长非编码RNA-UCA1调节循环肿瘤细胞抵抗NK细胞杀伤机制的研究

In order to distant metastasis, circulating tumor cells (CTC), riching in tumor metastasis stem cells, should ont noly have the high metastatic proclivity but also be well-shielded from immune attack . In this project we succeed in constructing multiple metastatic nude mouse model of both mesenchymal derivative osteosarcoma and epithelial originated breast cancer.We innovatively developed the method that efficiently separated CTC. Compared to parental cells, in the both models, CTC high expressed MHCI molecules, and lcnRNA-UCA1, secreted TGFβ, and primary immunotolerance to NK killing effect. Bioinformatics analysis shew that the transcription factor CREB maight be the key factor in this pathway,and participate in the regulation of HSPs. We have proved HSPs play an important role in breast cancer cell acquired immunotolerance induced by NK killing repeatedly in our previors study. On this basis, we put forward a hypothesis, that UCA1 regulates the CTC immunotolerance. Our study maily focus on the following aims: 1)To detect killing ratio of NK92 against CTC upon different expression level of UCA1, and the concentration of cytokines in supernatant and the level of MHC-I molecules will be also quantified; 2) To explore the regulation mechanism of UCA1 self-expression and its regulation role in CREB pathway; 3) Clarify these mechanism in nude mouse modle. In conclusion, the study provides a theoretical basis for clarifying the role of lncRNAs in tumor immunotolerance, and find the new specific target antigen for more effective biological treatment.

富含肿瘤转移干细胞的循环肿瘤细胞(CTC)在具有高转移潜能的同时，还要具备逃避免疫细胞杀伤的特性才能实现异位种植。为探求CTC共性，课题构建了多发转移的间叶来源骨肉瘤及上皮来源乳腺癌的裸鼠模型，应用磁免液相仪高效分离CTC，与亲本细胞相比，两模型CTC均抗NK 杀伤、高分泌TGFβ、高表达长非编码lncRNA-UCA1及NK抑制受体KIR的配体MHCI类分子。生物信息学分析显示CREB为调控节点，与前期NK多次杀伤诱导瘤细胞免疫逃逸研究crosstalk于HSPs。故提出"UCA1调控CTC免疫逃逸"的假说。本研究拟：1) 调节瘤细胞UCA1水平，检测细胞因子分泌，MHC表达及NK92对其杀伤，证实UCA1参与免疫耐受；2）探寻UCA1自身表达及其调节CREB通路的机制；3)通过裸鼠模型验证相关机制。本研究为阐明LncRNA调节CTC免疫耐受的提供理论基础，为免疫治疗寻找新靶标提供思路。

通过多次NK细胞杀伤诱导获得耐NK杀伤的乳腺癌MD-MBA-231细胞同时用经典TGF-β诱导免疫耐受模型对比lncRNA表达谱差异，发现UCA1均显著提高，在乳腺癌MD-MBA-231细胞过表达UCA1检测mRNA表达谱变化，发现UCA1通过抑制转录因子CREB表达抑制NK细胞活化受体ULBP-2蛋白表达，乳腺癌组织UCA1过表达，血浆UCA1水平与乳腺癌预后负相关。我们还探讨了UCA1对肿瘤细胞增殖及转移的调控。进一步探讨长非编码RNA, UCA1, 的功能，应用青蒿素处理前列腺癌细胞，发现青蒿素可显著抑制前列腺癌细胞增殖及迁移，通过长非编码表达谱检测发现UCA1显著下调，并发现UCA1是miR-184的海绵分子，青蒿素通过抑制UCA/miR-184/Bcl-2通路抑制前列腺癌增殖及转移。我们研究了与免疫密切相关的抗原递呈细胞，肿瘤相关巨噬细胞，首先发现骨肉瘤原发灶分泌CCL18的TAM（CCL18/CD68双染阳性细胞）表达与骨肉瘤预后密切相关，同时发现肺转移灶CCL18/CD68双染远高于原发灶，收集血浆检测CCL18也证实该结论。我们将外源性的CCL-18加入骨肉瘤培养体系，发现EP300显著上调，骨肉瘤细胞增殖及迁移能力显著增加，同时UCA1表达也明显升高，其下游靶标β-catenin和GSK3β上调。我们在裸鼠动物实验中也验证了该结果。.通过本研究我们发现UCA1调节乳腺癌细胞NK活化受体ULBP2表达，抑制NK细胞杀伤乳腺癌细胞，同时UCA1也在多种肿瘤中促进肿瘤细胞增殖及迁移，与前列腺癌、骨肉瘤预后密切相关。