【作者】 顾奕琦；

【导师】 续哲莉；

【作者基本信息】 吉林大学 ， 外科学， 2014， 博士

【摘要】 目的：乳腺癌发病率日益增高，渐成为威胁女性生命的第一位恶性肿瘤。绝大多数的乳腺癌变起源于导管上皮细胞，上皮细胞具有在哺乳期产生并排射乳汁的作用，但临床显示在哺乳期过后数年乃至数十年仍在大量患者的乳管内发现积乳且泌乳素在正常范围。已知肿瘤微环境对肿瘤的形成和发展起着重大作用，积乳可被认为是乳管上皮细胞的微环境，它存在和变化产生的作用还没有人研究。近年来在人类体液如血液、哺乳期乳汁、唾液等中被发现含有多种miRNA，这些miRNA与细胞的生理病理活动直接相关，尤其肿瘤微环境中的miRNA对于肿瘤的形成和细胞间的关联起着重要作用。因此，通过研究导管内积乳及合并肿物的miRNA表达谱将会引领我们发现导管上皮细胞微环境的变化，有助于发现特异miRNA在致瘤过程中的作用。另外与血清学对比，探讨乳管内液特异性miRNA作为新的肿瘤标志物的优势。方法：临床中有大量患者在乳管镜检查前后被发现有乳管内积乳，部分患者镜检同时发现合并有乳管内肿物，我们采集单纯积乳和积乳合并乳管内肿物患者的导管灌洗液。分别提取小RNA，质检后做高通量测序，将全长度的小RNA筛选后得到miRNA做进一步差异分析，得到两组的miRNA表达谱并预测出新miRNA，对比miRBase找到发生碱基突变的miRNA。之后做候选miRNA的靶基因预测及代谢通路富集分析可能的生物学过程。在随机的20个独立样本中做real-time RT-PCR验证miRNA的表达量，最后在随机7对样本中对比积乳和血清中同种miRNA的表达量，并做统计分析。结果：我们在积乳和乳管内肿物合并积乳组的导管内液中成功提取出纯度较高的小RNA，通过高通量测序，我们在单纯积乳的样本中发现了266种已知的miRNA和271种新的miRNA，在积乳合并肿瘤的样本中发现了271种已知的miRNA和140种新的miRNA，同时得到了差异表达的miRNA。积乳合并肿瘤组对比单纯积乳组，我们发现了10种显著下调的miRNA，如let-7、miR-29a、 miR-146、miR-223等；7种显著上调的miRNA，如miR-451、 miR-185、miR-107、miR-92、miR-10等。接下来用生物信息学富集分析差异性miRNA靶基因所处的细胞位置、分子功能和参与的生物学过程，值得一提的是肿瘤组显著上调的7种miRNA的靶基因所处的细胞位置显著富集于细胞膜上。再进一步分析得出与细胞间信号转导和肿瘤形成有关的信号通路，其中单纯积乳组3种信号通路，积乳合并肿物组9种信号通路。另外有13种miRNAs在两组中表达量均很高但无明显差异，其中miR-140归一化后的表达量在两组中十分接近。接下来，我们选择了三种已知的miRNAs用real-time RT-PCR方法证实它们在随机选取的积乳及积乳合并肿物组均有高表达，且这三种miRNAs在积乳中的表达量要明显高于血清中的表达量。结论：本实验第一个得出了人单纯积乳和积乳合并肿物时的miRNA表达谱。表达量高的积乳miRNAs不同程度地参与了“癌症中转录误调信号”通路。表达量高且有明显差异的miRNAs所靶向的基因显著富集于“粘附连接”信号通路，可见在肿瘤发生前后，上皮细胞微环境中的miRNA的种类和表达量有显著变化。乳管内肿物合并积乳组表达显著下调的10种miRNA中，既往对乳腺癌的研究中被认为6种有抑癌作用，1种有致癌作用，3种在癌发展中有两面性，它们的靶基因显著富集于与细胞间信号转导有密切关系的“MAPK信号通路”，暗示可能通过调节某些靶基因的表达来抑制上皮细胞增殖而发挥作用。乳管内肿物合并积乳组表达显著上调的7种miRNAs中，既往对肿瘤的研究中被认为5种有致癌作用，1种有抑癌作用，1种有两面性。它们的靶基因富集于细胞膜且显著富集在9种参与细胞间信号传递和致瘤作用的信号通路中，在微环境中可能起到促进肿瘤形成的作用。miR-140和miR-21在积乳中的表达量较稳定，尤其是前者或可作为积乳性乳管内液miRNA表达水平的内参。积乳可以作为乳腺上皮细胞的微环境，其中富含大量的miRNAs，且表达量明显高于血清，可以作为潜在的乳腺肿瘤标志物。更多还原

