Persistent microRNA changes in serum of cancer-free breast cancer patients

描述（由申请人提供）：临床适用的癌症生物标志物，特别是微创生物标志物的开发多年来一直是一个难以克服的挑战。最近发现的循环microRNA提供了一些希望;这些microRNA被认为是乳腺癌、肺癌和前列腺癌的生物标志物。这些循环microRNA的来源尚不清楚，但据信是通过分泌的外泌体或由垂死的癌细胞释放而来自癌症。有趣的是，与健康人相比，癌症患者血清中几种microRNA的水平较低，这表明癌症要么导致这些microRNA正常分泌的相邻正常组织的破坏，要么影响这些microRNA从远处器官的表达和分泌。我们最近的研究支持后一种可能性;来自临床上无疾病的乳腺癌患者的血清显示miR-451和miR-101水平升高，但miR-370、miR-574- 3 p、miR-342- 3 p和miR-197水平较低。此外，患者血清中U6和5S水平升高，这是由RNA聚合酶III转录的小RNA。 本提案将测试以下假设：癌症患者的慢性炎症样疾病导致远处器官中microRNA/小RNA表达的永久性变化，特别是在具有再生能力的器官（如肝脏）中。癌症或宿主对癌症的反应导致循环细胞因子水平升高，如白细胞介素6（IL-6），这可能介导癌症对远处器官中microRNA表达的长期影响。因此，即使在这些患者临床上没有疾病之后，不同的microRNA和U6 RNA水平仍然存在于癌症患者的血清中。在两个特定目的中描述的实验将测试上述假设：I）确定对应于在携带癌症的动物的血清中差异表达的微小RNA/小RNA的基因是否在肝和肺中显示改变的表达、组蛋白修饰和RNA聚合酶占据。将使用乳腺癌动物模型（包括MMTV-PyMT、MMTV-neu和ER 1阳性和ER 1阴性乳腺癌细胞异种移植物）沿着适当的对照来测量血清microRNA谱以及肝脏和肺中microRNA、U6和5S表达的癌症相关变化。将在对照和荷癌动物中测量修饰的组蛋白和RNA聚合酶II/III向U6、5S的调控区和肝和肺中选择的微小RNA基因的募集。将分析血清中的小鼠IL-6。II）前瞻性研究血清U6，5S和选择的miRNA水平在乳腺癌治疗期间是否变化。在开始新辅助治疗之前、在完成治疗之后但在手术之前立即以及在手术之后，将检查来自乳腺癌患者的血清的U6，5S RNA和选择的microRNA。如果癌症是血清中上述microRNA/ small RNA的主要来源，则其水平应在新辅助治疗和/或手术后下降。如果特异性微小RNA是宿主来源的，并且其在远端器官中的表达变化是永久性的，则治疗不应影响其表达。长期目标是确定癌症引起的对其他器官的附带损害以及这种损害如何影响癌症患者的整体健康。 公共卫生相关性：该提案将检查乳腺癌患者血清中检测到的微小RNA和小RNA是来自癌症还是来自远处器官。癌症患者的慢性炎症样病症可能会导致肝脏和肺部的永久性基因表达变化，从而导致血清microRNA和小RNA水平的改变。循环microRNA可能是癌症的无形长臂，可以到达远处器官，因为循环microRNA可以与异型细胞融合并影响基因表达。

DESCRIPTION (provided by applicant): The development of clinically applicable biomarkers for cancer, particularly minimally invasive biomarkers, has been an insurmountable challenge for a number of years. Recent discovery of circulating microRNAs provided some hope; these microRNAs have been suggested to be biomarkers of breast, lung, and prostate cancer. The source of these circulating microRNAs is unknown but is believed to be derived from cancer either through secreted exosomes or released by dying cancer cells. Intriguingly, the levels of several microRNAs are lower in the serum of cancer patients compared to healthy suggesting that cancer is either causing destruction of the adjoining normal tissues from which these microRNAs are normally secreted or it is influencing the expression and secretion of these microRNAs from distant organs. Our recent studies support the latter possibility; serum from breast cancer patients, who are clinically disease-free, displayed elevated levels of miR-451 and miR-101 but lower levels of miR-370, miR-574-3p, miR-342-3p, and miR-197. Additionally, serum from patients contained elevated levels of U6 and 5S, which are small RNAs transcribed by RNA polymerase III. The following hypotheses will be tested in this proposal: Chronic inflammation-like condition in cancer patients lead to permanent changes in microRNA/small RNA expression in distant organs, particularly in organs with regenerative capacity such as liver. Cancer or host-response to cancer causes elevated levels of circulating cytokines such as interleukin 6 (IL-6), which may mediate the long-term effects of cancer on microRNA expression in distant organs. Consequently, distinct microRNA and U6 RNA levels persist in the serum of cancer patients even after these patients are clinically free of the disease. Experiments described in two specific aims will test the above hypotheses: I) Determine whether genes corresponding to microRNAs/small RNA differentially expressed in the serum of cancer bearing animals show altered expression, histone modifications, and RNA polymerase occupancy in liver and lungs. The animal models of breast cancer including MMTV-PyMT, MMTV-neu and xenografts with ER1- positive and ER1-negative breast cancer cells along with appropriate controls will be used to measure serum microRNA profile and cancer-associated changes in microRNA, U6 and 5S expression in liver and lungs. Recruitment of modified histones and RNA polymerase II/III to regulatory regions of U6, 5S, and select microRNA genes in liver and lungs will be measured in control and cancer bearing animals. Serum will be analyzed for mouse IL-6. II) Investigate prospectively whether serum U6, 5S and select miRNA levels change during breast cancer treatment. Serum from breast cancer patients prior to starting neoadjuvant therapy, immediately after completing therapy but before surgery, and after surgery will be examined for U6, 5S RNA and select microRNAs. If cancer is the primary source of the above microRNA/ small RNAs in the serum, their levels should drop after neoadjuvant therapy and/or surgery. If specific microRNA is host -derived and changes in its expression in distant organ is permanent, therapy should not influence its expression. Long-term goal is to determine cancer-induced collateral damage to other organs and how this damage may influence overall health of cancer patients. PUBLIC HEALTH RELEVANCE: This proposal will examine whether microRNAs and small RNAs detected in the serum of breast cancer patients originate from cancer or from the distant organs. It is likely that the chronic inflammation-like condition in cancer patients may cause permanent gene expression changes in liver and lungs, which leads to altered levels of serum microRNAs and small RNAs. The circulating microRNAs may be the invisible long-arm of cancer that reaches distant organs as circulating microRNAs can fuse with heterotypic cells and influence gene expression.