Li Jian, Wang Yunchao, Ge Jiayun, Li Wenhua, Yin Liangyu, Zhao Zhiping, Liu Songsong, Qin Huan, Yang Jiali, Wang Lijiang, Ni Bing, Liu Yongkang, Wang Huaizhi
Institute of Hepatopancreatobiliary Surgery, Southwest Hospital, Third Military Medical University, Chongqing, China.
Department of General Surgery, 452 Hospital of PLA, Chengdu, China.
Cell Physiol Biochem. 2018;51(1):262-277. doi: 10.1159/000495228. Epub 2018 Nov 19.
BACKGROUND/AIMS: Cancer stem cells (CSCs) are largely responsible for tumor relapse and metastatic behavior. Doublecortin-like kinase 1 (DCLK1) was recently reported to be a biomarker for gastrointestinal CSCs and involved in the epithelial-mesenchymal transition (EMT) and tumor progression. B cell-specific Moloney murine leukemia virus insertion site 1 (Bmi-1) is a crucial regulator of CSC self-renewal, malignant transformation and EMT, and a previous study from our group showed that Bmi-1 is upregulated in pancreatic cancer progression and participates in EMT. However, it remains unclear whether DCLK1 is involved in pancreatic cancer or whether DCLK1 is associated with the altered level of Bmi-1 expression.
The correlation of DCLK1 expression and clinical features of pancreatic cancer was analyzed in 210 paraffin-embedded archived pancreatic cancer specimens by immunohistochemical analysis. The biological effects of DCLK1 siRNA on cells were investigated by examining cell proliferation using a cell counting kit and cell colony assays, cell migration by wound healing assay and cell invasion by Transwell invasion assay. We further investigated the effect of therapeutic siRNA targeting DCLK1 on pancreatic cancer cell growth in vivo. Moreover, the molecular mechanism by which DCLK1 upregulates Bmi-1 expression was explored using real-time PCR, western blotting and Co-immunoprecipitation assay.
DCLK1 is overexpressed in pancreatic cancer and is related to metastasis and prognosis. Knockdown of DCLK1 markedly suppressed cell growth in vitro and in vivo and also inhibited the migration and invasion of pancreatic cancer cells. Furthermore, we found that DCLK1 silencing could inhibit EMT in cancer cells via downregulation of Bmi-1 and the mesenchymal markers Snail and Vimentin and upregulation of the epithelial marker E-cadherin. Moreover, high DCLK1 expression in human pancreatic cancer samples was associated with a mesenchymal phenotype and increased cell proliferation. Further co-immunoprecipitation indicated that DCLK1 did not interact with Bmi-1 directly.
Our data suggest that upregulation of DCLK1 may contribute to pancreatic cancer metastasis and poor prognosis by increasing Bmi-1 expression indirectly. The findings indicate that inhibiting DCLK1 expression might be a novel strategy for pancreatic cancer therapy.
背景/目的：癌症干细胞（CSCs）在很大程度上导致肿瘤复发和转移行为。最近有报道称，双皮质素样激酶1（DCLK1）是胃肠道CSCs的生物标志物，并参与上皮-间质转化（EMT）和肿瘤进展。B细胞特异性莫洛尼鼠白血病病毒插入位点1（Bmi-1）是CSC自我更新、恶性转化和EMT的关键调节因子，我们团队之前的一项研究表明，Bmi-1在胰腺癌进展过程中上调并参与EMT。然而，尚不清楚DCLK1是否参与胰腺癌，或者DCLK1是否与Bmi-1表达水平的改变有关。
通过免疫组织化学分析，对210份石蜡包埋的存档胰腺癌标本中DCLK1表达与胰腺癌临床特征的相关性进行分析。通过使用细胞计数试剂盒和细胞集落试验检测细胞增殖、伤口愈合试验检测细胞迁移以及Transwell侵袭试验检测细胞侵袭，研究DCLK1 siRNA对细胞的生物学效应。我们进一步研究了靶向DCLK1的治疗性siRNA对体内胰腺癌细胞生长的影响。此外，使用实时PCR、蛋白质印迹法和免疫共沉淀试验探索DCLK1上调Bmi-1表达的分子机制。
DCLK1在胰腺癌中过表达，与转移和预后相关。敲低DCLK1可显著抑制体外和体内细胞生长，并抑制胰腺癌细胞的迁移和侵袭。此外，我们发现DCLK1沉默可通过下调Bmi-1、间充质标志物Snail和波形蛋白以及上调上皮标志物E-钙黏蛋白来抑制癌细胞中的EMT。此外，人胰腺癌样本中高DCLK1表达与间充质表型和细胞增殖增加相关。进一步的免疫共沉淀表明，DCLK1不直接与Bmi-1相互作用。
我们的数据表明，DCLK1的上调可能通过间接增加Bmi-1表达而促进胰腺癌转移和不良预后。这些发现表明，抑制DCLK1表达可能是胰腺癌治疗的一种新策略。
