微小RNA（miRNA）对肿瘤的影响是肿瘤研究的热点。已知miR-10a在多种肿瘤中表达上调，起原癌基因作用。至今，有关miR-10a上游的调控机制尚不清楚。我们通过下一代测序技术（Next-Generation Sequencing）结合实时荧光定量PCR发现，PIWIL2过表达的乳腺癌细胞，miR-10a表达下调5倍，提示PIWIL2可能调控miR-10a表达。前期研究发现PIWIL2能抑制肿瘤细胞体外成球和体内成瘤，进一步提示PIWIL2可能通过下调miR-10a水平影响肿瘤发生发展。本项目将进一步：(1)确立PIWIL2对miR-10a的调节作用。(2) 研究PIWIL2调控miR-10a的分子机制。(3) 确立miR-10a在PIWIL2介导肿瘤抑制中的关键作用。本项目有望阐明PIWIL2调控miR-10a表达影响肿瘤发生发展的分子机制，为发现和开发新的抗肿瘤靶点和药物提供可能。

miRNA dysregulation is one of the mechanisms in the pathogenesis of cancer. It's well known that the oncogene,miR-10a, is upregulated in many types of cancer, but the upstream regulator of miR-10a remains elusive. Combining the Next-Generation Sequencing technology with the TaqMan Real-time PCR, we demonstrated that miR-10a level was decreased 5 times in breast cancer cells with PIWIL2 overexpression, suggesting that PIWIL2 might be the upstream regulator of miR-10a. Our previous study showed that PIWIL2 inhibited tumor spheroid formation in vitro and tumor formation in a xenograft mouse model.According to these findings, we hypothesize that PIWIL2 is the upstream suppressor of miR-10a and inhibits miR-10a level resulting in tumor suppression. To test our hypothesis, we will futher examine the PIWIL2-mediated miR-10a suppression, dissect the underlying mechanism of how PIWIL2 suppresses miR-10a expression, and evaluate the roles of miR-10a in PIWIL2 mediated tumor suppression. This study will reveal the mechanisms of PIWIL2 mediated tumor inhibition through suppression of the oncogene miR-10a and provide us the possibilities for discovering new targets and developing novel drugs for cancer therapy.

微小RNA（miRNA）对肿瘤的影响是肿瘤研究的热点。已知miR-10a在多种肿瘤中表达上调，起原癌基因作用。至今有关miR-10a上游的调控机制尚不清楚。我们研究发现PIWIL2全长能抑制肿瘤细胞体外成球和体内成瘤，但其具体作用机制尚不清楚。通过下一代测序技术（Next-Generation Sequencing）结合实时荧光定量PCR，我们发现PIWIL2过表达的乳腺癌细胞中miR-10a表达下调5倍，提示PIWIL2可能通过调控miR-10a表达实现抑制肿瘤的目的。通过在乳腺癌细胞系MCF-7、MDA-MB-231和非小细胞肺癌细胞系A549中诱导表达PIWIL2，我们发现PIWIL2仅在乳腺癌细胞而不在肺癌细胞中抑制miR-10a的表达。通过对miR-10a启动子区域DNA甲基化测序，我们进一步发现PIWIL2过表达能增强miR-10a启动子的第三个CpG岛的甲基化修饰水平，提示PIWIL2对miR-10a表达的抑制很可能通过调控其启动子区域DNA的甲基化水平。但由于ChIP实验证实PIWIL2与miR-10a的启动子区域并无结合，提示PIWIL2对miR-10a启动子区域甲基化水平的影响很可能是间接地。为了寻找连接PIWIL2和miR-10a启动子区域DNA甲基化之间的关键因子，我们分析了PIWIL2过表达后的RNA-seq数据，发现PIWIL2过表达后能显著抑制一个关键DNA去甲基化酶GADD45b的表达，提示PIWIL2很可能通过抑制GADD45b进而增强miR-10a启动子区域DNA甲基化水平。为了证明该假说，我们在PIWIL2过表达的MCF-7和MDA-MB-231细胞中导入GADD45b过表达载体，并对miR-10a启动子区域DNA甲基化水平进行分析。结果显示，GADD45b过表达能显著改善PIWIL2引起的miR-10a启动子区域DNA甲基化水平上升并解除PIWIL2对miR-10a的抑制。更为重要的是, ChIP实验结果证实PIWIL2能与GADD45b启动子结合。我们的研究首次揭示PIWIL2-GADD45b-miR-10a的调控轴在乳腺癌发生发展中的作用，为乳腺癌治疗提供新的靶点。