血浆/血清异常表达miR-17, miR-20a, miR-29c, 和miR-223在鼻咽癌中的临床意义和功能研究

Circulating miRNAs have many of the essential characteristics of good biomarkers as several authoritative articles reported. First, they are stable in the circulation and resistant to storage handling. Serum miRNAs are resistant to RNase digestion and other harsh conditions such as extreme pH, boiling, extended storage, and multiple freeze-thaw cycles. Second, changes in miRNA levels in circulation have been associated with different diseases as well as certain biological or pathological stages. Third, sample collection is invasive. Finally, miRNAs levels can easily be determined by various methods. Based on these advantages, miRNA is expected to become a new type of molecular markers of cancer and other diseases. However, it is still a new field with much to be explored, such as the origin, function, transportation, etc. In our previous study, it was found that miR-17, miR-20a, miR-29c, and miR-223 have abnormal levels in the serum of nasopharyngeal carcinoma patients compare to the healthy control serum. Based on preliminary results, we are trying to further explore the clinical references in early diagnosis, clinical staging, therapeutic effect, and prognosis of 4 miRNAs in nasopharyngeal carcinoma. The binding protein of 4 miRNAs in serum and culture media supernant will be identified by immunoprecitation and Western Blot. The transportation (exosome, microvesicles, shedding bodies, etc) of 4 miRNAs will be explored. The function and molecular mechanism of 4 miRNAs playing in the proliferation\invasion\apoptosis\metatasis of nasopharyngeal carcinoma cells will be studied. The association of the levels in tissue and serum of 4 miRNA will be further studied by large scale samples and in animal model. The results of this program will provides a new idea for further elucidation of miRNA function in the circulation, and provides the reliable experimental basis for development and application of circulating miRNAs as nasopharyngeal carcinoma biomarker, so it has important theoretical, clinical value and application prospect.

权威期刊论文证实，循环中miRNA稳定性好，能耐受RNase酶和严峻条件保存，特定miRNA的表达水平与疾病状态相关，基本无创，检测技术已经成熟，基于这些优点，循环miRNA已成为肿瘤等疾病中的一类新型分子标志物。目前循环中miRNA的生物学功能研究刚刚开始，其来源、运输方式、作用途径等尚不清楚。本项目在前期研究基础上，拟从鼻咽癌患者血清中异常表达的miR-17、miR-20a 、miR-29c、和miR-223入手，深入研究它们在鼻咽癌的早期诊断、临床分期、疗效跟踪、预后判断中的临床意义；通过检测血浆/血清、血细胞和鼻咽癌细胞培养上清中的4个miRNAs结合蛋白和运输形式探求循环miRNA的来源、分泌、运输途径；建立动物模型和在临床大样本查明4个miRNAs对鼻咽癌细胞的生物学功能及分子机制；利用组织和血样中miRNA的表达水平分析组织和外周血的miRNA表达的相关规律。

循环中miRNA稳定性好，能耐受RNase酶和严峻条件保存，特定miRNA 的表达水平与疾病状态相关，基本无创，检测技术已经成熟，基于这些优点，循环miRNA 已成为肿瘤等疾病中的一类新型分子标志物。本研究基于前期研究中鼻咽癌患者血清中筛选出来的差异miRNAs，进一步研究了miR-29c，miR-223，miR-486，miR-17的表达水平，并深入研究了miR-29c和miR-223在鼻咽癌发生发展中作用的分子机制。通过本研究发现了miR-29c直接通过靶基因DNMT3a调控miR-34c和mir-449a表达，后者的启动子区存在高甲基化位点，受表观遗传学调控，甲基转移酶抑制剂5-Aza-2'-deoxycytidine（5’-氮杂胞苷）处理能解除高甲基化的抑制而表达上调。miR-34c和miR-449a在多种上皮性肿瘤中表达下调，在鼻咽癌癌也表达下调。我们的研究发现了miR-29c/DNMT3a/miR-34c/miR-449a的调控轴参与鼻咽癌发生发展的分子机制。通过免疫组化检测了DNMT3a在鼻咽癌组织中高表达，与患者5年生存率呈显著负相关，因此，本研究发现了miR-34c和miR-449a在鼻咽癌中表达的新的表观调控机制。同时，鉴定了miR-29c的靶基因HBP1，一个在其他恶性肿瘤中下调的转录因子，却发现在鼻咽癌中显著上调。免疫组化检测发现，其表达与患者5年生存率明显负相关，因此HBP1可能参与了鼻咽癌的发生，其作用的分子机制还在进一步研究中。发现了与正常人血清中表达上调的miR-223直接调控靶基因FBXO8和STK39调控鼻咽癌细胞的生存和增殖，其分子机制还在深入探讨中。本研究深入研究了miR-29c和miR-223在鼻咽癌发生发展中的作用，对鼻咽癌的发生机制以及防治均具有重要的意义。发表SCI论文3篇，均标注基金资助，参编专著1项，培养硕士研究生2名。