miR-107调控WNK3影响宫颈癌细胞增殖、侵袭的相关机制研究

The metastatic invasion of cancer cells from the primary lesion into the adjacent stroma is a key step in cancer progression, and is associated with poor prognosis. Emerging evidences indicate that K-Cl cotransporter sub (KCC) is closely related to the invasion of tumor, but its mechanism has not been elucidated. Our previous study found that miR-107 is highly expressed in cervical carcinoma, and its predicted target WNK3 can regulate KCC activity. We thereby assume that in regulating WNK3 expression in cervical cancer, miR-107 might indirectly mediate the KCC activity, thus leading to the change of the cell volume, the activation of downstream signaling, resulting in the promotion of cell proliferation and tumor invasion. In the proposal, we will apply the report gene system and crosslinking-immunoprecipitation technique to verify WNK3 is the target of miR-107. Thereafter, we will investigate whether the abnormally elavated expression of miR-107 in Human Cervical Cancer Samples, in turn, decreases the level of its target WNK3. Following the alternation of WNK3 in the cells, we will measure the related changes of KCC activity, cell volume and the cell proliferation and invasion. Finally, we will use the xenograft model to evaluate the effectivaness of transfected LNA-miR-107 in the treatment of cervical cancer. The implementation of this study might elucidate the mechanism by which miRNA-107 mediates KCCs regulated-cell proliferation and invasion in the cells, thus providing novel strategies in developing potential therapeutics for the treatment of cervical cancer.

肿瘤侵润是宫颈癌进展的关键步骤。K-Cl协同转运子（KCC）与肿瘤细胞的侵袭密切相关，但其作用机制尚未阐明。我们前期研究发现miR-107在宫颈癌组织中高表达，其预测的靶基因WNK3具有调节KCC活性的功能。我们假设在宫颈癌中miR-107调节WNK3 表达，导致KCC活性改变，从而调控细胞体积，激活下游信号传导，影响肿瘤细胞侵袭生长。我们将观察宫颈癌组织中miR-107与WNK3含量的相互关系；运用报告基因系统及CLIP-seq技术验证WNK3是miR-107的作用靶点；通过测定KCC活性，细胞体积改变和其下游相关蛋白的活性变化，探讨miR-107通过WNK3调控KCC，影响宫颈癌细胞侵袭生长的可能机制；利用移植瘤模型，研究抑制miR-107表达对宫颈癌的治疗作用。本研究的实施有助于阐明miR-107影响癌细胞侵袭生长的作用机制，为临床开发针对宫颈癌全新有效的治疗策略提供重要的实验依据。

肿瘤侵润是宫颈癌进展的关键步骤，然而，其具体分子机制至今尚未阐明。我们前期研究发现miR-107在宫颈癌组织中高表达，其预测的靶基因WNK3具有调节KCC活性的功能。本课题从患者、动物模型及相关细胞水平探讨miR-107在宫颈癌肿瘤增殖、侵袭中的作用。研究表明宫颈癌临床组织标本中miR-107与WNK3蛋白水平的负相关。且与临床分期及淋巴结转移正相关。通过突变实验证实WNK3为miR-107的靶基因，在SiHa细胞中调节WNK3的表达量可直接影响细胞的迁移、侵袭能力。WNK3表达下降，其下游Rb及cdc2的磷酸化程度上调。同时，整联蛋白αvβ3和α6β4的表达亦升高， MMP-2、MMP-9的活性较对照组增强。进一步研究显示，在低渗条件下，下调WNK3蛋白表达，KCCs活性增加，SiHa细胞RVD调控能力增强，下游Rb及cdc2的磷酸化程度均有所上升；整联蛋白αvβ3和α6β4的表达却较前升高。加用KCCs抑制剂DIOA处理后，虽WNK3表达下降，但KCCs活性丧失，对SiHa细胞RVD的调控减弱，下游Rb及cdc2的磷酸化程度下降；整联蛋白αvβ3和α6β4的表达下调。实验证明通过WNK3基因的表达水平的变化，可以直接调节KCC活性，调控细胞体积以及KCC下游信号传导相关蛋白的表达活性，从而影响宫颈癌细胞增殖、侵袭转移。宫颈癌异位移植瘤模型亦证实下调miR-107，促进WNK3蛋白表达 可以显著抑制肿瘤的生长。本课题综合运用生物信息学、细胞分子生物学、生物化学方法及现代成像技术等技术，系统研究miR-107及其靶基因WNK3在宫颈癌肿瘤生长、侵袭中的作用及其分子机制。通过在分子、细胞和临床等多个层面的研究，系统认识miR-107在宫颈癌中的作用机制，阐明癌细胞的调节性体积改变影响肿瘤细胞的增殖、浸润过程的作用机制，解析宫颈癌发生、发展的分子机理具有重要的理论意义，为临床开发针对宫颈癌患者全新有效的治疗策略提供重要的实验依据。