Role of miRNA-375 in Invasive Phenotype of Head and Neck Squamous Cell Carcinoma

描述（由申请人提供）：我们的研究小组评估了头颈部鳞状细胞癌（HNSCC）患者的肿瘤和正常样本中miRNA的总体表达模式;与配对的正常样本相比，我们将miR-375鉴定为肿瘤样本中最一致下调的miRNA。miR-375表达最低四分位数的患者疾病特异性生存率显著降低，局部区域复发和远处转移的频率增加。我推测miR-375通过抑制特定蛋白靶点的水平来抑制HNSCC的侵袭性表型。miR-375的低表达与预后不良和远处转移的高度相关性使我想研究miR-375的表达如何导致HNSCC侵袭性的改变。我已经观察到前体miR-375转导子在体外具有降低的侵袭。此外，初步的基因表达微阵列分析的转导已经确定了下调表达的基因具有相关的功能与细胞运动性和侵袭;这些基因的表达的一些也已经与HNSCC患者的生存。本研究的目的是：1）利用稳定的慢病毒转导子研究miR-375对HNSCC细胞系侵袭特性的影响。稳定的转导细胞系将用于评估miR-375表达水平是否影响体外生长、侵袭、趋化性和基质降解，以及三维嵌入生长、三维侵袭与反向侵袭和器官型侵袭测定。此外，从UMSCC 47转导细胞系产生的HNSCC口底异种移植物将用于评估miR-375表达水平对肿瘤生长、体内侵袭和转移的影响。2)确定HNSCC中特定的miR-375靶基因和改变的信号通路。稳定的慢病毒转导子将用于通过用全基因组微阵列分析RNA表达和通过SILAC（细胞培养物中细胞的稳定同位素标记）分析miR-375的抑制性抑制的推定靶标来鉴定miR-375的潜在靶基因。免疫途径分析将用于鉴定miR-375的潜在靶点所涉及的细胞功能和信号传导途径。将通过qRT-PCR和/或蛋白质印迹分析确认推定靶标的表达水平。miR-375与推定的靶mRNA转录物之间的相互作用将通过荧光素酶报告基因测定来证实。我们的小组已经生成了一个mRNA微阵列数据和来自患者的原发性肿瘤的全球蛋白质组学数据沿着每个样本的临床数据的数据库。在我的分析中确定的任何候选miR-375靶点的疾病相关性将通过细胞系数据与原发性肿瘤患者来源数据的相关性进行评估。下调miR-375推定靶标的表型效应的功能验证将通过siRNA敲减和转导细胞中靶标的拯救来进行。 公共卫生相关性：头颈部鳞状细胞癌是全球第六大常见恶性肿瘤，在过去30年中，HNSCC患者的五年生存率几乎没有改善。预后生物标志物的鉴定可能会导致改善治疗策略，从而改善患者的预后。从这个项目中，我们期望通过探索miR-375在HNSCC细胞系中的分子机制，深入了解miR-375表达水平如何影响HNSCC患者的预后和远处转移的发生率。

DESCRIPTION (provided by applicant): Our research group has assessed the global expression patterns of miRNAs in both tumor and normal samples from patients with head and neck squamous cell carcinoma (HNSCC); we identified miR-375 as the most consistently down-regulated miRNA in tumor samples when compared to paired normal samples. Patients in the lowest quartile of miR-375 expression had significantly decreased disease-specific survival, increased frequency of local regional recurrence and distant metastasis. I hypothesize that miR-375 suppresses the invasive phenotype of HNSCC through repression of the levels of specific protein targets. The highly significant association of lower miR-375 expression in patients with poor prognosis and distant metastasis led me to want to investigate how miR-375 expression causes alterations in the invasive properties of HNSCC. I have observed that precursor miR-375 transductants have reduced invasion in vitro. Furthermore, preliminary gene expression microarray analysis of the transductants has identified the down-regulated expression of genes having functions associated with cell motility and invasion; the expression of some of these genes also have been previously been correlated with HNSCC patient survival. This proposal addresses the invasive phenotype hypothesis with the following aims: 1) Utilize stable lentiviral transductants to investigate the effects of miR-375 on the invasive properties of HNSCC cell lines. Stable transductant cell lines will be used to assess whether miR-375 expression levels affects growth, invasion, chemotaxis and matrix degradation in vitro, as well as, three-dimensional embedded growth, three-dimensional invasion with inverted invasion and organotypic invasion assays. In addition HNSCC floor-of-mouth xenografts generated from UMSCC47 transductant cell lines will be used to evaluate the consequence of miR-375 expression levels on tumor growth, in vivo invasion and metastasis. 2) Identify specific miR-375 target genes and altered signaling pathways in HNSCC. Stable lentiviral transductants will be used to identify potential target genes of miR-375 by analyzing both RNA expression with whole genome microarrays and translationally repressed putative targets of miR-375 by SILAC (stable isotope labeling of cells in cell culture). Ingenuity Pathway Analysis will be used to identify cellular functions and signaling pathways that the potential targets of miR-375 are involved. The expression levels of the putative targets will be confirmed by qRT-PCR and/or western blot analysis. The interaction between miR-375 and the putative target mRNA transcripts will be confirmed by luciferase reporter assays. Our group has generated a database of mRNA microarray data and global proteomic data on primary tumors from patients along with clinical data on each sample. The disease relevance to any candidate miR-375 target identified in my analysis will be assessed by correlations of cell line data with patient-derived data from primary tumors. Functional validation of the phenotypic effects of the down-regulation of putative targets of miR-375 will be performed by siRNA knockdown and rescue of the targets in the transductant cells. PUBLIC HEALTH RELEVANCE: Head and neck squamous cell carcinoma is the sixth most common malignancy worldwide and the five-year survival rate for patients with HNSCC has shown little improvement over the last 30 years. The identification of prognostic biomarkers could lead to improved therapeutic strategies that may improve patient outcomes. From this project we anticipate gaining insight into how miR-375 expression levels can impact HNSCC patient outcome and incidence of distant metastasis through the exploration of its molecular mechanisms in HNSCC cell lines.