Hsa_circ_0003915抑制IMP2蛋白泛素化的分子机制及其在结肠癌转移中作用的实验研究

Colon cancer is one of the most frequently diagnosed cancer in our country. Circular RNAs(circRNAs) are recently identified as a naturally occurring family of noncoding RNAs that play an important role in tumor progression. However, the expression profile and function of circRNAs in human colon cancer remain to be investigated. In our previous study,we have analyzed the expression profile of human circRNAs in colon tissues and identified hsa_circ_0003915 as one circRNA significantly increased in colon cancer tissues. Interestingly,.upregulated hsa_circ_0003915 in colon cancer tissues was associated with distal metastasis.Transwell assay in colon cancer cells suggested that hsa_circ_0003915 promoted cell migration.More importantly, hsa_circ_0003915 could bind to IMP2 protein directly and increase the expression of IMP2 and its target genes,such as RhoA,CD44,SP1 and c-Myc. Proteasome inhibition assay revealed this circRNA might regulate IMP2 degradation.Further study suggested Cul3 could induce ubiquitination of IMP2,and hsa_circ_0003915 might interfere this process.Based on these findings,we hypothesise that hsa_circ_0003915 might promote colon cancer metastasis by inhibiting Cul3 induced IMP2 ubiquitination. Our research will further detect the expression of hsa_circ_0003915 in clinical tissue array and clarify its correlation to clinical indicators. In addition, we will confirm the mechanism of Cul3 on the ubiquitination of IMP2 protein and reveal the role of hsa_circ_0003915 in this process. Also, the function of hsa_circ_0003915 will be demonstrated in vivo.Thereby,this study is expected to provide a new target for the clinical therapy in colon cancer.

结肠癌是我国高发的恶性肿瘤之一。环状RNA是一种具有共价闭合环状结构的非编码RNA，对肿瘤发展具有重要调控作用。课题组前期通过环状RNA芯片筛选与RT-PCR验证发现hsa_circ_0003915在结肠癌组织中显著高表达，并且与结肠癌的侵袭、转移密切相关。RNA pull-down与质谱等检测发现hsa_circ_0003915与IMP2存在结合，并在翻译后水平增强IMP2的蛋白稳定性。结合文献与预实验结果，我们推测hsa_circ_0003915可能通过抑制E3泛素连接酶Cul3对IMP2的泛素化降解，进而上调其下游靶基因CD44、c-Myc、RhoA与SP1的表达，促进结肠癌的侵袭、转移。据此，课题组拟采用Co-IP与位点突变等方法并结合体内外实验验证上述假说。本课题有望加深对结肠癌发展机制的理解，为结肠癌治疗提供新证据。

项目背景：结肠癌是消化系统最常见的恶性肿瘤之一，侵袭转移是患者死亡的主要原因，但具体的分子机制仍不完全清楚。因此针对结肠癌侵袭转移相关分子机制的研究是结肠癌防治的重点攻克内容。环状RNA（circular RNA，circRNA）是一种新型闭合非编码RNA，环状RNA的差异表达在肿瘤的发生、发展中发挥重要作用，但其在结肠癌恶性进展中的具体机制仍未完全明确。.主要研究内容：1）筛选出结肠癌中差异表达的环状RNA；2）hsa_circ_0003915与结肠癌临床病理的关系；3）hsa_circ_0003915对结肠癌细胞侵袭转移的影响；4）hsa_circ_0003915促进结肠癌恶性进展机制的研究。.重要结果：利用环状RNA芯片比较结肠癌与癌旁组织中环状RNA的差异表达。通过分析芯片结果与qRT-PCR验证，我们发现hsa_circ_0003915在结肠癌组织中高表达，并与结肠癌转移和不良预后相关，提示其在结肠癌中发挥促癌因子的作用。我们进一步利用RNase R、免疫荧光、核浆分离等实验证实hsa_circ_0003915具备环状RNA的特性。接下来通过质粒和CRISPR-Cas13系统过表达或敲降hsa_circ_0003915，在体内和体外实验中证实该环状RNA促进结肠癌细胞的增殖及侵袭转移。在机制研究部分，利用RNA pulldown联合质谱分析找到与其结合的蛋白IGF2BP2，并通过RIP、截短实验明确hsa_circ_0003915结合于GF2BP2蛋白的KH-1\2结构域；进一步研究二者调控作用，我们发现hsa_circ_0003915与RNA结合蛋白IGF2BP2结合，抑制IGF2BP2蛋白在S162/164位点的磷酸化，而S162/164位点的磷酸化是IGF2BP2发挥RNA结合作用的关键。另外，蛋白芯片的结果及WB验证发现hsa_circ_0003915可促进IGF2BP2与FGFR2以及ITGA6的mRNA的结合，增强其蛋白水平的表达，从而促进结肠癌的恶性进展。.科学意义：阐明hsa_circ_0003915通过结合IGF2BP2上调FGFR2以及ITGA6表达进而促进结肠癌恶性进展的机制，为hsa_circ_0003915作为结肠癌的治疗靶点提供有力的理论支持。