应用宫内孕激素上调miR-197、影响子宫肌瘤细胞增殖与凋亡功能的机制研究

Previous research has demonstrated via microarray technique that the micro RNA miR-197 is significantly up-regulated in the uterine leiomyoma cells (UtLMCs) after progestin treatment. The research reveals higher concentration of progestin up-regulates miR-197 expression in UtLMCs and over-expression of miR-197 induces UtLMC's apoptosis. Bio-infomation suggests E2F7 is the target gene for miR-197 and over-expression of miR-197 will down-regulate E2F7. We proposes the progestin-induced miR-197 plays a significant role on leiomyoma cells proliferation and apoptosis via the regulation of E2F7. The research will study these influential mechanisms on UtLMCs in relation with the change of E2F7 under the circumstances of over-expression or down-regulation of miR-197. The influential role on E2F7 by miR-197 will also be examined by the construction of specific vectors and RNA interfering technique. The results are to be verified by using luciferase report system. The concept that miR-197 influences UtLMCs apoptosis or proliferation after progestin treatment has not been reported elsewhere. The innovative proposal by this study could possibly find a new target gene for the treatment of uterine leiomyoma.

miR-197是应用miRNA芯片筛选的一条在孕激素干预UtLMCs中显著上调的miRNA。预研发现：miR-197过表达促进UtLMCs凋亡；高浓度孕激素促进UtLMCs中 miR-197表达上调；生物信息学预测E2F7是miR-197靶基因， 高表达miR-197后引起E2F7表达下调。我们提出miR-197通过调控E2F7参与孕激素影响子宫肌瘤增殖、凋亡的学术思想。本研究拟观察miR-197过表达、表达沉默时对孕激素干预的UtLMCs增殖、凋亡的影响及E2F7的变化；采用表达载体构建或RNAi技术，判断miR-197对 E2F7的作用；最后采用荧光素酶验证其调控作用。由于miR-197受孕激素调控、影响UtLMCs增殖凋亡功能的研究目前尚未见报道，因此具有源头创新性。研究若成功，可为子宫肌瘤防治提供新靶标。

子宫肌瘤是育龄期女性最常见的盆腔肿瘤，过早切除会严重影响女性健康，因此，保守治疗（如左炔诺酮宫内缓释系统）受到人们的重视。孕激素在子宫肌瘤发生发展的进程中扮演着双重角色，从孕激素在子宫肌瘤细胞中的调控机制着手，将有利于探寻子宫肌瘤的发病机制及子宫肌瘤保守治疗潜在的新靶标。首先我们原代分离的子宫肌瘤细胞，采用miRNA芯片技术筛选子宫肌瘤细胞与孕激素干预后的子宫肌瘤细胞中差异表达的miRNAs，芯片结果显示：与未加药物比较，孕激素干预后的子宫肌瘤细胞上调miRNAs723个、下调miRNAs739个；运用实时定量PCR验证与肿瘤细胞恶性相关的miRNAs，发现孕激素干预后，细胞中共有19个miRNAs的表达有显著差异，其中miR-197, miR-1260b等9个基因在孕激素干预组中显著高表达、miR-377-3p, miR-29c-3p等10个基因在孕激素组中显著低表达。其次，我们证实miR-197在孕激素干预UtLMCs 中显著上调；同时证明miR-197过表达显著抑制子宫肌瘤细胞的增殖、诱导子宫肌瘤细胞凋亡、降低细胞的侵袭转移能力；为初步探索miR-197通过何种分子机制调控子宫肌瘤细胞的生物学功能，我们运用高通量基因芯片技术检测miR-197过表达的子宫肌瘤细胞中的基因表达谱，结果显示与空载对照相比，8个基因(TRIM75, HBA2, VPREB1, OR5IL, ATP8A2, FDFR2, Cxorf51A, TSTD1)在miR-197过表达的子宫肌瘤细胞中显著上调，另外9个基因(CDRT7, SLC549, SFMBT2, FLJ37956, FBLN2, C10orf35, HOXD12, CACNG7, LOC100134279)的表达水平在miR-197过表达的子宫肌瘤细胞中显著下降。最后，我们采用lncRNA芯片技术筛选子宫肌瘤组织与癌旁组织中差异表达的lncRNAs。芯片结果显示：与癌旁组织相比，在子宫肌瘤组织中异常表达的lncRNA共有11条，其中2条lncRNAs的表达水平显著降低、9条lncRNAs的表达水平显著升高。以上结果提示，miR-197可能是有效治疗子宫肌瘤的一个潜在作用靶点；11条差异表达的lncRNAs也可能为子宫肌瘤的防治提供新的理论依据和潜在的干预靶标。