宫颈癌中microRNA调控淋巴结转移的机制研究及其预测淋巴结转移模型的建立

Cervical cancer is the most common cancer that affects female genital tract. Lymph node metastasis is one of the most important prognostic factors. Correctly predicting the status of lymph nodes would be helpful for personalized treatment. Our prior study found that overexpression of EMMPRIN was positively related to lymph node metastasis. We hypothesize that miRNAs interconnect with targeted mRNAs and thus influence the downstream effector proteins. In this study, we try to further investigate the mechanism of lymph node metastasis from this perspective, and adopt metastasis-related miRNAs to establish a model to predict lymph node metastasis in cervical cancer. We planned to obtain the miRNAs associated likely with lymph node metastasis through screening miRNAs expression profiling in primary cervical cancers with and without positive lymph node using microarray. Furthermore, we try to obtain lymph node metastasis-related miRNAs by screening miRNAs expression profiling in the same patient's 'primary lesion' and 'metastatic lymph node'. Those differentially expressed miRNAs come from the same ecological background and may have direct relationship with lymph node metastasis. Transwell experiment in cell strains and metastatic lymph node models in mice would be employed to verify the relationship between miRNAs expression and lymph node metastasis. Furthermore, two prediction algorithms PicTar and TargetScan will be used to analyze the possible trget gene. RT-PCR and Western Blot can be used to verify the expression of targerted mRNAs and effector proteins. This study is geared to figure out the mechanism of miRNA-regulated lymph node metastasis, to establish an miRNA model to predict lymph node metastasis in cervical cancer and thus offer evidence for personalized treatment and possible gene therapy.

宫颈癌是女性生殖道最常见的恶性肿瘤，准确预测患者的淋巴结状态有助于个体化治疗的选择。本课题在既往发现"EMMPRIN过表达与淋巴结转移正相关"的基础上，拟从"miRNA通过调节靶基因mRNA而作用于下游功能蛋白"的角度深入研究淋巴结转移的调控机制。本课题从"有淋巴结转移"和"无淋巴结转移"的两组宫颈癌原发灶标本中筛选差异miRNA；此外还尝试从同一患者的宫颈"原发灶"与"淋巴结转移灶"筛选差异miRNA，由此获得同一生物背景下的差异miRNA可能与淋巴结转移有最直接关联，能更准确揭示miRNA调节转移的机制。课题拟用体外实验和动物实验验证差异miRNA与淋巴结转移的关系；软件预测miRNA下游靶基因，RT-PCR和Western Blot验证靶基因mRNA及功能蛋白表达。课题旨在通过推测和验证miRNA调控淋巴结转移的机制，建立miRNA预测转移的模型，为个体化治疗及可能的基因治疗提供依据

宫颈癌是女性生殖道最常见的恶性肿瘤。淋巴结转移是影响患者预后的重要因素。准确预测患者的淋巴结状态有助于个体化治疗的选择。本研究从“miRNA通过调节靶基因mRNA而作用于下游功能蛋白”的角度，研究淋巴结转移的调控机制，建立预测淋巴结转移的模型。研究从8例“伴淋巴结转移的宫颈癌原发灶组织”和9例“无淋巴结转移的宫颈癌原发灶组织”研究和筛选差异表达的miRNA，发现7个差异表达的miRNA:miR-142-3p，has-miR-196b，has-miR-320b，has-miR-424，has-miR-550a，has-miR-652，has-miR-H18。用PCR方法验证以上七个miRNA在“伴”和“不伴”淋巴结转移的宫颈癌组织中的表达，发现与基因芯片筛查结果基本一致。用宫颈癌Caski和SiHa细胞株进行transwell实验和细胞划痕试验，发现用has-miR-652-mimic转染细胞株后，细胞株的侵袭转移能力较转染前显著增强；用has-miR-196b－mimic，miR-142-3p－mimic，has-miR-424－mimic转染细胞株后，细胞株的侵袭转移能力较前减弱；用has-miR-320b－mimic转染细胞株后，细胞株的侵袭转移能力变化不明显。上述研究结果提示has-miR-652在宫颈癌中起促进淋巴结转移的作用，而has-miR-196b，miR-142-3p和has-miR-424可能起着抑制宫颈癌淋巴结转移的作用。下一步的研究将以上述结果为基础，选取has-miR-652深入研究，揭示miRNA通过“靶基因mRNA和功能蛋白”调控淋巴结转移的机制，并以此建立宫颈癌淋巴结转移预测模型。