Effects of siRNA on RET/PTC3 junction oncogene in papillary thyroid carcinoma: from molecular and cellular studies to preclinical investigations.
Effects of siRNA on RET/PTC3 junction oncogene in papillary thyroid carcinoma: from molecular and cellular studies to preclinical investigations.
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DOI：
10.1371/journal.pone.0095964
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Massaad-Massade L
中科院分区：
文献类型：
--
作者：
Ali HM;Urbinati G;Chapuis H;Desmaele D;Bertrand JR;Couvreur P;Massaad-Massade L
RET/PTC3 连接癌基因是放射诱发的儿童乳头状甲状腺癌 (PTC) 的典型特征，潜伏期较短。由于 RET/PTC3 仅存在于肿瘤细胞中，因此代表了小干扰 RNA (siRNA) 特异性治疗的有趣靶标。我们的目的是在体外和体内证明 siRNA 对 RET/PTC3 敲低的分子和细胞作用，以用于治疗应用。首先，我们建立了一种稳定表达 RET/PTC3 连接癌基因的新型细胞系，命名为 RP3，与 NIH/3T3 小鼠成纤维细胞相比，该细胞系在裸鼠中具有致瘤性。在针对 RET/PTC3 测试的四种 siRNA 和五个浓度中，选择了有效的 siRNA RET/PTC3 和适当的剂量 (50 nM)，其显示出对基因 (RT-qPCR) 和蛋白质 (Western blot) 表达的显着抑制 (p<0.001)。该 siRNA 在 RP3 细胞（含有 RET/PTC3）中有效，但在 BHP10-3 SCmice 细胞系（含有 RET/PTC1）中无效，表明需要针对融合序列的特异性 siRNA 来靶向连接癌基因。体外 siRNA RET/PTC3 对 RP3 细胞活力（MTT 测定）和侵袭/迁移（IncuCyte 划痕试验）显示出显着（p<0.001）的抑制作用，阻断细胞周期在 G0/G1 期（流式细胞术），并通过 caspase-3 和 PARP1 裂解（WB）诱导细胞凋亡。裸鼠静脉注射后，siRNA RET/PTC3 的角鲨烯 (SQ) 纳米颗粒 (NPs) 显着 (p<0.001) 降低 RP3 肿瘤生长、癌基因和癌蛋白表达、诱导细胞凋亡并部分恢复分化（Ki67 降低）。因此，我们的研究结果高度支持使用 siRNA RET/PTC3-SQ NP 作为一种新的有前景的治疗方法，用于治疗受表达 RET/PTC3 的 PTC 影响的患者。
RET/PTC3 junction oncogene is typical of radiation-induced childhood papillary thyroid carcinoma (PTC) with a short latency period. Since, RET/PTC3 is only present in the tumour cells, thus represents an interesting target for specific therapy by small interfering RNA (siRNA). Our aim is to demonstrate in vitro and in vivo molecular and cellular effects of siRNA on RET/PTC3 knockdown for therapeutic application.First, we established a novel cell line stably expressing RET/PTC3 junction oncogene, named RP3 which was found tumorigenic in nude mice compared to NIH/3T3 mouse fibroblasts. Among four siRNAs and five concentrations tested against RET/PTC3, an efficient siRNA RET/PTC3 and an appropriate dose (50 nM) were selected which showed significant inhibition (p<0.001) of gene (RT-qPCR) and protein (Western blot) expressions. This siRNA was found efficient in RP3 cells (harbouring RET/PTC3) but non-efficient in BHP10-3 SCmice cell line (harbouring RET/PTC1) showing that a specific siRNA against fusion sequence is required to target the junction oncogene. In vitro siRNA RET/PTC3 showed significant (p<0.001) inhibitory effects on RP3 cell viability (MTT assay) and on invasion/migration (IncuCyte scratch test) with blockage of cell cycle at G0/G1 phase (flow cytometry) and induced apoptosis by caspase-3 and PARP1 cleavage (WB). After intravenous injection in nude mice, respective squalene (SQ) nanoparticles (NPs) of siRNA RET/PTC3 significantly (p<0.001) reduced RP3 tumour growth, oncogene and oncoprotein expressions, induced apoptosis and partially restored differentiation (decrease in Ki67). Hence, our findings highly support the use of siRNA RET/PTC3-SQ NPs as a new promising treatment for patients affected by PTC expressing RET/PTC3.
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