lncRNA H19在多柔比星和他莫昔芬耐药中的作用、分子机制与临床应用研究

Doxorubicin (DOX) and tamoxifen (TAM) are the basic components of chemotherapy and endocrine therapy for breast cancer, respectively. A large number of patients receiving these therapies encounter recurrence and poor prognosis. In our preliminary study, we found that lncRNA H19 could induce DOX and TAM resistance in the breast cancer cells through regulation of the methylation and expression of ABCB1 and NAT1 genes. More interestingly, the regulation of H19 presented bi-directional effect on these two genes. However, the mechanism of this specific and bi-directional regulation is yet to be explored. Based on our hypothesis, we will first identify the interacting trans-acting factors of H19 through in vitro transcription and RNA-pulldown/protein profiling; then illustrate the mechanism of the specific and bi-directional regulation of H19 on the methylation and expression of ABCA1 and NAT1 using over-expression or knock-down techniques; and finally reveal the effect of H19 expression on DOX and TAM resistance/curative effect from animal models and clinical studies. This work will provide research basis for discovery of new biomarkers for predicting the resistance/efficacy of DOX/TAM, promotion of personalized chemotherapy or endocrine therapy for breast cancer, and development of new drug targets.

多柔比星(DOX)和他莫昔芬(TAM)分别是乳腺癌化疗和内分泌治疗基础成分，大量患者接受治疗时因耐药复发而预后更差。我们前期研究发现lncRNA H19介导乳腺癌细胞DOX和TAM耐药，并通过调控ABCB1或NAT1基因甲基化和表达改变而实现；同时发现H19对此两个基因甲基化和表达改变调控呈双向性。但是，H19特异性、双向性调控ABCB1和NAT1甲基化和表达变化具体机制不明。依据我们的假说，本项目将采用体外转录和RNA-pulldown/蛋白质谱等明确与H19互作的反式作用因子；过表达或敲减等技术阐明H19特异性、双向性调控ABCB1或NAT1甲基化和表达改变具体机制；利用动物实验和临床研究揭示H19表达对DOX和TAM耐药/疗效的影响。本项目将为发现预测DOX或TAM耐药/疗效的新生物标志物、推动个体化乳腺癌化疗或内分泌治疗新方案，开发新治疗药物靶点提供研究基础。

耐药是癌症临床系统性治疗中一个长期存在的现象和难题，耐药的发生可以导致肿瘤治疗失败。前期研究发现lncRNA H19是雌激素受体下游调控的目的基因，可以部分介导雌激素信号激活的雌激素受体阳性乳腺癌细胞增殖。本项目进一步探讨lncRNA H19在雌激素受体阳性乳腺癌细胞化疗及内分泌治疗耐药中的作用及其机制。本项目课题组自行构建了多药耐药MCF-7细胞株和他莫昔芬耐药MCF-7细胞株，利用遗传学和药理学研究方法，在这些细胞模型中揭示了H19在耐药株的高表达介导细胞耐药的作用，进一步分别阐明了H19通过调控CUL4A/ABCB1分子通路介导化疗药物多柔比星耐药；H19通过调控TPD52表达，抑制细胞自噬引起多西他赛耐药；H19还通过调控NAT1基因甲基化水平，抑制内分泌治疗药物他莫昔芬代谢成生物活性产物导致他莫昔芬耐药。本项目取得的研究结果充分显示lncRNA H19在雌激素受体阳性乳腺癌细胞系统性治疗抵抗过程发挥了重要的调控作用，居于核心调控地位，从表观遗传学角度为临床乳腺癌耐药问题提供了新的机制解释，也为解决雌激素受体阳性乳腺癌系统性治疗耐药问题提供了新的靶点，具有一定的临床及转化意义。