miR-302-367基因簇调控高迁移率族蛋白A2影响子宫内膜癌干细胞生物学行为的分子机制

Our previous study has found that of the endometrial cancer stem cells,miR-302-367 gene cluster members were low expression,but HMGA2 was high expression. Through a series of work,we speculate that miR-302-367 gene cluster members of low expression may regulate the expression of HMGA2 in two ways,one is that the miR-302-367 gene cluster members can directly regulate the expression of HMGA2 negatively,the other is that the down-regulated transcription factor RUNX1 indirectly regulate the expression of HMGA2, thereby affecting the biological behavior of endometrial cancer stem cells.The project intends to verify the binding and binding sites where the miR-302-367 gene cluster members combined with the HMGA2 and RUNX1; clear that if the miR-302-367 gene cluster members directly regulate the expression of HMGA2 or regulate the HMGA2 expression by RUNX1 indirectly, clarify the RUNX1 transcriptional regulation mechanism for HMGA2; analyze if the changes of the HMGA2 expression can affact the biological functions and mechanisms in the endometrial cancer stem cells; finally study the miR-302-367 gene cluster members individually or in combination on the inhibitory effect of the endometrial cancer stem cells in nude mice, the findings could provide a new target for the treatment of endometrial cancer.

前期研究发现子宫内膜癌干细胞中miR-302-367基因簇成员低表达，HMGA2高表达。通过系列工作推测低表达的miR-302-367基因簇成员可能通过两种方式调节HMGA2的表达，一种是miR-302-367基因簇成员能够直接负性调控HMGA2的表达，另一种是下调转录因子RUNX1的表达间接调控HMGA2的表达，进而影响子宫内膜癌干细胞的生物学行为。本项目拟先验证miR-302-367基因簇成员与HMGA2和RUNX1的靶向结合作用和结合位点；明确miR-302-367基因簇成员直接调节HMGA2的表达或通过RUNX1间接调节HMGA2的转录和表达，阐明RUNX1对HMGA2的转录调节机制；分析HMGA2表达变化对子宫内膜癌干细胞生物学功能的作用与机制；最后研究miR-302-367基因簇成员单独或联合应用对子宫内膜癌干细胞裸鼠移植瘤的抑瘤效应，研究结果能为子宫内膜癌的治疗提供新靶点。

子宫内膜癌是女性生殖系统最常见的三大恶性肿瘤之一，探寻较为有效的子宫内膜癌分子靶向治疗方法与策略，已成为子宫内膜癌药物治疗研究的热点。近年来的研究表明，人子宫内膜组织是一种动态、周期性再生的组织，干细胞相关学说提出，侧群细胞具有将其移植给裸鼠后能诱发肿瘤和基质样组织的形成，衍生自侧群细胞的基质细胞通过上皮细胞间质转化过程(EMT)影响肿瘤的迁移、侵袭等生物学行为。因此，深入研究调控子宫内膜癌细胞特性的相关分子机制以及干细胞的特征，将有助于探寻有效的子宫内膜癌分子治疗的新靶点。我们的研究发现在子宫内膜癌组织中HMGA2的表达高于正常子宫内膜组织，具有高表达HMGA2的子宫内膜癌患者有较差的预后。在子宫内膜癌组织中检测miR-302-367基因簇成员表达，结果表明miR-302-367基因簇成员在子宫内膜癌组织中低表达，且具有低表达miR-302-367基因簇成员的子宫内膜癌患者有较差的预后。利用无血清悬浮培养法分离出子宫内膜癌干细胞。通过体外实验分析其参与子宫内膜癌发生、发展的可能机制，发现miR-302-367基因簇成员通过靶向结合HMGA2影响子宫内膜癌细胞恶性生物学行为，并调控子宫内膜癌细胞EMT进程。在体内实验中，构建子宫内膜癌裸鼠移植瘤模型，明确过表达miR-302-367基因簇成员和（或）下调HMGA2的表达能够抑制裸鼠移植瘤的生长。利用JASPAR软件预测了RUNX1在HMGA2启动子区的结合位点，通过ChIP实验，确定RUNX1与HMGA2启动子区的特异结合位点相结合。进一步通过体外实验分析其参与子宫内膜癌发生、发展的可能机制，发现miR-302通过靶向结合RUNX1影响子宫内膜癌细胞恶性生物学行为。体内实验明确下调 RUNX1的表达能够抑制裸鼠移植瘤的生长。本研究结果提示miR-302/367-HMGA2-EMT轴以及miR-302-RUNX1-HMGA2 -EMT轴对子宫内膜癌细胞的发生、发展影响，为子宫内膜癌的早期发现及早期防治提供了一个新的视角。