Exosomes携带miRNAs参与结直肠癌细胞对耐奥沙利铂耐药的机制研究

It is not clear about the mechanisms of exosome uptake and drug resistance induced by exosomal shuttle miRNA. In our present study, we found that expression of miR-1299 in exosome released by oxaliplatin (L-OHP)-resistant colorectal cancer (CRC) was significantly higher than that in L-OHP sensitive CRC. Knockdown the expression of miR-1299 in exosome/R remarked inhibited the CRC drug resistance. By bioinformatics software, SEPT10 was predicted to be a potential target gene of miR-1299. Thus, we propose a hypothesis that exosomal/miR-1299 released by L-OHP-resistant CRC cells is endocytosed by recipient cells. In recipient cells, exosomal/miR-1299 could lead to L-OHP resistance by regulating the expression of SEPT10 and subsequently affect tubuline formation in parental CRC cells. To validate the hypothesis, we plan to determine the routes of exosome uptake by uptake routes inhibitors and immunofluorescence localization. Next, dual-luciferase reporter system will also be used to confirm the target gene of exo/miR-1299. Additionally, the mechanism concerning exo/miR-1299 induced drug resistance by regulating the expression of SEPT10 will be assessed by miRNA inhibitor and lenti-overpression of SEPT10. This study might provide a new potential biomarker for improvement the L-OHP-resistance in CRC.

Exosomes携带miRNAs进入细胞的方式和诱导耐药的机制不清楚。课题组前期发现miR-1299在结直肠癌奥沙利铂(L-OHP)耐药细胞所释放的exosome/R中高表达，沉默其表达后，结直肠癌细胞耐药能力显著下降。采用生物信息学软件预测miR-1299调控的靶基因为SEPT10。因此提出“结直肠癌L-OHP耐药细胞释放的exosomes携带miR-1299经内吞途径进入靶细胞调控SEPT10蛋白的表达从而影响微管形成而诱导结直肠癌敏感细胞耐药”这一科学假说。本项目拟采用摄取通路抑制剂和免疫荧光共定位方法确定细胞摄取exosome/miRNA方式，双荧光素酶报告系统验证exo/miR-1299调控SEPT10，采用miRNA抑制剂和慢病毒过表SEPT10技术研究exo/miR-1299影响SEPT10表达诱导耐药的分子机制，为逆转结直肠癌细胞对L-OHP耐药提供新靶标。

奥沙利铂（OX）是抑制DNA复制和转录并导致细胞死亡的第三代铂化合物，已被证明可有效改善III期和高危II期结直肠癌患者的预后。尽管取得了有效的临床结果，仍有90％的OX敏感性CRC逐渐变成难治性的。目前对其背后机制的理解仍然有限。. 近年来研究发现，外泌体(Exo)miRNA与多种癌症的化学耐药有关。我们通过超速离心从OX敏感（Exo-S）和耐药性CRC细胞（Exo-R）中分离了外泌体，并进行了表征。结果显示，主要的颗粒表现出典型的外泌体形态。接下来，我们分别与Exo-S和Exo-R共培养亲代CRC细胞。结果表明外泌体可以在CRC细胞之间转移OX耐药特性。. 近年来的研究报道，外泌体miRNA参与化学耐药性，我们进一步从Exo-S和Exo-R筛选了miRNA，以分别表征亲本和OX耐药细胞中的差异miRNA。我们发现新型miRNA miR-46146在HCT116-R和HT29-R的Exo-R中显著上调。. 考虑到外泌体可能是从OX耐药细胞传递到敏感细胞，我们进一步研究了外泌体miR-46146对其受体细胞的影响。结果显示，共培养Exo-R的受体细胞中miR-46146水平显著增加。通过cy3荧光标记技术，证明miR-46146通过外泌体转移到HCT16细胞中。接下来我们通过敲低OX耐药细胞中miR-46146的表达来评估外泌体miR-46146对OX耐药的影响。结果显示，与对照相比，外泌体miR-46146的水平显著降低。在有OX的情况下，与Exo-R相比，与miR-46146-KD-Exo-R共培养时，敏感细胞的细胞活力显著下降。细胞凋亡试验显示，与Exo-R相比，与miR-46146-KD-Exo-R共培养后敏感细胞的OX耐药能力减弱。这些数据表明Exo-R miR-46146介导了OX耐药性转移。. 我们随后利用生物信息学软件和双重萤光素酶报告基因实验验证miR-46146的PDCD10是miR46146的直接功能靶标，PDCD10表达的增加可能会逆转Exo-miR-46146驱动的化学耐药的作用。. 上述结果表明，外泌体miR-46146通过靶向调控PDCD10表达诱导细胞对OX耐药，本项目结果为使CRC细胞对OX重新敏感的提供潜在的分子靶标。