HMGA2作为ceRNA调控子宫肌瘤增殖的机制研究

Uterine leiomyomas (ULs), related to hormone levels, are the most common indication for hysterectomy in female. As one of the surgical indications，the proliferation mechanism of ULs larger than 5cm is still unclear. HMGA2 overexpression might be one of the crucial factors contribute to the growth of ULs. Our previous study showed that HMGA2 expressed highly in ULs larger than 5cm; whereas knockdown of HMGA2 led to a significantly decrease in proliferation ability of UL cells，meanwhile miRNA let-7b increased and ERαreduced in UL cells. Software dissects that 3’UTR of HMGA2 exist binding sites with let-7, and ERα is proved to be one of the downstream genes of let-7 previously. It seemed to hint that HMGA2 might function as a competing endogenous RNA (ceRNA) to inhibit expression or activity of let-7, thereby affecting ERα expression. We intend to make use of lots of clinical samples, primary UL cells and animal models, and take HMGA2 as a starting point to do followings：(1) experiments in vivo and vitro to determine if HMGA2 impacts ERα expression to promote UL cells proliferation; (2) tissue microarray analysis to examine let-7 members associated with HMGA2; (3) alleles construction to confirm whether HMGA2 functions as a ceRNA to inhibit let-7, clarifying how HMGA2-let-7-ERα axis regulates ULs growth. It will provide an important supplement theory of abnormal proliferation in ULs, but also develop a new target of therapy for ULs.

子宫肌瘤是与女性激素水平相关的最常见的子宫切除术指征。体积>5cm肌瘤为手术指征之一，其增殖机制尚不明确。HMGA2过表达很可能是肌瘤增殖关键因素之一。我们预实验发现>5cm肌瘤HMGA2高表达，下调后细胞增殖减弱，miRNA let-7b升高、ERα减少；软件分析HMGA2 3’UTR与let-7存结合位点，既往证实ERα是let-7的下游基因之一。提示HMGA2可能作为竞争性内源RNA(ceRNA)抑制let-7并影响ERα表达。课题拟用大量临床样本、原代肌瘤细胞及动物模型，以HMGA2为切入点:(1)利用体内外实验确定HMGA2影响ERα表达促肌瘤增殖；(2)利用组织芯片分析与HMGA2相关的let-7成员；(3)构建等位基因证实HMGA2作为ceRNA抑制let-7，以阐明HMGA2-let-7-ERα轴对肌瘤增殖的调控。这将为肌瘤增殖理论提供重要补充，更为肌瘤靶向治疗开辟新途径。

子宫肌瘤是与女性激素水平相关的最常见的子宫切除术指征。体积>5cm肌瘤为手术指征之一，其增殖机制尚不明确。HMGA2亚型是子宫肌瘤的第二大分子分型亚型，与肌瘤体积大小相关，提示HMGA2过表达很可能是肌瘤增殖关键因素之一。本课题研究HMGA2类型子宫肌瘤中HMGA2如何通过调控ERα促进肌瘤增殖。利用免疫组化、蛋白质印迹和细胞增殖实验等，证实HMGA2在肌瘤中表达明显高于正常肌层，且促进原代肌瘤细胞增殖；通过免疫组化、蛋白质印迹和qPCR技术等，我们发现HMGA2与ERα蛋白水平呈正相关，且可调控ERα蛋白，而非mRNA；再加入自噬抑制剂和蛋白酶体抑制剂等进一步实验发现，自噬途径和重要自噬载体蛋白p62/SQSTM1参与该调控网络。此外，ChIP和双荧光素酶报告基因检测证明HMGA2可直接结合p62启动子抑制其转录，而肌瘤标本组织的免疫组化分析也进一步验证了HMGA2与p62的表达负相关性；同时工具细胞中的免疫共沉淀结果显示p62与ERα存在明显的相互作用，进而揭示了HMGA2-p62-ERα轴对肌瘤增殖的调控。在肾包膜下成瘤模型中，免疫组化结果显示，过表达HMGA2后，体内肌瘤中ERα和Ki67表达增多，进一步证实HMGA2在体内对肌瘤增殖的重要作用。另外，自噬激动剂雷帕霉素可阻断HMGA2-p62-ERα轴对肌瘤细胞的增殖作用，使肌瘤原代细胞增殖能力明显减弱。本项目充分阐明HMGA2-p62-ERα轴在HMGA2型子宫肌瘤中的重要作用，并发掘了自噬途径及自噬激动剂雷帕霉素在其中发挥的重要作用。这将为肌瘤增殖理论提供重要补充，更为肌瘤靶向治疗开辟新途径。