miRNA-195调控DCLK1/ERK通路参与的胰腺癌生长转移机制研究

The small RNA and protein in the tumors interacting together play a huge decisive role for the activities of tumor cells. The protein kinase superfamily is widely involved in variable process of tumors. Through exon capture sequencing, miRNA expression profile sequencing, bioinformatics analysis and molecular biology research, our preliminary studies found that DCLK1 which is a member of protein kinase superfamily promote the proliferation and metastasis of pancreatic cancer cells, and is possibly regulated by miR-195, our further studies suggest that miR-195 and DCLK1 play their roles through the Ras-Raf-MEK1-ERK pathway. However, it is unclear the regulatory mechanism of miR-195 for DCLK1/ERK signaling pathway. Therefore, this study will identify the role of miR-195 for DCLK1 in vitro and in vivo, explore the characteristic molecular mechanisms which miR-195 through the protein kinase network regulating the proliferation and metastasis of pancreatic cancer. This study will solve: 1) to explore the relationship between miR-195 and DCLK1; 2) to study the possible mechanisms of regulatory role of miR-195 for the expression of DCLK1 affecting the function of the cells; 3) to illustrate miR-195 through regulating the expression of DCLK1 affect the ERK pathway, which can ultimately regulate the proliferation and metastasis of pancreatic cancer.

小分子RNA与蛋白分子相互作用影响肿瘤细胞的生命活动。蛋白激酶超家族广泛参与肿瘤的发展过程。前期研究中，我们通过外显子组捕获测序、miRNA测序、生物信息学和分子生物学研究发现蛋白激酶DCLK1促进胰腺癌的生长转移，并可能被miR-195调控，进一步研究发现可能通过Ras-Raf-MEK-ERK通路发挥作用。然而关于miR-195对DCLK1/ERK通路的调控机制尚不清楚。因此，本研究拟对调控DCLK1的miR-195进行体内外功能验证，对miR-195通过DCLK1调控的分子机制予以深入研究，并对miR-195和DCLK1在临床中的关系和意义进行探讨。拟解决：1）探寻miR-195与DCLK1之间的联系；2）研究miR-195调控DCLK1影响胰腺癌细胞功能的可能机制；3）阐明miR-195通过调控DCLK1参与的ERK通路，最终调节胰腺癌的生长转移。

Doublecortin-like激酶1（DCLK1） 在胰腺癌（PC）中作为一个特定的肿瘤干细胞标记物。microRNA-195（miR-195）在许多肿瘤细胞中发挥着重要作用。然而，通过microRNA抑制DCLK1的作用并没有被直接研究过。本项目旨在研究miR-195对DCLK1的抑制作用并阐明miR-195-DCLK1在胰腺癌细胞中的调节机制。首先，荧光素酶分析表明DCLK1是miR-195直接靶标。免疫组织化学染色和实时定量PCR分别检测DCLK1和miR-195在胰腺癌组织和细胞中的表达，结果表明DCLK1和miR-195有显著负相关，它们均与更高的TNM分期、更高淋巴结转移和较差的预后有关。体外和体内相关实验表明改变miR-195和DCLK1的表达水平可以影响PC细胞的增殖、迁移侵袭和转移能力。miR-195的过表达可以抑制PC细胞的增殖和迁移，而降低miR-195表达作用相反。这与DCLK1敲低和过表达产生的影响相一致。miR-195通过靶向调控DCLK1而调控与增殖和侵袭相关基因的表达，如p-ERK1/2、p-p38、p21、vimentin、E-cadherin、RhoA和MMP-9。这些结果表明，miR-195通过靶向调控DCLK1在PC中发挥肿瘤抑制作用。因此，miR-195-DCLK1途径可以为将来PC的诊断和治疗提供新的前景。