基于分泌型microRNAs差异表达谱的结直肠癌转移预测模型构建及应用

Tumor metastasis is the main cause of death in colorectal cancer （CRC） patients. Notably, extensive metastasis renders the current treatment ineffective. Therefore, the prediction of the potential metastasis in CRC can provide valuable information as well as opportunities for enhanced intervention, leading to an improved prognosis and increased survival rate. Recently, it is reported microRNAs are associated with tumor metastasis, and can be secreted into serum by cells, which open up a new avenue for noninvasive cancer prediction. SW480 and SW620 cell lines are derived from primary colon tumor and lymph node metastasis of the same patient, respectively. Based on the study strategy of "Secretome", the secrete microRNAs profiles of these two CRC cell lines with different metastatic potentials have been analyzed by Solexa sequencing technique in our previous studies. However, lymph node metastasis is only the intermediate step in CRC metastasis. In an effort to gain further insight, we will establish a distant metastatic cell line via serial passage of SW480 in nude mice, and then analyze its secrete microRNAs profile based on the above methods. Compared with the microRNAs profiles of SW480, the differentially expressed microRNAs in the two metastatic cell lines are selected. Next, we will establish a high throughput xMAP technology to confirm the above microRNAs with serum samples, screen the significant microRNAs, and further construct predictive model using support vector machines. Finally, the predictive value of this model will be further assessed in clinic, which may show us medically values on prediction of CRC metastssis and eventually guide therapeutic choices.

转移是导致结直肠癌患者死亡和治疗失败的最主要原因，如何对其进行有效预测是临床亟待解决的难题。近期研究发现，microRNAs与肿瘤转移密切相关，并可经细胞代谢分泌到血清中，为无创性预测肿瘤转移提供了一条新的途径。课题组基于"分泌蛋白质组学"的研究思路，前期已利用Solexa技术完成了相同遗传背景但不同转移潜能的人结直肠癌原发灶和淋巴结转移灶细胞株SW480、SW620的分泌型microRNAs表达谱，现拟采用裸鼠体内连续传代法进一步筛选SW480的远处转移细胞株，再利用上述方法分析其分泌型microRNAs表达谱，通过三者的比较，初步获得差异microRNAs，进而建立针对上述microRNAs的xMAP高通量检测方法进行临床验证分析，最后筛选差异有统计学意义者，运用支持向量机构建结直肠癌转移预测模型，探讨其在结直肠癌转移预测中的价值，为结直肠癌转移的有效预测及临床治疗方案的选择提供依据。

结直肠癌是一种发病率和死亡率较高的恶性肿瘤，发生转移是直肠癌患者治疗失败和死亡的最主要原因。研究显示，超过半数的结直肠癌患者最终会发生转移，即使早期结直肠癌患者（TNMⅠ期、Ⅱ期）体内也存在一定的微转移，导致术后肿瘤的转移复发。从临床治疗效果来看，对转移或转移高危人群的准确预测，积极采取预防性治疗措施，是降低结直肠癌复发转移、改善预后的重要环节。本课题收集30例结直肠腺癌、25例结直肠腺瘤和30例健康对照血清，采用二代测序技术对三组混合样本进行测序，选择在三组样本中表达无差异的microRNAs作为候选内参基因，筛选差异表达microRNAs作为候选标志物，后期通过大量临床样本验证，发现miR-191-5p和 U6的组合可作为结直肠腺癌、结直肠腺瘤和健康对照血清中microRNAs RT-qPCR检测的最适宜内参基因，血清miR-19a-3p，miR-92a-3p，miR-223-3p和miR-422a可作为结直肠腺癌潜在的肿瘤标志物，构建的4-microRNAs panel能够准确区分结直肠腺癌、腺瘤和健康对照者，为结直肠腺癌早期诊断提供了一条新途径。通过对40例淋巴结转移结直肠癌患者和40例未发生淋巴结转移结直肠癌患者血清exosomal microRNAs差异表达谱进行筛选，建立exosomal microRNAs检测方法，随后分别选取上调表达的miR-483-5p和下调的miR-451a进行验证，发现miR-483-5p、miR-451a具有较好的结直肠癌淋巴结转移的鉴别诊断效能，为结直肠癌转移的有效判断以及治疗方案的选择提供了重要的参考依据。同时建立直接RT-qPCR定量检测方法，为临床准确、简便、可靠的检测血清microRNAs提供了一种新的方法。对miR-218、miR-210、miR-203、miR-133a和miR-223在结直肠癌发生发展中的作用进行了相关研究，发现其均参与了结直肠癌的转移过程。