长非编码RNA对成瘤性乳腺癌干细胞分化和自我更新的调控作用研究

Breast cancer stem cells (BCSCs) are the source of oncogenesis, development, metastasis and recurrence of breast cancer. Our previous studies revealed that a panel of microRNAs regulated many aspects of the stem cell-like properties of breast cancer cells,such as self-renewal, multipotent differentiation,epithelial-mesenchymal transition(EMT), and chemotherapeutic resistance. Recently, long non-coding RNA (lncRNA) has been reported to mutually regualte miRNA,and closly corralated with the development of stem cell and tumor occurrence. Therefore, our research is proposed to further explore the function of lncRNAs which regulate the biological characteristics of BCSCs and mutual regulation mechanism with the expression of miRNA. We will first analysis the difference expression of lncRNA between BCSCs and non-cancer stem cells, and compare with miRNA expression spectrum in the previous study, look for specific expression of lncRNA. Then we will explore whether lncRNA regulate the self-renewal, multi-differentiation potential, oncogenesis and resistance to chemotherapy of BCSCs in vivo and in vitro, when the level of lncRNA was incresed or decreased. Further, we will study the regulatory mechanism between lncRNA and miRNA, elucidate the signaling pathways of lncRNA in participating in the regulation of the stemness of BCSCs.Investigating the machiner that govern lncRNA expression in BCSCs will not only help us to extend the knowledge of molecular basis for the stemness of BCSCs, but also guide us to develop novel approaches that may manipulate lncRNA expression for therapeutic purposes.

乳腺癌干细胞是乳腺癌发生、发展、转移和治疗后复发的根源。申请人前期研究发现微小RNA（miRNA）对乳腺癌干细胞自我更新、多向分化和治疗抵抗等具有调控作用。近年发现长非编码RNA（lncRNA）可与miRNA相互调控，并与干细胞的发育和肿瘤的发生等有紧密的联系。因此，本课题拟进一步探索调控乳腺癌干细胞生物学特性的lncRNA的功能及与miRNA表达的相互调控机制。首先分析乳腺癌干细胞和非癌干细胞lncRNA的表达差异，对比前期研究中乳腺癌干细胞的miRHA表达谱，寻找特异表达的目标lncRNA。探索干预lncRNA水平对乳腺干细胞自我更新能力、多向分化潜能、体内成瘤能力及化疗耐药等生物学特性的调控作用，从而观察它们对乳腺癌干细胞成瘤和转移的影响。进一步研究lncRNA与miRNA之间的互相调控关系，阐明lncRNA参与调控乳腺癌干细胞特性的分子信号通路的机制。

研究背景.乳腺癌干细胞是乳腺癌发生、发展、转移和治疗后复发的根源。大量研究认为肿瘤干细胞表达大量的耐药基因，故对常规的化疗、放疗和内分泌治疗不敏感。申请人前期研究发现微小RNA（miRNA）对乳腺癌干细胞自我更新、多向分化和治疗抵抗等具有调控作用，近年发现长非编码RNA（lncRNA）可与miRNA 相互调控，并与干细胞的发育和肿瘤的发生等有紧密的联系。但是调控乳腺癌干细胞生物学特性和化疗耐药特性的lncRNA与miRNA以及相互调控机制尚不明确。.研究目标.建立具有干细胞特性的乳腺癌细胞株；乳腺癌干细胞和非癌干细胞lncRNA 的表达差异，对比前期研究中乳腺癌干细胞的miRHA 表达谱，寻找特异表达的目标lncRNA，探索干预lncRNA 水平对乳腺干细胞自我更新能力、体内成瘤能力及化疗耐药等生物学特性的调控作用，从而观察它们对乳腺癌干细胞成瘤转移和耐药的影响。.研究结果.基于前期工作基础发现的非编码小分子RNA let-7分别通过Ras 和HMGA2两条分子通路，调控乳腺癌干细胞自我增殖和多向分化的能力，在功能方面，我们通过体外实验研究证实非编码RNA let-7a减少与原发性乳腺癌化疗耐药相关，诱导let-7a表达可以通过增强化疗药物诱导的凋亡提高乳腺癌对化疗药物的敏感性；长非编码RNA H19表达水平提高与乳腺癌化疗耐药相关，敲低H19表达后通过诱导凋亡而恢复乳腺癌细胞对化疗药物紫杉醇的敏感性；长非编码RNA G6.3通过结合并稳定转录共激活因子YAP1维持乳腺癌干细胞“干性”特性和自我更新能力，并通过介导Hippo通路调控乳腺癌对化疗药物表阿霉素的敏感性；在临床应用方面，通过对H19和let-7a分析建立了一个组合风险评分，该评分对接受新辅助化疗的乳腺癌患者具有指导预后价值；对临床上单侧乳腺导管原位癌患者对对侧乳房预防性切除术的发展趋势和因素分析。.结论.本项目验证了多种长非编码RNA在乳腺癌细胞“干性”特性和自我更新能力，以及化疗药物敏感性中的作用和临床意义，这为逆转乳腺癌耐药提供了新靶点和新的预测指标。