Department of Otolaryngology-Head and Neck Surgery, New York University School of Medicine, New York, NY, USA.
Otolaryngol Head Neck Surg. 2010 Oct;143(4):525-30. doi: 10.1016/j.otohns.2010.06.912.
Radiotherapy, an essential modality in cancer treatment, frequently induces fibrotic processes in the skin, including accumulation of extracellular matrix. Transforming growth factor-β is essential in regulating extracellular matrix gene expression and is dependent on Smad3, an intracellular mediator/transcription factor. Our study characterized the genetic expression involved in extracellular matrix accumulation during radiation-induced fibrosis. We performed Smad3 gene silencing in an attempt to abrogate the effects of radiation.
Laboratory research.
University laboratory.
C57 murine dermal fibroblasts were irradiated with 20 Gy RNA isolated (0, 6, 12, 24, 48, 72 hours postirradiation) and mRNA analyzed (reverse transcriptase polymerase chain reaction) for known regulators (Smad3, interleukin-13 [IL-13]), tumor necrosis factor-α [TNF-α]) and mediators of fibrosis (collagen 1A1 [Col1A1]), TGF-β, matrix metalloprotease-1 and -2 (MMP-1, MMP-2), and tissue inhibitor of metalloprotease-1 (TIMP-1). Smad3 gene expression was silenced using siRNA in an effort to restore an unirradiated gene profile.
Following irradiation, there was a steady increase in mRNA expression of Smad3, IL-13, TGF-β, Col1A1, MMP-2, TIMP-1, with peak at 12 to 24 hours and subsequent decline by 72 hours. TNF-α expression remained elevated throughout. MMP-1 showed minimal expression initially, which decreased to negligible by 72 hours. Inhibition of Smad3 significantly decreased expression of Col1A1, TGF-β, MMP-2, and TIMP-1. IL-13 and TNF-α expression was not affected by Smad3 silencing.
We have characterized the early-phase mRNA expression profiles of the major mediators of radiation-induced fibrosis. Smad3 siRNA effectively abrogated the elevation of Col1A1, TGF-β, TIMP-1, and MMP-2. IL-13 and TNF-α were unaffected by Smad3 silencing and appear to be minor regulators in fibrosis. These findings suggest a therapeutic rationale for Smad3 silencing in vivo.
放射治疗是癌症治疗中不可或缺的一种方法，它经常会导致皮肤纤维化过程，包括细胞外基质的积累。转化生长因子-β在调节细胞外基质基因表达中起着至关重要的作用，并且依赖于细胞内介质/转录因子 Smad3。我们的研究描述了放射诱导纤维化过程中细胞外基质积累所涉及的遗传表达。我们进行了 Smad3 基因沉默实验，试图阻断放射的影响。
实验室研究。
大学实验室。
用 20 Gy 射线照射 C57 鼠真皮成纤维细胞，分别在照射后 0、6、12、24、48、72 小时提取 RNA 并进行 mRNA 分析（逆转录聚合酶链反应），检测已知的调节剂（Smad3、白细胞介素-13 [IL-13]）、肿瘤坏死因子-α[TNF-α]）和纤维化介质（胶原 1A1[Col1A1]）、TGF-β、基质金属蛋白酶-1 和 -2（MMP-1、MMP-2）和金属蛋白酶组织抑制剂-1（TIMP-1）。通过 siRNA 沉默 Smad3 基因，以恢复未照射的基因谱。
照射后，Smad3、IL-13、TGF-β、Col1A1、MMP-2、TIMP-1 的 mRNA 表达水平持续增加，在 12 至 24 小时达到峰值，随后在 72 小时下降。TNF-α 的表达一直升高。MMP-1 的初始表达水平较低，72 小时后降至可忽略水平。Smad3 抑制显著降低 Col1A1、TGF-β、MMP-2 和 TIMP-1 的表达。Smad3 沉默对 IL-13 和 TNF-α 的表达没有影响。
我们已经描述了放射诱导纤维化的主要介质的早期 mRNA 表达谱。Smad3 siRNA 有效阻断了 Col1A1、TGF-β、TIMP-1 和 MMP-2 的升高。Smad3 沉默对 IL-13 和 TNF-α 的表达没有影响，并且在纤维化中似乎是次要调节剂。这些发现为 Smad3 体内沉默提供了治疗依据。
