外泌体介导的microRNAs传递在乳腺癌耐药中的过程及其机制研究

发布时间：2018-04-16 08:38

本文选题：外泌体 + 耐药　；参考：《南京医科大学》2016年博士论文

【摘要】：第一部分耐药乳腺癌细胞外泌体横向转运microRNAs传递化疗耐药多西他赛和阿霉素是乳腺癌辅助化疗的两种基本药物,然而治疗过程中会不可避免地出现耐药现象,进而影响患者临床化疗疗效。近期研究表明外泌体能在异质性肿瘤细胞之间充当传播媒介来交换遗传物质,传递耐药性,加快肿瘤进展。目前,关于乳腺癌细胞来源的外泌体的特异性作用还尚不明确。本研究发现人敏感乳腺癌细胞系MCF-7/S可以从共育的耐药突变株MCF-7/Doc及MCF-7/Adr上获得增强的存活潜能。此外,耐药突变株分泌的外泌体(D/exo和A/exo)相较于敏感株外泌体(S/exo),能够显著调控细胞周期和药物诱导的凋亡。外泌体经RNA酶处理后,其对细胞周期和凋亡耐受的作用明显下调,提示外泌体的功能是部分依赖RNA的。利用微阵列和聚合酶链反应对D/exo、A/exo和S/exo内的微小RNA(microRNAs)表达谱分析证实这些外泌体选择性地装载microRNAs。当D/exo和A/exo被受体MCF-7/S细胞摄取后,某些microRNAs的表达水平在受体细胞中显著上调。通过靶基因预测和通路富集分析表明miR-100、miR-222和miR-30a与肿瘤发病机理、膜囊泡化和化疗失败等进程相关。最后,D/exo共育实验和microRNA模拟物转染实验证实富含miR-222的D/exo能够调节MCF-7/S细胞内靶基因的表达。我们的研究结果提示耐药乳腺癌细胞可以通过释放外泌体进而传递耐药性至敏感细胞,并且该作用可能与细胞间横向转移的某些特异性microRNAs有关。第二部分外泌体传递microRNAs减少乳腺癌细胞对阿霉素敏感性阿霉素是乳腺癌临床化疗的基石,显著提高了患者总生存,然而治疗过程中耐药现象会不可避免地出现。越来越多证据表明外泌体能充当遗传物质的载体,并在异质性肿瘤细胞之间不断转运生物活性分子例如microRNAs,为肿瘤进展提供促耐药环境。我们前期研究发现多西他赛耐药株来源的外泌体能通过传递microRNAs调节乳腺癌细胞化疗敏感性。因此,我们将实验进一步拓展,探索外泌体介导的microRNAs转运在阿霉素耐药株中的耐药传递现象。结果证实阿霉素耐药株来源的外泌体(A/exo)选择性包裹microRNAs,并且当A/exo与荧光敏感株细胞(GFP-S)融合后,A/exo中高表达的microRNAs在受体细胞中也显著增加。对A/exo中含量最丰富的30个microRNAs的预测靶点行基因本体论分析发现它们参与许多关键性生物进程。此外,A/exo不仅装载与自身形成、释放有关的microRNAs,还携带与Wnt信号通路有关的microRNAs。最后,共育实验表明富含microRNAs的A/exo能提高GFP-S细胞对阿霉素的耐受,改变GFP-S细胞内基因表达。我们的实验再次充实了前期研究,证明阿霉素耐药乳腺癌细胞通过转运外泌体内特异性microRNAs,传递耐药特性并形成促癌微环境。
[Abstract]:The first part is the translocation of transmembrane transporter of chemotherapeutic agents (microRNAs) in drug-resistant breast cancer cells. Doxetaxel and doxorubicin are two essential drugs for adjuvant chemotherapy in breast cancer. However, drug resistance will inevitably occur in the course of treatment.And then affect the clinical efficacy of chemotherapy.Recent studies have shown that exocrine can act as a transmission medium between heterogeneous tumor cells to exchange genetic material, transfer drug resistance, and accelerate tumor progression.At present, the specific role of exocrine derived from breast cancer cells remains unclear.In this study, we found that human breast cancer cell line MCF-7/S could obtain enhanced survival potential from cocultured drug-resistant mutants MCF-7/Doc and MCF-7/Adr.In addition, D- and A- exo secreted by drug-resistant mutants could significantly regulate cell cycle and drug-induced apoptosis compared with those of sensitive strains.After treated with RNA, the function of exocrine on cell cycle and apoptosis tolerance was down-regulated, suggesting that the function of exocrine was partly dependent on RNA.MicroRNAs were selectively loaded with these exosomes by microarray and polymerase chain reaction.When D/exo and A/exo were ingested by receptor MCF-7/S cells, the expression of some microRNAs was up-regulated in recipient cells.Target gene prediction and pathway enrichment analysis showed that miR-100 miR-222 and miR-30a were associated with tumor pathogenesis, membrane vesicle formation and chemotherapy failure.Finally, the results of D / exo co-breeding and microRNA mimic transfection showed that D/exo rich in miR-222 could regulate the expression of target genes in MCF-7/S cells.Our results suggest that drug-resistant breast cancer cells can transfer drug resistance to sensitive cells by releasing exocrine, and this effect may be related to some specific microRNAs transversely transferred between cells.The second part of the exocrine transmission of microRNAs to reduce the sensitivity of breast cancer cells to doxorubicin is the cornerstone of clinical chemotherapy of breast cancer, significantly improved the total survival of patients, but drug resistance will inevitably occur during the treatment process.There is increasing evidence that exocrine acts as a carrier of genetic material and transports bioactive molecules such as microRNAsbetween heterogeneous tumor cells providing a pro-drug resistant environment for tumor progression.Our previous study found that the exocrine of docetaxel-resistant strain regulates the chemosensitivity of breast cancer cells by transmitting microRNAs.Therefore, we further expand our experiment to explore the drug resistance transfer of microRNAs mediated by exocrine in adriamycin-resistant strains.The results showed that the exosomes of adriamycin-resistant strains were selectively encapsulated in microRNAss, and the overexpression of microRNAs in the receptor cells was also significantly increased after the fusion of A/exo with fluorescent sensitive cell line (GFP-S).The analysis of the predicted target genes of the 30 most abundant microRNAs in A/exo revealed that they were involved in many key biological processes.In addition, A / P / exo not only carries microRNAs related to the formation and release of microRNAs, but also carries microRNAs related to the Wnt signaling pathway.Finally, co-breeding experiments showed that A/exo rich in microRNAs could increase the tolerance of GFP-S cells to adriamycin and change the gene expression in GFP-S cells.Our experiment enriches the previous studies and proves that doxorubicin resistant breast cancer cells transmit drug resistance characteristics and form a tumor promoting microenvironment by transporting in vivo specific microRNAss.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.9

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