miR-485-5p作为一种新的结直肠癌诊断生物标志物及其调控机制研究

Studies have shown that miRNAs are involved in tumor occurrence and development by regulating the target gene expression, and could be a new biomarker. Preliminary studies of our group found that the expression of miRNA-485-5p significantly reduced in cancer tissue and peripheral blood serum of colorectal cancer (CRC) patients. The high expression of CD147 which was one of the miRNA-485-5p target gene promoted the proliferation, invasion and metastasis of tumor cells. We intend to construct miRNA-485-5p expression/gene knockout vector, and observe its role in the regulation of CD147 and its downstream signaling pathway, detect the proliferation, invasion and metastasis of CRC cells in vivo and in vitro, to explore the regulatory mechanism of miRNA-485-5p via the CD147 on the occurrence and development of CRC. We want to detect miR-485-5p expression levels by Realtime-PCR in different pathological grading and clinical staging of CRC patients and validate the methodology, and analysis its correlation with the traditional experimental diagnostic indicators to explore miR-485-5p, as a new biomarker, the value in the diagnostic and prognosis of CRC. This study will provide new targets and theoretical basis for clinical diagnosis and pathogenesis research of CRC.

研究表明，miRNA可通过调控靶基因的表达而参与肿瘤的发生、发展等行为，可作为一种新的生物标志物。本课题组前期研究发现，miR-485-5p在结直肠癌（CRC）患者癌组织及外周血清中表达明显降低，而其靶基因CD147在CRC中异常高表达，且促进肿瘤细胞的增殖、侵袭及转移。本项目拟构建miR-485-5p表达/基因敲除载体，观察其对CD147及下游信号通路的调控作用，体内外水平检测其对CRC细胞增殖、侵袭转移及凋亡的影响，探讨miR-485-5p经由CD147在CRC发生发展中的作用及其机制；进一步建立Realtime-PCR方法检测不同病理分级和临床分期的CRC患者血清中miR-485-5p水平，进行方法学验证，并与传统诊断指标进行相关性分析，以探讨其作为生物标志物在CRC辅助诊断及预后判断中的价值。本研究可为CRC的临床诊治及发病机制研究提供新的靶点和依据。

结直肠癌（CRC）是临床常见的恶性肿瘤，研究显示CRC不仅发病率高，且呈上升趋势。由于 CRC 患者早期症状不明显，大多数患者在确诊时已属中晚期，因此进一步研究 CRC 发病机制并及时发现早期诊断 CRC 的理想生物标志物具有重要的临床应用前景。本项目实验结果显示miR-485-5p在结直肠癌组织及结直肠癌细胞株中均低表达；构建miR-485-5p表达载体并稳定转染结直肠癌细胞，结果表明高表达miR-485-5p可抑制CRC细胞的增殖、侵袭迁移能力，并促进细胞凋亡；裸鼠移植瘤模型结果显示高表达miR-485-5p可抑制结直肠癌细胞的体内成瘤能力；结直肠癌组织中miR-485-5p与CD147表达存在负相关；双荧光素酶报告基因检测结果显示miR-485-5p可与CD147 3'UTR部分结合，进一步证实了CD147是miR-485-5p的靶基因；在结直肠癌细胞中miR-485-5p上调后，CD147的mRNA和蛋白的表达均降低，并下调ERK1/2信号通路；挽救实验表明过表达CD147能够部分恢复miR-485-5p高表达对结直肠癌细胞生物学功能的影响，提示在结直肠癌中miR-485-5p发挥了抑癌基因的功能，可通过调控CD147的表达抑制结直肠癌细胞的增殖、侵袭迁移能力并促进细胞凋亡。.建立SYBR GreenⅠrealtime-PCR法检测CRC患者及健康对照组外周血中miR-485-5p的表达，结果表明CRC患者外周血miR-485-5p水平显著低于健康体检组，通过构建ROC曲线评估外周血miR-485-5p水平对CRC患者潜在的诊断价值，其诊断灵敏度为61.7％，特异性为68.3％；统计结果显示外周血miR-485-5p表达与CRC患者肿瘤分期和淋巴结转移显著相关，提示血清miR-485-5p对于CRC的临床诊断具有一定的应用价值。