miR-101靶向FMNL3/CDC42调控丝状伪足形成影响宫颈癌侵袭迁移及转移的作用和机制

Cervical cancer in Xinjiang Uighur women has a high morbidity and mortality. The patients with advanced stage of cervical cancer in Uighur women are more than other patients. A large part present with metastatic disease will have poor prognosis. Filiform pseudopodia is one of the cytologic foundamental structure to adapt to a new environment and migration. FMNL3 and CDC42 can promote pseudopodia stretching of endothelial cells. Previous studies have found that low levels were present of miR-101 in cervical cancer tissue and cell lines, and miR-101 could suppress the invasion ability of cervical cancer cells. FMNL3 may be one of miR-101’s target genes. FMNL3 and CDC42 protein levels were down-regulated after miR-101 up-regulated. We speculate that miR-101 targeted FMNL3 / CDC42 to regulate filiform pseudopodia formation may be explain the high metastasis of cervical cancer of Uighur women. This research by in vitro and in vivo experiments : Firstly, verify miR-101 regulate to FMNL3 in post-translational level by luciferase reporter gene , the interaction with FMNL3 and CDC42 by Immunoprecipitation .Sencondly,to observe the filiform pseudopodia formation and invasion and metastasis by Transwell, nude mouse lung metastasis tumor, and so on. Thirdly,to study the mechanism of miR-101-FMNL3 / CDC42 regulate filiform pseudopodia formation by detecting activity of CDC42, downstream signaling molecule , the location and quantification of F-actin in cervical cancer cells after up-regulated FMNL3. Lastly, analysis the relationship of miR-101 expression in peripheral blood and prognosis of cervical cancer to provide theoretical and experimental basis for prediction, treatment, prognosis of cervical cancer .

维吾尔族妇女宫颈癌晚期（转移）患者多，预后差。丝状伪足形成是肿瘤迁移的起始步骤，FMNL3 和CDC42 均可促进内皮细胞伪足延伸。前期工作：miR-101在宫颈癌组织和细胞系中低表达，可抑制细胞侵袭；FMNL3是其可能靶基因；上调miR-101后FMNL3和CDC42蛋白水平降低。推测：miR-101通过FMNL3/CDC42 调控丝状伪足形成可以解释宫颈癌的高转移。本项目应用荧光标记鬼笔环肽、Transwell、裸鼠肺转移瘤等观察miR-101对丝状伪足形成和侵袭转移的作用；双荧光素酶报告基因实验等验证miR-101对FMNL3的转录后调控，免疫共沉淀等验证FMNL3与CDC42的作用；上调FMNL3后，检测CDC42活性、下游信号分子、F-actin等，探讨上述途径调控丝状伪足的机制；结合宫颈癌病例循环miR-101与预后的关系，为宫颈癌转移的预测、治疗及预后判定提供理论和实验基础。

新疆地区维吾尔族妇女宫颈癌发病率高，死亡率高，中晚期患者较多，被列为新疆特高发肿瘤之一，转移是导致患者预后差和死亡的重要原因。丝状伪足形成是肿瘤迁移的起始步骤。前期研究发现：miR-101在宫颈癌组织和细胞系中低表达，FMNL3是其可能靶基因；上调miR-101后FMNL3和CDC42蛋白水平降低。本研究发现1. miR-101高表达可抑制宫颈癌细胞丝状伪足的形成及其侵袭迁移能力：宫颈癌细胞转染miR-101后，细胞丝状伪足数量减少、增殖能力下降、侵袭和迁移能力减弱。2. miR-101通过RAC1/CDC42途径调控宫颈癌细胞丝状伪足形成：双荧光素酶报告基因实验显示，RAC1 mRNA 3`UTR部位有miR-101结合位点，RAC1是miR-101的靶基因；免疫共沉淀实验显示，RAC1与CDC42在蛋白水平上有相互作用，CDC42和RAC1在SiHa细胞内结合。过表达miR-101后CDC42及其下游信号蛋白NWASP、Arp2/3表达量降低，同时Rac1过表达后以上三个蛋白表达量升高。F-actin表达量无明显变化，即RAC1对其下游的N-WASP和Arp2/3有调控作用。CDC42活性检测显示，miR-101过表达组CDC42蛋白活性降低。免疫荧光显示，miR-101过表达组F-actin团簇状结构消失，RAC1和PDGF处理组F-actin呈团簇状，即miR-101过表达引起丝状伪足结构的解聚和破坏。3. miR-101抑制宫颈癌HeLa细胞裸鼠皮下移植瘤的生长及肺转移：转染miR-101宫颈癌细胞，裸鼠皮下移植瘤体积较空白组及对照组缩小；肿瘤细胞有显著异型性，移植瘤呈乳头状，与周围组织界限清晰；部分实验组移植瘤可见坏死细胞灶。转染miR-101宫颈癌细胞肺转移瘤裸鼠体质量高于空白组及对照组；肺转移瘤呈小灶状分布，可见肿瘤细胞坏死灶。4. 循环miR-101可以作为宫颈癌病例临床预后评价指标：与对照组相比，宫颈癌患者组血浆miR-101明显低表达，并与宫颈癌患者淋巴结转移和分化程度正相关。本项目将为维吾尔族妇女宫颈癌转移的预测、治疗及预后判定提供理论和实验基础。