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

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

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(mitogenactivated 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Cyclin D1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。

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 isoform1 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 Cyclin D1 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.