HOTTIP作为ceRNA调控WNT1/TCF4介导的乳腺癌干细胞特性的分子机制

It is still unclear what specific mechanism involves in mediating breast cancer stem cells (BCSCs) biological characteristics. We have successfully enriched mammary sphere cells MCF-7 MS with characteristics of BCSCs, and found that they highly expressed HOXA distal transcript antisense RNA（HOTTIP）, a long non-coding RNA (LncRNA), and increased the expression of WNT1 and TCF4, but decreased the express of miR-148a. Furthermore, in breast cancer tissues, we also found that the high expression of HOTTIP was positively correlated with the expression of CD44+CD24- phenotype and the high expression of WNT1, but negatively correlated with the miR-148a expression. Bioinformatic analysis showed that miR-148a combined with HOTTIP, WNT1 and TCF4 depending on the same microRNA response elements (MREs). So, Can we presume that HOTTIP regulates BCSCs characteristics mediated by WNT1 and TCF4 as a ceRNA? We plan to detect and analyze the changes and differences of the BCSCs characteristics and the expression of the expression of WNT1 and TCF4, even the Wnt signal pathway activation after we interfere the expression HOTTIP and miR-148a respectively and alternately, after confirming the direct binding between HOTTIP and miR-148a at endogenous levels. Our study will clarify the mechanism of HOTTIP regulating BCSCs biological characteristics mediated by WNT1 and TCF4 as a ceRNA, and will propose a new target for deep researching the mechanism of maintaining the BCSCs characteristics.

介导BCSCs生物学特性的相关机制尚不明确。课题组成功富集具有BCSCs特性的MCF-7 MS细胞，检测发现其高表达HOTTIP及WNT1/TCF4，低表达miR-148a；乳腺癌组织HOTTIP的高表达与BCSCs干性表型及WNT1表达正相关，与miR-148a表达负相关；信息学预测miR-148a以相同的MRE与HOTTIP和WNT1/TCF4结合。那么HOTTIP是否作为ceRNA竞争性调控WNT1/TCF4介导的BCSCs干性？本课题拟在明确HOTTIP与miR-148a特异结合基础上，检测比较分别干预及相互干预HOTTIP与miR-148a时BCSCs干性与WNT1/TCF4表达及其介导的Wnt通路活性改变的相关性，阐明HOTTIP作为ceRNA竞争性参与miR-148a调控WNT1/TCF4介导的BCSCs干性的作用与机制。为深入研究BCSCs干性维持机制提供新思路和新靶点。

长链非编码RNA（Long-chain non-coding RNA，lncRNA）在调控肿瘤发生发展中起到重要的作用。长链非编码RNA HOTTIP（HOXA transcript at the distal tip）被报道在多种肿瘤中异常表达，但其是否参与乳腺癌干细胞（Breast Cancer stem cells, BCSCs）的干性维持目前尚无相关报道。本研究中我们发现HOTTIP在BCSCs中高表达，其与乳腺癌患者预后不良相关；功能学研究揭示了HOTTIP过表达明显增加了乳腺癌细胞的克隆形成能力，增加了干性标志物OCT4和SOX2表达，而降低了分化蛋白CK14和CK18表达。相反，沉默HOTTIP明显抑制了BCSCs的干性表型，也降低了BCSCs荷瘤鼠的瘤体积和瘤重。机制研究证实了HOTTIP可与miR-148a-3p直接结合，抑制WNT1的表达，导致Wnt/β-catenin信号通路失活。总之，本研究首次发现HOTTIP在BCSCs中高表达，并通过系列实验证实了HOTTIP在促进BCSCs干性维持中的关键作用，并进一步明确了其对BCSCs干性的调控作用可能是通过调控miR-148a-3p/wnt1通路实现的。为靶向BCSCs治疗乳腺癌提供了新靶点和新的治疗策略。