长链非编码RNA n385229吸附miR-497对胰腺癌化疗耐药表型的调控作用

Chemoresistance is one of the key and the difficult subject of pancreatic cancer research. The mechanism of drug-resistance of pancreatic cancer remains unclear. Our previous study indicated that miR-497 re-sensitized pancreatic cancer cells to gemcitabine through direct downregulation of IGF-1R and FGFR1 protein expression, which had been partly published on Oncotarget. Bioinformatic analysis revealed the putative complementary sequences for the seed region of mir-497 in lncRNA-n385229. In addition, downregulation of lncRNA-n385229 significantly increased miR-497 expression levels, decreased the protein levels of IGF-1R and FGFR1, and re-sensitized cells to gemcitabine treatment. Based on our previous studies and literatures review, we speculated that lncRNA-n385229 might be a ceRNA and acted as a miR-497 "sponge" to involve in regulating chemoresistance of pancreatic cancer. This study will investigate the roles and mechanisms of lncRNA-n385229/miR-497/(IGF-1R/FGFR1）axis in regulating chemoresistance of pancreatic cancer by in vitro and in vivo experiments. We will also evaluate clinical values of lncRNA-n385229/miR-497 in predicting sensitivity to chemotherapy and prognosis of pancreatic cancer patients. This study will help us further investigate the mechanisms of chemoresistance in pancreatic cancer, and provide a new molecular target for assessing chemosensitivity and reversing drug resistance.

胰腺癌化疗耐药机制是目前研究的热点和难点。我们前期研究发现miR-497负向调控IGF-1R和FGFR1，参与胰腺癌化疗敏感性调控，部分成果已发表至Oncotarget杂志；生物信息学分析发现lncRNA-n385229序列上有多个miR-497种子区的结合位点，下调lncRNA-n385229，促进miR-497表达，降低IGF-1R和FGFR1的蛋白表达水平。因此，我们推测：lncRNA-n385229作为ceRNA，竞争性结合miR-497，进而调控miR-497靶基因（IGF-1R、FGFR1）的表达水平，从而参与胰腺癌化疗耐药表型的调控。本课题拟通过体内外实验探索lncRNA-n385229/miR-497/（IGF-1R、FGFR1）对胰腺癌吉西他滨化疗敏感性的调控作用及调控机制；并结合临床标本，明确lncRNA-n385229/miR-497评估化疗敏感性、及判断预后的价值。

胰腺癌化疗耐药机制是目前研究的热点和难点。项目计划对lncRNA-n385229/miR-497/靶基因对胰腺癌化疗敏感性的调控作用进行研究，但是在执行过程中，及时发现了原课题假说的局限性，并调整了研究内容。本课题主要通过体内外实验探索了长链非编码RNA UCA1调控胰腺癌化疗耐药的作用及机制。.首先，对胰腺癌组织（94例）及癌旁组织（73例）中UCA1表达水平进行了原位杂交检测。胰腺癌组织UCA1表达水平与癌旁组织无统计学差异（p=0.734）。根据评分情况，将随访信息完整的88例胰腺癌患者分为低表达组（72例）和高表达组（16例）。临床病理参数与组织UCA1表达水平并无显著相关性，但多因素生存分析发现UCA1表达水平是独立预后因素。.然后，体外实验发现，下调UCA1显著抑制胰腺癌细胞增殖，增加吉西他滨化疗敏感性，抑制细胞迁移能力，下调胰腺癌干细胞标志物表达水平。上调UCA1，可观察到相反的效应。构建稳转细胞株，建立裸鼠皮下移植瘤模型，并进行吉西他滨化疗干预。实验发现，UCA1下调组与对照组相比：肿瘤体积无显著差异，体重变化具有统计学差异，移植瘤CD44表达水平有统计学差异。.最后，对UCA1调控机制进行了探索。通过生物信息学预测，UCA1序列上存在miR-16结合位点。RNA免疫共沉淀实验（RIP）证实，UCA1与miR-16存在结合作用。通过生物信息学数据库对UCA1与靶基因的结合作用进行了分析，共预测16条靶基因。qRT-PCR检测发现，上调及下调UCA1后，均出现统计学差异的基因包括：CXCL2，KLK6，KLK7，CEACAM1，GBP1，ICAM1，S100p；部分基因仅在上调或下调UCA1后出现表达异常，包括TRIM29，PHLPP1，LAMP3，KRT18。.我们的研究为胰腺癌耐药机制的研究提供了新的思路；有望为逆转胰腺癌化疗耐药，寻找新的分子治疗靶点奠定基础；该研究探索的分子，可能用于判断胰腺癌患者化疗敏感性及预后，指导个体化治疗。