双肾上腺素样激酶1不同剪接体通过激活JNK通路增强胰腺癌细胞的增生能力

目的 研究双肾上腺素样激酶1（doublecortin-like kinase 1，DCLK1）长、短亚型对人胰腺癌增生能力的影响，并探讨其分子机制。方法 分别将空载（PCMV6-AC-GFP）、DCLK1亚型1和DCLK1亚型4真核表达质粒转染胰腺癌PANC-1细胞，G418筛选法构建DCLK1不同亚型稳定过表达的细胞系；通过定量实时聚合酶链式反应（quantitative real time polymerase chain reaction，qRT-PCR）和Western blotting法鉴定DCLK1长、短亚型的表达情况；实时无标记细胞分析仪（real-time cell analysis，RTCA）技术检测DCLK1长、短亚型表达对PANC-1细胞增生能力的影响；Western blotting法检测DCLK1对丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）通路的影响；利用c-Jun氨基末端激酶（c-Jun N-terminal kinase，JNK）通路特异性抑制剂SP600125处理DCLK1稳转细胞，检测JNK通路抑制对胰腺癌细胞增生能力的影响。结果 DCLK1长、短亚型的过表达显著促进胰腺癌细胞的增生能力（P<0.05），并且促进MAPK通路中JNK的磷酸化水平以及JNK通路靶分子CMYC、CD44和CyclinD1的表达（P<0.05），而对MAPK通路中细胞外调节蛋白激酶（extracellular regulated protein kinases，ERK）和p38的磷酸化无明显影响（P>0.05）；SP600125抑制JNK的磷酸化，可以显著降低DCLK1对JNK通路的激活以及对胰腺癌细胞的促增生能力（P<0.05）。结论 DCLK1长、短亚型均可以通过激活JNK通路促进胰腺癌细胞的增生能力。

DCLK1 alternative variants enhance proliferation of pancreatic cancer cells by activating JNK pathway

Objective To investigate the effects of long-and short-isoform of doublecortin-like kinase 1 (DCLK1) on the proliferation of human pancreatic cancer and further to explore the molecular mechanism. Methods The control (PCMV6-AC-GFP), DCLK1 isoform 1 and DCLK1 isoform 4 eukaryotic expression plasmids were transfected into pancreatic cancer PANC-1 cells. G418 screening method was used to construct stable cell lines overexpressing long-and short-isoform of DCLK1. Expression of long-and short-isoform of DCLK1 were determined by quantitative real time polymerase chain reaction(qRT-PCR) and Western blotting. The effect of long-and short-isoform of DCLK1 on the proliferation of PANC-1 cells were detected with real-time cell analysis (RTCA) technology. The effect of DCLK1 on mitogen-activated protein kinase (MAPK) pathway was detected with Western blotting. DCLK1 stabilizing cells were treated with specific c-Jun N-terminal kinase (JNK) pathway inhibitor SP600125 to detect the effect of JNK pathway inhibition on the proliferation of pancreatic cancer cells. Results Overexpression of long-and short-isoform of DCLK1 remarkably promoted the proliferation of pancreatic cancer cells (P<0.05), increased the phosphorylation of JNK in MAPK pathway and the expression of target molecules CMYC, CD44 and CyclinD1 in JNK pathway (P<0.05). No significant influence on the phosphorylation of ERK and p38 were detected (P>0.05). SP600125 inhibited the phosphorylation of JNK and markedly decreased the activation of JNK pathway and the proliferation of pancreatic cancer cells by DCLK1 (P<0.05). Conclusion Both long-and short-isoform of DCLK1 promote the proliferation of pancreatic cancer cells via activating JNK pathway.