Department of Hepatobiliary Surgery, Shandong Provincial Hospital Affiliated to Shandong University, Jinan, China.
Key Laboratory of Hepatosplenic Surgery, Department of General Surgery, the First Affiliated Hospital of Harbin Medical University, Harbin, China.
Liver Int. 2018 Jan;38(1):125-135. doi: 10.1111/liv.13495. Epub 2017 Jul 5.
BACKGROUND & AIMS: Neuropilin-1 (NRP-1) activates signalling pathways as multifunctional co-receptors in cancer cells. However, its role and how it is regulated by miRNAs in cholangiocarcinoma (CCA) have not yet been investigated.
The expression of NRP-1, miR-320 and key molecules involved in cell proliferation, migration and related signalling pathways were detected by immunohistochemistry, immunoblotting and qRT-PCR. Stable transfectants depleted of NRP-1 were generated. The regulatory effect of miR-320 on NRP-1 was evaluated by luciferase reporter assays. Cell proliferation, cell cycle distribution and migration were examined. Xenograft tumour models were established to assess tumourigenesis, tumour growth and lung metastasis.
Cholangiocarcinoma tissues expressed higher levels of NRP-1 than adjacent normal biliary tissues, and its expression negatively correlated with miR-320. NRP-1 depletion inhibited cell proliferation and induced cell cycle arrest in the G1/S phase by upregulating p27, and downregulating cyclin E and cyclin-dependent kinase 2; and reduced cell migration by inhibiting the phosphorylation of focal adhesion kinase. NRP-1 depletion suppressed tumourigenesis, tumour growth and lung metastasis by inhibiting cell proliferation and tumour angiogenesis in experimental animals. Depletion of NRP-1 inhibited the activation of VEGF/VEGFR2, EGF/EGFR and HGF/c-Met pathways stimulated by respective ligands. MiR-320 negatively regulated the expression of NRP-1 by binding to the 3'-UTR of NRP-1 promoter, and miR-320 mimics inhibited cell proliferation and migration, and the growth of established tumours in animals by downregulating NRP-1.
The present results indicate that NRP-1 is negatively regulated by miR-320, and both of them may be potentially therapeutic targets for CCA.
神经纤毛蛋白-1（NRP-1）作为多功能共受体在癌细胞中激活信号通路。然而，其在胆管癌（CCA）中的作用及其如何被 miRNA 调控尚未得到研究。
通过免疫组织化学、免疫印迹和 qRT-PCR 检测 NRP-1、miR-320 和参与细胞增殖、迁移及相关信号通路的关键分子的表达。生成 NRP-1 耗尽的稳定转染细胞系。通过荧光素酶报告基因实验评估 miR-320 对 NRP-1 的调控作用。检测细胞增殖、细胞周期分布和迁移。建立异种移植肿瘤模型以评估肿瘤发生、肿瘤生长和肺转移。
与相邻的正常胆管组织相比，胆管癌组织表达更高水平的 NRP-1，其表达与 miR-320 呈负相关。NRP-1 耗竭通过上调 p27、下调细胞周期蛋白 E 和细胞周期蛋白依赖性激酶 2，抑制细胞增殖并诱导细胞周期停滞在 G1/S 期；并通过抑制黏着斑激酶的磷酸化来减少细胞迁移。在实验动物中，NRP-1 耗竭通过抑制细胞增殖和肿瘤血管生成来抑制肿瘤发生、肿瘤生长和肺转移。NRP-1 耗竭抑制了相应配体刺激的 VEGF/VEGFR2、EGF/EGFR 和 HGF/c-Met 通路的激活。miR-320 通过结合 NRP-1 启动子的 3'-UTR 负调控 NRP-1 的表达，miR-320 模拟物通过下调 NRP-1 抑制细胞增殖和迁移以及动物体内已建立肿瘤的生长。
本研究结果表明，NRP-1 受 miR-320 负调控，两者均可能成为 CCA 的潜在治疗靶点。
