circRNA104084经吸附miR-506上调FLOT1促进胰腺癌侵袭转移的分子机制

It was discovered recently that circRNA is involved in tumor progression, but its mechanism is unclear. We previously found that circ104084 expression is higher in pancreatic adenocarcinoma compared with normal pancreatic tissues, and promotes invasion and metastasis of pancreatic adenocarcinoma. It is known that circRNA exerts its biological effects via complementary based binding miRNA to reduce its activity. Under the support of previous NSFC, we found that miR-506 could inhibit invasion and metastasis of pancreatic adenocarcinoma. We further performed preliminary experiments and found that there is complementary nucleotide binding sites between circ104084 and miR-506 seed region, and their expressions were negatively correlated; miR-506 can regulate FLOT1, which may promote pancreatic adenocarcinoma invasion and metastasis through NF-κB singaling pathway. Thus, it is reasonable for us to speculate: circ104084 can bind to miR-506 by complementary base pairing and reduce its activity, upregulate FLOT1, activate NF-κB signaling pathway, and finally promote invasion and metastasis of pancreatic adenocarcinoma. We are planning to do the following experiments: to identify circ104084 and miR-506 seed binding region sequence by reporter gene, Pull-down and fluorescence in situ hybridization; to detect invasion and metastasis of pancreatic adenocarcinoma in cell lines and animal models and examine FLOT1 expression and its downstream molecules using lentiviral vector; to verify reversely. This study is expected to clarify the molecular mechanisms regarding circ104084 promoting invasion and metastasis of pancreatic adenocarcinoma, and provide new ideas for diagnosis and treatment of pancreatic adenocarcinoma.

新近发现circRNA参与肿瘤发生发展，但其作用机制不清。我们前期发现circ104084在胰腺癌中高表达并促进其侵袭转移。已知circRNA可经碱基互补配对吸附miRNA降低其活性从而发挥生物学效应。前一项国家基金资助下发现：过表达miR-506可抑制胰腺癌侵袭转移。预实验发现：circ104084与miR-506种子区存在碱基互补结合位点，二者表达呈负相关；miR-506可调控FLOT1，后者可经NF-κB通路促进胰腺癌侵袭转移。故推测：circ104084经碱基互补配对吸附miR-506并降低其活性，上调FLOT1，活化NF-κB通路促进胰腺癌侵袭转移。拟采用报告基因、Pull-down鉴定二者的结合序列；通过慢病毒载体在细胞及动物模型中检测胰腺癌侵袭转移、FLOT1及下游分子表达并行反向验证。本研究有望阐明circ104084促进胰腺癌侵袭转移的分子机制，为胰腺癌的诊治提供新思路。

最新研究发现circRNAs参与调控肿瘤发生发展，但其作用机制不清。本课题组前期通过芯片分析发现circ104084和circ100331均在胰腺癌中高表达，随后采用qRT-PCR、IHC等实验明确了circRNA104084、FLOT1在胰腺癌组织中的高表达而miR-506的低表达，以及三者与患者淋巴转移、分期分级、生存时间等临床资料的相关性。本课题组进一步验证了miR-506可作为上游分子调控FLOT1的表达，促进胰腺癌细胞侵袭转移。同时采用qRT-PCR验证了circ100331在胰腺癌中高表达，且EdU、划痕和Transwell实验表明下调circ100331可抑制胰腺癌细胞的增殖迁移。通过生物信息学分析发现circ100331与miR-491具有吸附作用，且miR-491与UBE2C具有结合位点。采用qRT-PCR、EdU、划痕和Transwell实验证明了miR-491在胰腺癌中低表达，且抑制胰腺癌细胞的增殖迁移。UBE2C是重要泛素偶联酶，介导多种肿瘤的发生和发展。本课题通过IHC实验发现UBE2C在PDAC患者组织中高表达，并且与临床分期，淋巴结转移，神经浸润呈正相关相关，且其高表达是PDAC患者不良预后的独立危险因素。随后采用EdU、Transwell和WB等实验表明在PDAC细胞系CFPAC-1和PANC-1中，UBE2C的表达下调可诱导cyclingD1下调，使细胞阻滞在G1 / S期，抑制增殖，也可下调EMT蛋白的表达，抑制细胞迁移。RNAseq分析表明，在CFPAC-1细胞中使UBE2C下调后，cyclingD1和vimentin分别下调了约3.5倍和2.6倍，并且主要的富集通路与细胞周期进程有关。通过小鼠荷瘤实验表明，UBE2C表达下调可显著抑制体内肿瘤的生长。本研究有望阐明circ100331经吸附miR-491上调UBE2C的表达促进胰腺癌增殖迁移的分子机制，为胰腺癌的诊治提供新思路。通过相关的实验成功培养了硕士生3名，博士生3名，发表相关论文12篇，获得国家重点研发计划1项（项目编号2017YFC1308600）。