Dysregulation of MicroRNA-34a Expression in Head and Neck Squamous Cell Carcinoma Promotes Tumor Growth and Tumor Angiogenesis

BackgroundMicroRNAs (miRs) are small non-coding RNAs that play an important role in cancer development where they can act as oncogenes or as tumor-suppressors. miR-34a is a tumor-suppressor that is frequently downregulated in a number of tumor types. However, little is known about the role of miR-34a in head and neck squamous cell carcinoma (HNSCC). Methods and ResultsmiR-34a expression in tumor samples, HNSCC cell lines and endothelial cells was examined by real time PCR. Lipofectamine-2000 was used to transfect miR-34a in HNSCC cell lines and human endothelial cells. Cell-proliferation, migration and clonogenic survival was examined by MTT, Xcelligence system, scratch assay and colony formation assay. miR-34a effect on tumor growth and tumor angiogenesis was examined by in vivo SCID mouse xenograft model. Our results demonstrate that miR-34a is significantly downregulated in HNSCC tumors and cell lines. Ectopic expression of miR-34a in HNSCC cell lines significantly inhibited tumor cell proliferation, colony formation and migration. miR-34a overexpression also markedly downregulated E2F3 and survivin levels. Rescue experiments using microRNA resistant E2F3 isoforms suggest that miR-34a-mediated inhibition of cell proliferation and colony formation is predominantly mediated by E2F3a isoform. In addition, tumor samples from HNSCC patients showed an inverse relationship between miR-34a and survivin as well as miR-34a and E2F3 levels. Overexpression of E2F3a completely rescued survivin expression in miR-34a expressing cells, thereby suggesting that miR-34a may be regulating survivin expression via E2F3a. Ectopic expression of miR-34a also significantly inhibited tumor growth and tumor angiogenesis in a SCID mouse xenograft model. Interestingly, miR-34a inhibited tumor angiogenesis by blocking VEGF production by tumor cells as well as directly inhibiting endothelial cell functions. ConclusionsTaken together, these findings suggest that dysregulation of miR-34a expression is common in HNSCC and modulation of miR34a activity might represent a novel therapeutic strategy for the treatment of HNSCC.

背景：微小RNA（microRNAs, miRs）是一类小型非编码RNA，在癌症发生发展中发挥关键调控作用，可作为癌基因或抑癌基因行使功能。miR-34a作为一种抑癌miR，在多种肿瘤类型中均呈现频繁下调现象。然而，目前针对miR-34a在头颈部鳞状细胞癌（head and neck squamous cell carcinoma, HNSCC）中的作用机制仍知之甚少。
方法与结果：本研究通过实时定量PCR检测了肿瘤样本、HNSCC细胞系及内皮细胞中miR-34a的表达水平。采用Lipofectamine-2000转染试剂将miR-34a转染至HNSCC细胞系与人类内皮细胞中。分别通过MTT法、Xcelligence实时细胞分析系统、划痕实验及集落形成实验，检测细胞增殖、迁移能力与克隆存活能力。通过重症联合免疫缺陷（severe combined immunodeficiency, SCID）小鼠异种移植模型，探究miR-34a对肿瘤生长及肿瘤血管生成的影响。
本研究结果显示，miR-34a在HNSCC肿瘤组织及细胞系中均显著下调。在HNSCC细胞系中外源过表达miR-34a，可显著抑制肿瘤细胞的增殖、集落形成与迁移能力。miR-34a过表达还可显著下调E2F3与存活素（survivin）的表达水平。采用抗miR的E2F3异构体进行拯救实验的结果表明，miR-34a介导的细胞增殖与集落形成抑制作用，主要由E2F3a异构体介导。此外，HNSCC患者的肿瘤样本中，miR-34a的表达水平与survivin及E2F3的表达均呈负相关关系。过表达E2F3a可完全恢复过表达miR-34a的细胞中survivin的表达水平，由此提示miR-34a可能通过E2F3a调控survivin的表达。在SCID小鼠异种移植模型中，外源过表达miR-34a还可显著抑制肿瘤生长与肿瘤血管生成。值得注意的是，miR-34a可通过阻断肿瘤细胞产生血管内皮生长因子（vascular endothelial growth factor, VEGF），以及直接抑制内皮细胞功能，从而发挥抗肿瘤血管生成的作用。
结论：综上，本研究结果表明，miR-34a表达失调在HNSCC中十分普遍，调控miR-34a的活性或可成为治疗HNSCC的新型治疗策略。