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

描述(申请人提供)：我们的研究小组评估了来自头颈部鳞状细胞癌(HNSCC)患者的肿瘤和正常样本中miRNAs的全球表达模式；我们发现，与配对的正常样本相比，miR-375是肿瘤样本中下调幅度最大的。MiR-375表达最低四分位数的患者的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。我假设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。在预后不良和远处转移的患者中，miR-375表达降低的相关性非常显著，这促使我想要研究miR-375表达如何导致HNSCC侵袭性的改变。我观察到前体miR-375转导子减少了体外侵袭。此外，对转导基因的初步基因表达芯片分析发现，与细胞运动和侵袭相关的基因表达下调；其中一些基因的表达也与HNSCC患者的生存相关。该建议针对侵袭性表型假说提出了以下目标：1)利用稳定的慢病毒转导系统研究miR-375对HNSCC细胞侵袭性的影响。稳定的转导细胞株将用于评估miR-375表达水平是否影响体外生长、侵袭、趋化和基质降解，以及三维包埋生长、三维侵袭与反向侵袭和器官型侵袭分析。此外，从UMSCC47转导细胞系产生的HNSCC口底移植将被用于评估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miR-375的特异性靶基因并改变信号转导途径。稳定的慢病毒转导载体将通过全基因组芯片分析RNA表达和SILAC(细胞培养中细胞稳定同位素标记)抑制miR-375的翻译假定靶点来鉴定miR-375的潜在靶基因。独创性通路分析将用于确定miR-375的潜在靶点所涉及的细胞功能和信号通路。可能的靶点的表达水平将通过qRT-PCR和/或蛋白质印迹分析来确认。MiR-375与可能的靶mRNA转录本之间的相互作用将通过荧光素酶报告基因的分析得到证实。我们的团队已经生成了一个数据库，其中包括来自患者的原发肿瘤的mRNA微阵列数据和全球蛋白质组数据，以及每个样本的临床数据。我的分析中确定的任何候选miR-375靶点与疾病的相关性将通过细胞系数据与患者来自原发肿瘤的数据的相关性来评估。MiR-375可能靶标下调的表型效应的功能验证将通过siRNA击倒和挽救转导细胞中的靶标来进行。 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患者的五年存活率几乎没有改善。预后生物标记物的识别可能会导致改善治疗策略，从而可能改善患者的预后。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的表达水平如何影响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.