基于人源性异种移植小鼠模型的乳腺癌骨转移机制研究

Bone metastasis of breast cancer seriously harm to female life. But the clinical treatment and mechanistic study still need to be further improved. To solve the difficulty to obtain clinical metastatic bone specimens of breast cancer, we established a novel human-derived bone metastasis mouse model of breast cancer, in which the primary tumor, bone metastasis and breast cancer cells are all human-derived. Based on this model, we obtained the matching specimens of orthotopic breast cancer and bone metastasis. Then we had these breast cancer cells primary cultured and purified, and finally obtained the genome and miRNA data. Based on these data, we will study the mechanisms of breast cancer bone metastasis. MMP2 and SPARC genes were chosen as targets by scientific selection. Using technology of siRNA interference and lentiviral transfection, the effects of the two genes on bone metastasis of breast cancer will be studied in cell biology behavior, animal model validation, clinical specimen's validation and prognosis of patients. Based on software and literature search, their upstream regulatory miRNA and the potential moderating role of SPARC on MMP2 will be discussed. Otherwise, we will continue to analyze the data acquired from the precious high throughput comparative study. This study will deepen the understanding of the mechanism of breast cancer bone metastasis.

乳腺癌骨转移严重危害女性生命和生活质量，其临床治疗和机制研究有待进一步完善。针对难以获取临床骨转移标本的困境，我们在前期工作中构建了新型的人源性乳腺癌骨转移小鼠模型，保证了其原发灶、转移灶和乳腺癌细胞均为人源性。基于该模型，我们获得了匹配的乳腺癌原发灶和骨转移灶标本，并进行了乳腺癌细胞原代培养和提纯，得到了基因组和miRNA组的数据。本研究拟基于该数据对乳腺癌骨转移机制进行深入研究。通过科学甄选，MMP2和SPARC基因成为研究对象。项目通过siRNA干涉和慢病毒转染等基因操作，从细胞生物学行为、动物模型验证、临床标本检测和患者预后研究四个层次阐明候选基因在乳腺癌骨转移过程中的功能。通过软件计算和文献检索，探索它们的上游调控miRNA的作用以及SPARC对MMP2潜在的调节作用。同时以这些研究为基础，继续发掘前期高通量对比研究获得的数据。本研究将加深人们对乳腺癌骨转移的机制的理解。

我们运用人源性乳腺癌骨转移小鼠模型，对移植的人乳腺组织中的原发灶和远处移植人骨中的转移灶进行了基因组学分析，发现基质金属蛋白酶2（MMP2）在从乳腺组织到人骨组织的转移过程中发生了极高的上调，推断MMP2很可能在乳腺癌骨转移过程中发挥着重要作用。在对该组标本同时进行的miRNA分析中，发现MMP2的调控子miR-106b表达下调。而MMP2及其调控子miR-106b在乳腺癌骨转移中的研究甚少。.本研究从基因水平和蛋白水平证实：MMP2在侵袭能力强的乳腺癌细胞如SUM1315-bo中表达较高，而在侵袭能力较差的乳腺癌细胞如MCF-7中表达较低；这一表达趋势与miR-106b相反。运用免疫组织化学方法分析50例乳腺癌病人原位肿瘤标本切片，与未发生骨转移的乳腺癌病人相比，MMP2蛋白在乳腺癌骨转移病人原位肿瘤标本中表达较高。.超表达和干涉MMP2基因的表达证实MMP2能增强乳腺癌细胞的侵袭和迁移能力而miR-106b则显著抑制。荧光素酶报告基因试验证实miR-106b可以下调MMP2的表达。细胞迁移和侵袭实验证实：MMP2表达的下调可以逆转miR-106b抑制剂(miR-106bI)的功能。此外, Western Blot的方法证实：乳腺癌细胞SUM1315-bo中转染miR-106b模拟剂(miR-106b)或MMP2干涉片段后，其下游ERK/p-ERK表达减少。TRAP（抗酒石酸酸性磷酸酶）染色证实：乳腺癌细胞SUM1315-bo在诱导HMSC(人骨髓间充质干细胞)定向分化为成骨细胞的过程中，乳腺癌细胞SUM1315-bo能分泌某些细胞因子，从而导致最终分化为破骨细胞的数量增多。Western Blot方法证实：SUM1315-bo中下调MMP2基因后，会导致HMSC定向分化为成骨细胞中RANKL/OPG轴的失衡，即OPG（骨保护素）含量增加、RANKL（细胞核因子B受体活化因子配体）含量减少，最终导致破骨细胞分化数量的减少。.MMP2过表达是乳腺癌骨转移的一个危险因素，miR-106b失活是MMP2失控并促进乳腺癌骨转移的一条途径；MMP2通过调节ERK信号通路进而调控骨微环境促进乳腺癌溶骨性骨转移。miR-106b-MMP2-ERK信号通路有潜力成为乳腺癌骨转移预测因子和治疗靶点。