环状RNA-645以竞争性内源RNA方式结合miR-221调控GPX4影响卵巢癌转移侵袭的机制研究

The high tendency of metastasis is the main reason behind ovarian cancer’s low survival rate. Our previous study find that increased ROS and HIF1α in ovarian cancer is associated with its higher metastasis ability. Further analysis into this mechanism we find that GPX4，a critical anti-oxidant gene, is down-regulated in ovarian cancer cells. Increased ROS level then up-regulate HIF1α and VEGF-A, which then accelerate EMT related transcription factors expression, thus promoting ovarian cancer cell metastasis. Moreover, We are the first to find that in ovarian cancer cells the GPX4 targeted miRNA miR-221 is negatively correlated with circ-645, which harbors 11 conserved binding sites for miR-221. Therefore, we postulated that GPX4 regulated by miR-221 and circ-645 through competing endogenous RNAs a possible mechanism involved in ovarian cancer metastasis. Via clinical sample, in vitro cell experiment and animal experiment, our group will verify this regulating pathway: the lower level of circ-645 in ovarian cencer cell abrogates its function as miR-221 spongy, resulting increased free miR-221, which in turn targets GPX4 mRNA for degradation. Disturbed anti-oxidant mechanism increased ROS production, which then up-regulated VEGF-A and activated AKT/GSK-3β and EMT signaling pathway. In conclusion, we illustrate a novel mechanism involved in ovarian cancer cells metastasis will later facilitates finding new biomarkers and potential therapeutic targets for early stage ovarian cancer patients.

卵巢癌转移侵袭能力强是患者生存期短的主要原因。前期研究发现，卵巢癌细胞内ROS及HIF1α水平升高与其转移侵袭相关。通过预实验首次发现抗氧化关键基因GPX4下调可以使ROS升高，上调HIF1α及VEGF-A，增加EMT相关转录因子表达，促进卵巢癌细胞转移侵袭；还首次发现卵巢癌细胞中与GPX4相关的miR-221与circ-645表达量呈负相关，且二者之间有11个结合位点。因此我们首次提出miR-221和circ-645之间存在竞争性内源RNA方式调控GPX4促进卵巢癌转移侵袭的可能机制。为此本课题将从临床、细胞和动物三个层面揭示：卵巢癌细胞内circ-645↓→竞争结合miRNA能力↓→游离miR-221↑→GPX4↓→ROS↑→VEGF-A↑→AKT/GSK-3β信号激活→EMT相关转录因子及蛋白表达量改变→促进卵巢癌转移侵袭的通路，探寻卵巢癌患者早期转移相关分子标志物及靶向治疗药物。

卵巢癌是全世界死亡率最高的妇科肿瘤，卵巢癌转移侵袭能力强是患者生存期短的主要原因，近年来研究表明，上皮间质转化是肿瘤细胞转移侵袭的关键步骤，而氧化应激产物ROS可能参与了上皮间质转化的过程。本研究前期实验发现抗氧化关键蛋白GPX4在卵巢癌癌旁正常组织、原发灶组织及转移灶组织中呈逐渐降低趋势，而且其表达量与上皮标志物E-Cadherin表达量之间存在正线性相关， 与间质标志物Vimentin mRNA表达量之间存在负线性相关。通过基因芯片技术和序列对比，发现微小RNAmiR-221可能与GPX4 mRNA的3’-UTR相结合。本研究进一步在临床大样本上证实GPX4和miR-221与卵巢癌患者的FIGO分期及预后显著相关，提示GPX4和miR-221可以作为评估卵巢癌患者早期转移、临床预后的分子标志物。同时本研究利用细胞生物学实验发现，降低卵巢癌细胞中GPX4的表达可以显著升高细胞中的ROS水平，从而激活HIF1α/VEGF-A/AKT通路，诱导卵巢癌细胞的上皮间质转化，增强其侵袭转移能力，而增加GPX4的表达则结果相反，GPX4的这种抗卵巢癌转移的作用在小鼠原位瘤腹腔转移模型上也得到了证实。本研究还利用荧光素酶报告基因实验证实miR-221能与GPX4 mRNA的3’-UTR相结合，抑制后者的表达，负向调控GPX4的抗转移作用。. 尽管在我们的预实验中，环状RNAcirc-645与miR-221在少量卵巢癌样本中表现出较好的相关性，但是两者的结合及调控关系未能在细胞实验中证实。通过对9组卵巢癌和癌旁组织样本的RNA-seq结果进行生物信息学分析，我们发现circ-645在卵巢癌及癌旁组织样本中表达差异并不明显，因此我们选取差异性表达最明显的两条环状RNA: hsa-circ-0006473及hsa-circ-0078607进行进一步研究，发现两者均对卵巢癌的转移侵袭有明显的调控作用，但具体机制尚待后续研究。