcinoma Ying-Qin Li 0 1 2 Qing-Mei He 0 1 2 Xian-Yue Ren 0 1 2 Xin-Ran Tang 0 1 2 Ya-Fei Xu 0 1 2 Xin Wen 0 1 2 Xiao- Jing Yang 0 1 2 Jun Ma 0 1 2 Na Liu 0 1 2 Data Availability Statement: All relevant data are within the paper. 0 1 2 0 Funding: This work was supported by grants from the National Natural Science Foundation of China (81402516; NL), the Pearl River Science and Technology New Star of Guangzhou , China (2015; NL) , the National Science & Technology Pillar Program during the Twelfth Five-year Plan Period (2014BAI09B10; JM), the Health & Medical Collaborative Innovation Project of Guangzhou City (201400000001; JM), and the Key Laboratory Construction Project of Guangzhou City (121800085; JM). The funders had no role in the study design 1 Academic Editor: Zhiqian Zhang, Peking University Cancer Hospital & Institute , CHINA 2 Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center of Cancer Medicine , Guangzhou , People's Republic of China - These authors contributed equally to this work. * (NL); (JM) MiR-145 was obviously decreased in NPC cell lines and clinical samples (P<0.01). Ectopic overexpression of miR-145 significantly inhibited the migratory and invasive ability of SUNE-1 and CNE-2 cells. In addition, stably overexpressing of miR-145 in SUNE-1 cells could remarkably restrain the formation of metastatic nodes in the lungs of mice. Furthermore, fascin actin-bundling protein 1 (FSCN1) was verified as a target of miR-145, and silencing FSCN1 with small RNA interfering RNA could suppress NPC cell migration and invasion. Our findings demonstrated that miR-145 function as a tumor suppressor in NPC development and progression via targeting FSCN1, which could sever as a potential novel therapeutic target for patients with NPC. Based on our recent microarray analysis, we found that miR-145 was obviously downregulated in nasopharyngeal carcinoma (NPC) tissues. However, little is known about its function and mechanism involving in NPC development and progression. Quantitative RT-PCR was used to detect miR-145 expression in NPC cell lines and clinical samples. Wound healing, Transwell migration and invasion, three-dimension spheroid invasion assays, and lung metastasis model were performed to test the migratory, invasive, and metastatic ability of NPC cells. Luciferase reporter assay, quantitative RT-PCR, and Western blotting were used to verify the target of miR-145. Competing Interests: The authors have declared that no competing interests exist. Nasopharyngeal carcinoma (NPC) is a relatively common malignant tumor in Southeast Asia, especially in Southern China. Radiotherapy has been the primary treatment for patients with NPC because of its inherent anatomic constraints and high degree of radiosensitivity. Recently, more and more evidence indicates that chemoradiotherapy is the optimal treatment choice for NPC patients with advanced disease stage and its five-year survival rate has been significantly improved; however, distant metastasis is still one of the most common failure patterns [14]. Therefore, best understanding of the molecular mechanisms that involved in NPC metastasis is essential for the development of novel therapeutic strategies. Metastasis is the major feature of malignant tumors and the major cause of cancer-related deaths [5]. Metastasis is a multi-step process, including detachment of cancer cells from the primary sites, intravasation into and dissemination through the vasculature, and finally proliferation and formation of secondary tumors [6]. Recently, researches about genes and gene products that drive the metastasis have been performed; however, the underlying molecular mechanisms are still elusive. Increasing evidence has demonstrated that in addition to genetic alteration, miRNAs also take part in the regulation of cancer pathological processes, especially tumor metastasis [79]. MiRNAs are small non-coding RNAs that repress gene expression via mRNA degradation or translational inhibition based on base pairing to the 30 untranslated region (30 UTR) of their target mRNAs [10]. Abnormal expression of miRNAs has been reported in most types of human cancers [1112], including NPC [1316]. To date, several dysregulated miRNAs have been demonstrated to be involved in NPC cell migration, invasion and metastasis, such as miR-29c, miR-451, miR-10b, miR-93, and miR-124 [1721]. These results suggest that miRNAs play important roles in NPC tumorigenesis and progression, however, the roles of miRNAs involved in NPC carcinogenesis warrant further investigations. Based on our recent microarray analysis, we discovered that miR-145 was significantly downregulated in NPC tissues. A series of studies have reported that miR-145 is frequently decreased in many malignancies, including breast, colon, liver, prostate, and gastric cancers, and functions as a tumor suppressor by inhibiting tumor cell growth, invasion, metastasis, and tumorigenesis, regulating cell apoptosis, cell cycle, and epithelial to mesenchymal transition [2228]. However, the effect and mechanisms of miR-145 dysregulation involved in NPC development and progression remain unknown. Therefore, in this study, we investigated the effects of miR-145 on NPC cell migration, invasion, and metastasis. Moreover, fascin actinbundling protein 1 (FSCN1) was verified as a direct functional target of miR-145. Our findings demonstrated that miR-145 function as a tumor suppressor in NPC development and progression through targeting FSCN1, suggesting that miR-145/FSCN1 pathway may sever as a potential novel therapeutic target for patients with NPC. Six human NPC cell lines (CNE-1, CNE-2, C666-1, SUNE-1, HNE-1, and HONE-1) were maintained in RPMI-1640 (Invitrogen) supplemented with 10% FBS, the immortalized human nasopharyngeal epithelial cell line NP69 was maintained in Keratinocyte/serum-free medium (Invitrogen) supplemented with bovine pituitary extract (BD Biosciences), and 293FT cell line was grown in DMEM (Invitrogen) supplemented with 10% FBS. Thirty-six freshly-frozen biopsy NPC and fourteen normal nasopharyngeal epithelium tissue samples were collected from the pathology archives of Sun Yat-sen University Cancer Center. The protocol of this study was approved by the Institutional Ethical Review Committee of Sun Yat-sen University Cancer Center (L201402052), and written informed consent was obtained from each patient for the use of their tissue samples. RNA isolation and quantitative RT-PCR Total RNA was isolated from NPC cell lines and clinical samples using TRIzol reagent (Invitrogen). 2 g of total RNA was reverse-transcribed using M-MLV reverse transcriptase (Promega) and Bulge- Loop specific RT-primers (RiboBio) or random primers (Promega) for detecting miRNA or mRNA expression, respectively. Quantitative PCR reactions we (...truncated)