环状RNA circ-0046263调控鼻咽癌侵袭转移的机制研究

We identified and verified an circular RNA, circ-0046263 (CI) which is highly expressed in human metastatic tissues of nasopharyngeal carcinoma (NPC), by the application of high-throughput microarray technology and the validation of expression level. Our pre-experiments suggested that CI was significantly correlated with advanced N stage and poor prognosis in NPC patients. We found that CI can inhibit invasion and metastasis of NPC cell lines by silencing siRNA. Further bioinformatics analysis and experimental results revealed that CI could used as a “sponge” which competing with miR-133. In turn, miR-133 upregulates the expression of the downstream IGFBP3 gene and promotes the invasion and metastasis of NPC. Therefore, we propose the scientific hypothesis that “CI promotes the invasion and metastasis of NPC through a competitive endogenous combination of miR-133 which upregulating IGFBP3 expression. Our project will evaluate the biological function of CI in NPC both in vivo and in vitro in over-expression and knockdown manner. Along with rescue experiment, RIP and dual-luciferase reporter system, we aimed to clarify the molecular mechanism that CI competitively adsorbs miR-133 as a competing endogenous RNA，which leading to upregulating IGFBP3 expression. Finally, with clinical samples and cell lines, we will further evaluate the relationship between CI/miR-133/IGFBP3 regulatory networks and NPC invasion and metastasis. There are no report about function and mechanism of CI in NPC, this study may provide a new therapeutic target for the diagnosis and treatment of NPC.

我们应用高通量芯片技术筛选并验证获得了一条差异高表达于人鼻咽癌转移组织、功能未知的环状RNA，circ-0046263（简称CI）。预实验提示：CI高表达与鼻咽癌N分期及不良预后显著正相关；沉默CI后鼻咽癌细胞侵袭迁移能力下降；生物信息学预测及实验提示CI可作为“分子海绵”吸附miR-133上调促转移基因IGFBP3的表达。故我们提出“CI竞争性结合miR-133上调IGFBP3表达促进鼻咽癌侵袭转移”的科学假说。本研究拟通过体内外实验，验证CI促进鼻咽癌侵袭转移的生物学功能；通过拯救实验、RIP及荧光素酶基因等实验，阐明CI作为分子海绵通过竞争性内源RNA机制封闭miR-133，从而上调IGFBP3表达促进鼻咽癌侵袭转移的分子机制；最后结合临床样本验证CI/miR-133/IGFBP3通路与鼻咽癌侵袭转移的关系。CI促进鼻咽癌侵袭转移的功能机制未见报道，本研究可为鼻咽癌诊治提供新靶标。

研究背景 环状RNA（circRNA）作为非编码RNA的亚类，在调节真核生物的基因表达中起重要作用。但是，它们在鼻咽癌中的作用知之甚少。本研究旨在明确环状RNA circ-0046263在鼻咽癌中的生物学功能，并初步探讨其可能的分子机制。.研究内容 我们使用qRT-PCR检测circ-0046263在鼻咽癌组织和细胞中的相对表达量。Sanger测序、RNase R消化实验、琼脂糖凝胶电泳来鉴定环状RNA。核质分离和原位杂交用来确定circ-0046263的亚细胞定位。萤光素酶报告基因和RNA结合蛋白免疫沉淀用来确认circ-0046263的miR-133a-5p的相互作用。克隆形成和CCK-8检测细胞增殖能力。Transwell和伤口愈合试验检测细胞的侵袭和迁移能力。Western bolt和免疫组化检测EMT相关蛋白的变化。通过裸鼠模型观察circ-0046263在动物体内对肿瘤生长及淋巴结转移的影响。.研究结果 qRT-PCR结果显示，与正常鼻咽上皮细胞NP69相比，circ-0046263在鼻咽癌细胞系呈高表达。临床组织样本检测结果显示，circ-0046263在鼻咽癌组织中的表达上调，且与分期成正相关。体外细胞功能实验表明circ-0046263促进细胞的增殖、迁移和侵袭，而且促进细胞上皮间充质转化（EMT）。体内裸鼠实验同样验证circ-0046263的生物学功能。FISH及核质分离实验表明circ-0046263主要定位于细胞质。生物信息学预测miR-133a-5p存在与circ-0046263的结合位点，其可能是circ-0046263下游靶分子。双萤光素酶报告基因和RNA结合蛋白免疫沉淀验证了circ-0046263和miR-133a-5p的相互结合。生物信息学预测IGFBP3可能为miR-133a-5p的下游靶标。双萤光素酶报告基因结果显示miR-133a-5p靶向结合IGFBP3。挽救实验结果显示敲低miR-133a-5p或者过表达IGFBP3能够逆转circ-0046263沉默引起的细胞表型。circ-0046263通过抑制miR-133a-5p的活性，上调IGFBP3蛋白水平，促进EMT表型，从而促进鼻咽癌的增殖、迁移和侵袭。.研究意义 circ-0046263可能作为一个生物标记物或为鼻咽癌治疗提供一个潜在靶点。