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

描述（由申请人提供）：开发临床适用的癌症生物标志物，特别是微创生物标志物，多年来一直是一个不可逾越的挑战。最近循环microrna的发现带来了一些希望；这些微小rna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的癌症来源。有趣的是，与健康患者相比，癌症患者血清中几种microrna的水平较低，这表明癌症要么破坏了正常分泌这些microrna的邻近正常组织，要么影响了远端器官中这些microrna的表达和分泌。我们最近的研究支持后一种可能性；临床无病乳腺癌患者血清中miR-451和miR-101水平升高，但miR-370、miR-574-3p、miR-342-3p和miR-197水平较低。此外，患者血清中U6和5S水平升高，U6和5S是RNA聚合酶III转录的小RNA。以下假设将在本提案中进行验证：癌症患者的慢性炎症样疾病导致远端器官，特别是具有再生能力的器官（如肝脏）中microRNA/小RNA表达的永久性变化。癌症或对癌症的宿主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端器官microRNA表达的长期影响。因此，不同的microRNA和U6 RNA水平持续存在于癌症患者的血清中，即使这些患者在临床上已经摆脱了疾病。两个具体目的描述的实验将检验上述假设：1)确定在患癌动物血清中差异表达的microRNAs/小RNA对应的基因是否在肝脏和肺部表现出表达改变、组蛋白修饰和RNA聚合酶占用。乳腺癌动物模型包括MMTV-PyMT、MMTV-neu和ER1阳性和ER1阴性乳腺癌细胞的异种移植，并辅以适当的对照，用于测量血清microRNA谱和肝脏和肺部microRNA、U6和5S表达的癌症相关变化。在对照动物和荷瘤动物中，检测修饰组蛋白和RNA聚合酶II/III在肝脏和肺部U6、5S调控区域的募集情况，以及选择的microRNA基因。分析小鼠血清中IL-6的含量。II)前瞻性研究乳腺癌治疗期间血清U6、5S及选择性miRNA水平是否发生变化。对乳腺癌患者在新辅助治疗开始前、治疗结束后、手术前、手术后的血清进行U6、5S RNA和精选microrna的检测。如果癌症是血清中上述microRNA/小rna的主要来源，则在新辅助治疗和/或手术后，其水平应下降。如果特异性microRNA来源于宿主，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。

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.