【Abstract】 Objection: The incidence of breast cancer is rising,。Breast cancer gradually become thefirst malignant tumor in women. Breast cancer cells mostly change from epithelial cells. Inthe clinic, we find that many patients with milk remains in their breast instead of dischargingduring feeding, resulting in ‘milk stasis’. The duration required for milk stasis for milk stasisranges from several years to more than30years with nomal PRL. Tumor microenvironmentplays an important role in breast carcinogenesis. Milk acts as an important microenvironmentof breast cancer, but its role in breast carcinogenesis is largely unknown. Recently peoplefound miRNAs in human body fluid such as serum, milk,saliva and so on. These miRNAs aredirectly related to the cell activity. So, studying the miRNA profiling between milk stasis onlyand milk stasis plus tumor group can help us to find the changes of microenvironment forepithelial cell, and also find the function of special miRNAs in neoplasia.Methods: Samples were collected by lavaging the milk ducts from the two groups. Afterisolating total RNAs using miRNA Isolation kit, we did high-throughput sequencing andselected miRNAs from full lengh small RNAs. Then we got miRNAs profiles with differentexpression in the two groups, also got novel miRNAs. For the candidate of miRNAs, we didtarget genes prediction and metabolic pathway analysis of possible biological processes. Realtime RT-PCR was used to verify the expression of miRNAs in20samples. Finally wecompared the expression between milk stasis samples and the same patients’ serum in7pairsof samples.Results: We successfully extracted miRNAs from the two groups. We identified266known miRNAs and271novel miRNAs in10milk stasis only samples,271known miRNAsand140novel miRNAs in10milk stasis plus breast tumor samples by high-throughput sequencing. MiRNA profiles were different between the two groups. Furthermore,we foundten significantly down-regulated miRNAs such as let-7,miR-29a, miR-146and miR-223,while seven significantly up-regulated miRNAs such as miR-451,miR-185,miR-107,miR-92,miR-10were in the milk of milk stasis plus tumor patients. We analyzed the results of targetgenes prediction such as molecular function, cellular component and biological process, alsogot metabolic pathway analysis of possible biological processes,3in milk stasis only groupand9in milk stasis plus tumor group. We identified13miRNAs which high expressed in bothgroups with no signiftant difference, especially miR-140. Three kinds of the identifiedmiRNAs were selected using real time RT-PCR, confirming that these miRNAs were highlyexpressed. The results also showed that those three kinds of miRNAs were more abundant inthe milk than in the blood.Conclusion: We make the first miRNA profiling in milk stasis and we found thehigh-expression miRNAs all took part in the “regulation of cancer inordinately”. Significantlydifferent expression miRNAs could regulate the “adhesion junction” pathway. We found10miRNAs significantly down-regulated in milk stasis plus tumor group,6tumor suppressor,1oncogene and3dual character.There might be a close relationship between them andinformation transmission by ‘MAPK signaling’ pathways. Suggesting that they mightcontribute the microenvironment of epithelial cells as a role of suppressor.7miRNAs(5oncogene,1tumor suppressor and1dual character), most target genes are on themembrane,significantly up-regulated in milk stasis plus tumor group might act as oncogenein the microenvironment joined nine pathways about neoplasia and information transmissionamong epitheliar cells. miR-140might be internal reference.MiRNAs in the milk could be abetter biomarker for breast tumor than in the blood.更多还原