Department of Endocrinology, Heilongjiang Provincial Hospital, Harbin 150001, PR China.
Department of Endocrinology, Heilongjiang Provincial Hospital, Harbin 150001, PR China.
Biomed Pharmacother. 2018 Aug;104:686-698. doi: 10.1016/j.biopha.2018.05.078. Epub 2018 May 25.
Invasion and metastasis of papillary thyroid carcinoma (PTC) significantly affects prognosis and quality of life of patients. Herein, we explored the binding relationship of long noncoding RNA PVT1 as ceRNA to microRNA-30a (miR-30a), and their effect on the development of PTC through regulating insulin like growth factor 1 receptor (IGF1R).
PTC and adjacent normal tissues were collected, where the qRT-PCR and western blot assay were employed to evaluate the expression levels of PVT1, miR-30a and IGF1R. The correlation between PVT1 expression and clinicopathological characteristics of PTC patients was observed. PTC cell lines with the most/least significant difference from normal thyroid cells were selected and treated with siRNA PVT1 or overexpression PVT1 plasmids, miR-30a mimics or miR-30a inhibitors. Nucleus and cytoplasm segmentation was used to identify subcellular fractionation of PVT1. The binding relationship of PVT1 to miR-30a and the targeting relationship of miR-30a to IGF1R were confirmed by using bioinformatic prediction program, dual-luciferase reporter gene assay and RNA-pull down. Cell viability, cell cycle and apoptosis, invasion and migration capacities were assessed by MTT, flow cytometry, Transwell assay and scratch test, respectively. Western blot assay was employed to examine protein expression of IGF1R, apoptosis-related factors (caspase-3, cleaved capase-3) and epithelial-mesenchymal transition (EMT)-related factors (E-cadherin, Vimentin).
In the PTC tissues and cells, PVT1 and IGF1R were highly expressed and miR-30a was poorly expressed. PVT1 exerted its effects on PTC mainly in the cytoplasm. The PVT1 expression was correlated with TNM staging, LNM and tumor infiltration of PTC. The competitive binding of PVT1 to miR-30a enhanced expression of IGF1R. In the in vitro experiments, BCPAP and TPC-1 cells were selected. When subjected to siRNA PVT1 or miR-30a mimics, BCPAP and TPC-1 cells exhibited inhibited proliferation, cell cycle progression, invasion, migration, EMT (increased E-cadherin and reduced Vimentin) and promoted apoptosis (reduced caspase-3 and increased cleaved capase-3), and moreover, the expression of IGF1R was reduced.
This study provides evidence that long noncoding RNA PVT1 enhances the expression of IGF1R through competitive binding to miR-30a, whereby PVT1 facilitates the development of PTC.
甲状腺乳头状癌（PTC）的侵袭和转移显著影响患者的预后和生活质量。在此，我们通过研究长链非编码 RNA PVT1 作为 ceRNA 与 microRNA-30a（miR-30a）的结合关系及其对胰岛素样生长因子 1 受体（IGF1R）的调控作用，探讨其对 PTC 发生发展的影响。
收集 PTC 及癌旁正常组织，采用 qRT-PCR 和 Western blot 检测 PVT1、miR-30a 和 IGF1R 的表达水平，观察 PVT1 表达与 PTC 患者临床病理特征的关系。选择与正常甲状腺细胞差异最显著的 PTC 细胞系，用 siRNA PVT1 或过表达 PVT1 质粒、miR-30a 模拟物或 miR-30a 抑制剂处理。采用核质分离法鉴定 PVT1 的亚细胞定位。利用生物信息学预测程序、双荧光素酶报告基因检测和 RNA 下拉实验证实 PVT1 与 miR-30a 的结合关系以及 miR-30a 与 IGF1R 的靶向关系。采用 MTT、流式细胞术、Transwell 实验和划痕实验分别评估细胞活力、细胞周期和凋亡、侵袭和迁移能力，Western blot 检测 IGF1R、凋亡相关因子（caspase-3、cleaved caspase-3）和上皮间质转化（EMT）相关因子（E-cadherin、Vimentin）的蛋白表达水平。
在 PTC 组织和细胞中，PVT1 和 IGF1R 表达升高，miR-30a 表达降低。PVT1 主要在细胞质中发挥对 PTC 的作用。PVT1 表达与 PTC 的 TNM 分期、淋巴结转移和肿瘤浸润有关。PVT1 与 miR-30a 的竞争性结合增强了 IGF1R 的表达。在体外实验中，选择 BCPAP 和 TPC-1 细胞。当用 siRNA PVT1 或 miR-30a 模拟物处理时，BCPAP 和 TPC-1 细胞的增殖、细胞周期进展、侵袭、迁移、EMT（E-cadherin 增加，Vimentin 减少）和凋亡（caspase-3 减少，cleaved caspase-3 增加）受到抑制，IGF1R 的表达也降低。
本研究表明，长链非编码 RNA PVT1 通过与 miR-30a 的竞争性结合增强 IGF1R 的表达，从而促进 PTC 的发生发展。
