miR-760影响乳腺癌化疗耐药性转变的机制研究

Previous study had demonstrated via microarray technique that the microRNA miR-760 is significantly down-regulated in the drug resistance breast cancer cell MCF-7Adr than breast cancer cell MCF-7. The research reveals that the deregulating of miR-760 may be involved in the development of drug resistance in MCF-7. Bio-information suggests ABCA1 gene is the target gene for miR-760 and ABCA1 plays an important role in drug resistance of breast cancer. We propose that miR-760 developes drug resistance of breast cancer via the regulation of ABCA1. The research will study these influential mechanisms in the difference of miR-760 expression between MCF-7 and drug resistant cell lines. Using luciferase report system to analysis of the miR-760 directly regulates the function of ABCA1. Observe the change of drug resistance of breast cancer cell line and expression of ABCA1 under the circumstance of over-expression or down-regulation of miR-760. The influential role on ABCA1 by miR-760 will be examined by the construction of specific vectors and RNA interfering technique. We will also use model animals to evaluating the effect of miR-760 on drug resistance of breast cancer cells and expression of ABCA1. The concept that miR-760 influences drug resistance of breast cancer has not been reported before. The innovative proposal by this study could possibly find a new target gene to avoid drug resistance of breast cancer.

miR-760是我们通过芯片筛选发现的、差异低表达于乳腺癌耐药细胞MCF-7Adr、功能未知的miRNA。前期发现，抑制miR-760可增加乳腺癌细胞MCF-7耐药性；软件预测与化疗耐药相关的ABCA1是miR-760的靶基因；而抑制miR-760的MCF-7细胞中ABCA1表达显著升高，提示miR-760可能通过调控ABCA1而参与乳腺癌化疗耐药的新机制。研究拟采用过表达和敲低技术，评价miR-760对乳腺癌细胞耐药转变及ABCA1表达的影响；以萤光素酶实验论证 miR-760对ABCA1的直接作用；以ABCA1为机制线索，通过挽救实验论证miR-760致乳腺癌细胞耐药转变的分子机制；借助小鼠荷瘤模型在体观察miR-760与乳腺癌耐药性之间的关系。miR-760在乳腺癌耐药中的作用与机制未见报道，本研究将可能为临床乳腺癌化疗耐药提供潜在的干预靶标。

miR-760 是我们前期在乳腺癌组织和细胞中筛选发现的、不仅在耐药组织中，而且在耐药细胞株中均显著低表达，而且变化倍数最大的一条功能未知的microRNA。通过构建miR-760慢病毒表达/沉默载体，在MCF-7/Adr细胞中转染miR-760过表达慢病毒载体（空载为对照），定量验证PCRmiR-760过表达15.82倍；同时在MCF-7细胞中转染miR-760沉默慢病毒载体，定量验证miR-760表达下调2.7倍。升高miR-760的表达水平会使MCF-7/ADR细胞对药物的敏感性增强，耐药性降低；反之，降低miR-760的表达水平能够降低MCF-7细胞对药物的敏感性。通过高通量测序技术检测miR-760过表达后表达水平发生改变的基因，结合生物信息学分析，发现ABCA1可能是miR-760下游靶基因。定量PCR和western结果显示，miR-760过表达后，ABCA1的表达水平显著下调；miR-760表达沉默后，ABCA1表达水平显著上调；同时双荧光素酶报告基因实验证明miR-760能够结合到ABCA1-mRNA的3’-UTR区，干扰ABCA1的蛋白合成。以上结果表明ABCA1的确是miR-760调控的目的基因。ABCA1在调控乳腺癌细胞药物敏感性中发挥着重要作用，ABCA1过表达，能够显著增强乳腺癌细胞的化疗耐药性。通过挽救实验表明，miR-760通过调控ABCA1的表达，影响乳腺癌细胞对化疗药物的敏感性。将miR-760 过表达、沉默细胞分别进行裸鼠成瘤实验，结果发现miR-760过表达促进肿瘤细胞在裸鼠体内增殖，缩短裸鼠的寿命；奇怪的是，miR-760沉默与过表达效果相似，具体原因仍在研究中。综上所述，miR-760能够调控乳腺癌化疗耐药性，有可能为临床乳腺癌化疗耐药提供潜在的干预靶标。此外，我们通过lncRNA高通量测序技术检测了MCF-7与MCF-7/Adr中差异表达的lncRNAs，有望为进一步的机制研究提供必要的数据。