环状RNA-257以竞争性内源RNA方式结合miR-9调控FOXO1影响胰腺癌发生、发展的机制研究

Circular RNAs (circRNAs) are stable and conserved, and often exhibit tissue/developmental-stage-specific expression. CircRNAs can act as microRNA sponge or regulate gene transcription, and can play important roles in some tumors. However, the expressions and roles of circRNAs in pancreatic cancer are unclear for us. We have shown that has-circ-0000257 is downregulated via microarray screening and tissue detection. Moreover, we have found that overexpression of has-circ-0000257 in pancreatic cancer cell lines can significantly inhibit cell proliferation and migration, reduce miR-9 expression and promote FOXO1 expression. Bioinformatic analysis indicated that has-circ-0000257, acting as miR-9 sponge to regulate FOXO1 expression, plays roles in the development of pancreatic cancer. Nevertheless, the detailed mechanisms still need to be clarified. This study will reveal regulatory relationships among has-circ-0000257, miR-9 and FOXO1, and illustrate the mechanism of has-circ-0000257 serving as miR-9 sponge to regulate FOXO1 expression in vitro or in vivo. Meanwhile, we will evaluate their clinical significances in tissue level. This study would provide novel insights into the prevention and treatment of pancreatic cancer.

环状RNA具有较强地稳定性和保守性，表达具有组织时序性，可以发挥miRNA海绵或调控基因转录等功能，并在部分肿瘤等疾病中发挥重要作用，但在胰腺癌中的表达和作用机制尚不明确。前期利用芯片筛选并在胰腺癌组织中验证发现has-circ-0000257呈低表达。过表达has-circ-0000257后，可抑制胰腺癌细胞增殖和迁移，且发现miR-9表达显著下调而FOXO1表达升高。生物信息学预测其可通过竞争性内源RNA方式结合miR-9调控FOXO1表达，但具体机制仍待探究。本课题拟通过体内、外实验揭示has-circ-0000257、miR-9和FOXO1之间的调控作用，阐明has-circ-0000257充当miR-9海绵调控FOXO1对胰腺癌恶性表型影响的分子机制。从胰腺癌组织水平，评价has-circ-0000257/miR-9/FOXO1通路对胰腺癌的临床意义，最终为胰腺癌防治提供新思路。

探索circNOLC1(has-circ-0000257)在胰腺癌恶性进展中的作用机制。circRNA芯片检测胰腺癌中差异表达circRNA；qPCR检测circNOLC1在50例胰腺癌组织和细胞中的表达，并分析与临床病理资料间的相关性；CCK8和平板克隆实验检测对胰腺癌细胞增殖影响；Transwell实验检测对胰腺癌细胞侵袭转移能力影响；克隆悬浮成球实验检测对胰腺癌细胞干性表型影响。FISH和核质分离实验确认circNOLC1在胰腺癌细胞中定位分布。生信分析和荧光素酶报告基因实验确定circNOLC1和miR-9的关系。mRNA测序分析circNOLC1对下游基因通路影响，Western-blot确认对ERK和EMT相关通路影响。体内动物实验进一步确认circNOLC1对胰腺癌细胞生长影响。实验结果表明，部分circRNA在胰腺癌中差异表达，circNOLC1在胰腺癌组织和细胞中表达相对较低，且与肿瘤分化程度和TNM分期相关。细胞功能实验显示，在胰腺癌细胞中过表达circNOLC1可以抑制胰腺癌细胞增殖、侵袭、迁移和克隆悬浮成瘤能力。小动物实验显示过表达circNOLC1可以显著抑制胰腺癌细胞肿瘤生长，降低肿瘤细胞Ki67指数。FISH和核质分离实验显示circNOLC1在胰腺癌细胞质中表达相对较高，生信分析显示circNOLC1与miR-9存在三个结合位点，荧光素酶报告基因实验进一步确认两者调控作用。mRNA测序后KEGG和GSEA分析显示过表达circNOLC1后可以影响胰腺癌AMPK、ErbB和PI3K-Akt信号通路。Western-blot进一步确认过表达circNOLC1后可以抑制胰腺癌ERK、AKT和EMT等分子表达。综上，circNOLC1可以通过结合miR-9调控胰腺癌AKT和ERK通路抑制胰腺癌的恶性进展。