Zhou Bin, Sun Chuandong, Hu Xiao, Zhan Hanxiang, Zou Hao, Feng Yujie, Qiu Fabo, Zhang Shun, Wu Liqun, Zhang Bingyuan
Department of Hepatopancreatobiliary Surgery, the Affiliated Hospital of Qingdao University, Qingdao, China.
Department of General Surgery, Qilu Hospital, Shandong University, Jinan, China.
Cell Physiol Biochem. 2017;44(5):1867-1881. doi: 10.1159/000485876. Epub 2017 Dec 8.
BACKGROUND/AIMS: Doublecortin-like kinase 1 (DCLK1) is emerging as a tumor-specific stem cell marker in pancreatic cancer (PC). MicroRNA-195 (miR-195) plays an important role in many types of tumors. However, the roles of DCLK1 in cancer and miRNAs that directly regulate DCLK1 have not been elucidated. The goal of this study is to assess the effects of miR-195 on inhibiting DCLK1 and to clarify the regulating mechanism of miR-195-DCLK1 in PC cells.
The expression of DCLK1 protein and miR-195 in PC tissues and adjacent healthy pancreatic tissues was detected by Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively and the correlation between overall survival of PC patients and expression of DCLK1 was measured by Kaplan-Meier analysis. Bioinformatics tools were used to identify the target gene of miR-195. Effects of miR-195 and DCLK1 on proliferation and cell cycle of PC cells were analyzed by MTT, colony formation assays and flow cytometry. Transwell and wound-healing experiments were employed to examine the cellular migration and invasion. A xenograft mouse model was also used to test the effects of miR-195 on tumor growth and metastasis in vivo.
The expression level of DCLK1 and miR-195 shows an inverse correlation in PC tissues and cell lines. A higher DCLK1 level is associated with higher TNM (tumor, node, and metastasis) stage, higher rate of lymph node metastasis, and poor survival. Luciferase reporter assay shows that miR-195 directly targets DCLK1. Overexpression of miR-195 inhibits proliferation, migration and invasion of PC cells, whereas downregulation of miR-195 has an opposite role. These actions were similar to the effects of knockdown and overexpression of DCLK1, respectively.
These data suggest that miR-195 has tumor suppressor roles in PC by targeting DCLK1. MiR-195-DCLK1 pathway may provide insight into PC progression and represent a novel, promising diagnostic and therapeutic target for PC.
背景/目的：双皮质素样激酶1（DCLK1）正逐渐成为胰腺癌（PC）中一种肿瘤特异性干细胞标志物。微小RNA-195（miR-195）在多种肿瘤中发挥重要作用。然而，DCLK1在癌症中的作用以及直接调控DCLK1的微小RNA尚未阐明。本研究的目的是评估miR-195对抑制DCLK1的作用，并阐明miR-195-DCLK1在PC细胞中的调控机制。
分别采用蛋白质免疫印迹法和定量逆转录聚合酶链反应（qRT-PCR）检测PC组织及相邻正常胰腺组织中DCLK1蛋白和miR-195的表达，并通过Kaplan-Meier分析评估PC患者总生存期与DCLK1表达之间的相关性。利用生物信息学工具鉴定miR-195的靶基因。通过MTT法、集落形成实验和流式细胞术分析miR-195和DCLK1对PC细胞增殖和细胞周期的影响。采用Transwell实验和伤口愈合实验检测细胞迁移和侵袭能力。还利用异种移植小鼠模型在体内测试miR-195对肿瘤生长和转移的影响。
在PC组织和细胞系中，DCLK1和miR-195的表达水平呈负相关。较高的DCLK1水平与较高的TNM（肿瘤、淋巴结、转移）分期、较高的淋巴结转移率及较差的生存率相关。荧光素酶报告基因实验表明miR-195直接靶向DCLK1。miR-195的过表达抑制PC细胞的增殖、迁移和侵袭，而miR-195的下调则起相反作用。这些作用分别类似于DCLK1基因敲低和过表达的效果。
这些数据表明miR-195通过靶向DCLK1在PC中发挥肿瘤抑制作用。MiR-195-DCLK1通路可能为深入了解PC的进展提供线索，并代表一种用于PC的新型、有前景的诊断和治疗靶点。
