Ma Lixin, Zhai Bo, Zhu Huaqiang, Li Weidong, Jiang Wenjing, Lei Liwang, Zhang Shujun, Qiao Haiquan, Jiang Xian, Sun Xueying
1Department of General Surgery, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001 China.
2Department of General Surgery, The Fourth Affiliated Hospital of Harbin Medical University, Harbin, 150001 China.
Cancer Cell Int. 2019 Sep 27;19:248. doi: 10.1186/s12935-019-0963-2. eCollection 2019.
Neuropilin-1 (NRP-1) is a non-tyrosine kinase receptor interacting with multiple signaling pathways that underpin the biological behavior and fate of cancer cells. However, in pancreatic cancer, the mechanisms underlying the function of NRP-1 in cell proliferation and metastasis and the involvement of regulatory upstream miRNAs remain unclear.
Potential miRNAs were mined by using multiple bioinformatics prediction tools and validated by luciferase assays. The expression of NRP-1 and miRNA-141 (miR-141) in pancreatic tissues and cells was examined by immunohistochemistry, immunoblotting and/or real-time RT-PCR. Stable transfected cells depleted of NRP-1 were generated, and regulatory effects of miR-141 were investigated by transfecting cells with miR-141 mimics and anti-miR-141. Assays of cell viability, proliferation, cell cycle distribution, transwell migration and cell scratch were employed. Xenograft tumor models were established to assess the effects of NRP-1 depletion on tumorigenesis and liver metastasis, and therapeutic effects of miR-141 on tumor growth. The role of miR-141/NRP-1 axis in regulating epithelial-mesenchymal transition (EMT) by co-interacting the TGF-β pathway was examined.
In this study, of 12 candidate miRNAs identified, miR-141 showed the strongest ability to regulate NRP-1. In pancreatic cancer tissues and cells, the expression level of NRP-1 was negatively correlated with that of miR-141. NRP-1 was highly expressed in pancreatic cancer tissues compared with normal pancreatic tissues, and its expression levels were positively correlated with tumor grade, lymph metastasis and AJCC staging. NRP-1 depletion inhibited cell proliferation by inducing cell cycle arrest at the G0/G1 phase through upregulating p27 and downregulating cyclin E and cyclin-dependent kinase 2, and reduced cell migration by inhibiting EMT through upregulating E-cadherin and downregulating Snail and N-cadherin. Through downregulating NRP-1, miR-141 mimics showed a similar effect as NRP-1 depletion on cell proliferation and migration. NRP-1 depletion suppressed tumor growth and liver metastasis and miR-141 mimics inhibited the growth of established tumors in mice. NRP-1 depletion and/or miR-141 mimics inhibited the activation of the TGF-β pathway stimulated by TGF-β ligand.
The present results indicate that NRP-1 is negatively regulated by miR-141 and the miR-141/NRP-1 axis may serve as potentially valuable biomarkers and therapeutic targets for pancreatic cancer.
神经纤毛蛋白-1（NRP-1）是一种非酪氨酸激酶受体，可与多种信号通路相互作用，这些信号通路支撑着癌细胞的生物学行为和命运。然而，在胰腺癌中，NRP-1在细胞增殖和转移中的功能机制以及上游调控性微小RNA（miRNA）的参与情况仍不清楚。
使用多种生物信息学预测工具挖掘潜在的miRNA，并通过荧光素酶测定进行验证。通过免疫组织化学、免疫印迹和/或实时逆转录聚合酶链反应检测胰腺组织和细胞中NRP-1和miRNA-141（miR-141）的表达。构建稳定转染的NRP-1缺失细胞，通过用miR-141模拟物和抗miR-141转染细胞来研究miR-141的调控作用。采用细胞活力、增殖、细胞周期分布、Transwell迁移和细胞划痕试验。建立异种移植瘤模型以评估NRP-1缺失对肿瘤发生和肝转移的影响，以及miR-141对肿瘤生长的治疗作用。研究miR-141/NRP-1轴通过与转化生长因子-β（TGF-β）途径共同作用在调节上皮-间质转化（EMT）中的作用。
在本研究中，在鉴定出的12种候选miRNA中，miR-141显示出最强的调节NRP-1的能力。在胰腺癌组织和细胞中，NRP-1的表达水平与miR-141呈负相关。与正常胰腺组织相比，NRP-1在胰腺癌组织中高表达，其表达水平与肿瘤分级、淋巴结转移和美国癌症联合委员会（AJCC）分期呈正相关。NRP-1缺失通过上调p27并下调细胞周期蛋白E和细胞周期蛋白依赖性激酶2诱导细胞周期停滞在G0/G1期，从而抑制细胞增殖，并通过上调E-钙黏蛋白并下调Snail和N-钙黏蛋白抑制EMT，进而减少细胞迁移。通过下调NRP-1，miR-141模拟物在细胞增殖和迁移方面显示出与NRP-1缺失类似的作用。NRP-1缺失抑制肿瘤生长和肝转移，miR-141模拟物抑制小鼠体内已形成肿瘤的生长。NRP-1缺失和/或miR-141模拟物抑制TGF-β配体刺激的TGF-β途径的激活。
目前的结果表明，miR-141对NRP-1具有负调控作用，miR-141/NRP-1轴可能是胰腺癌潜在的有价值的生物标志物和治疗靶点。
