探寻miR-200a-3p作为结直肠癌的诊断及预后生物标志物的潜在价值

Potential value of miR-200a-3p as a biomarker for the diagnosis and prognosis of colorectal cancer

摘要

目的 阐明 miR-200a-3p及下游靶基因程序性细胞死亡4（programmed cell death 4，PDCD4）在结直肠癌（colorectal cancer，CRC）诊断及预后的潜在价值。 方法 从2017年1月至2019年9月在长沙市第一医院接受根治性切除术或姑息性切除术的CRC患者中收集包含120对CRC组织和相应的癌旁正常组织的队列。另1个队列包括50例诊断为CRC的患者；2019年1月至2020年1月从这些患者手术前后收集术前和术后血清样本。RT-qPCR检测CRC组织和细胞系中miR-200a-3p的表达。将靶向miR-200a-3p（si-miR-200a-3p）和siRNAs阴性对照（si-NC），以及miR-200a-3p过表达（miR-200a-3p-OE）和PDCD4过表达（PDCD4）质粒转染CRC细胞。通过CCK-8测定、集落形成和Transwell细胞侵袭测定来确定miR-200a-3p对CRC细胞生长的影响。雌性BALB/c裸鼠随机分为3组：载体组（Vector组）、miR-200a-3p过表达组（miR-200a-3p-OE组）和miR-200a-3p 过表达+PDCD4过表达组（miR-200a-3p-OE+PDCD4组）。构建小鼠远处转移模型，监测肺转移的荧光强度，计数肺转移结节的数量。 结果 miR-200a-3p在CRC组织和细胞系中上调。miR-200a-3p高表达与肿瘤大小、肿瘤转移和TNM分期显著相关。miR-200a-3p表达可以作为总生存率（overall survival，OS）（HR=2.077，95%CI=1.079~3.998，P=0.029）和无病生存率（disease free survival，DFS）（HR=2.927，95%CI=1.179~7.270，P=0.021）的独立预后生物标志物。功能研究发现miR-200a-3p结合 PDCD4的3'-UTR区，并降低PDCD4在CRC细胞中的表达。PDCD4过表达抑制miR-200a-3p过表达对CRC细胞增殖、集落形成、迁移和侵袭的促进作用。与体外结果一致，在转移的小鼠尾静脉注射模型中，miR-200a-3p-OE+PDCD4组SW480细胞的肺定植、肺转移灶较 miR-200a-3p-OE组明显减少（P<0.05）。 结论 miR-200a-3p在CRC细胞、组织和血清中表达显著增加，并可以作为诊断和预后的生物标志物。miR-200a-3p过表达通过抑制PDCD4表达促进CRC进展。

Abstract

Objective To investigate the potential value of miR-200a-3p and its downstream target gene programmed cell death 4 （PDCD4） in the diagnosis and prognosis of colorectal cancer（CRC）. Methods A cohort containing 120 pairs of CRC tissue samples and corresponding adjacent tissue samples were collected from CRC patients who underwent radical resection or palliative resection from January 2017 to September 2019. Another cohort included 50 patients diagnosed with CRC，and preoperative and postoperative serum samples were collected from these patients before and after surgery from January 2019 to January 2020. RT-qPCR was used to measure the expression of miR-200a-3p in CRC tissue and cell lines. CRC cells were transfected with plasmids targeting miR-200a-3p（si-miR-200a-3p） and siRNAs negative control（si-NC） and plasmids with miR-200a-3p overexpression（miR-200a-3p-OE） and PDCD4 overexpression（PDCD4），and Cell Counting Kit-8 assay，colony formation assay，and Transwell assay were used to observe the influence of miR-200a-3p on the growth of CRC cells. Female BALB/c nude mice were randomly divided into Vector group，miR-200a-3p overexpression group（miR-200a-3p-OE group），and miR-200a-3p overexpression+PDCD4 overexpression group（miR-200a-3p-OE+PDCD4 group）. A mouse model of distant metastasis was established，and the fluorescence intensity of lung metastasis was monitored，as well as the number of lung metastasis nodules. Results The expression of miR-200a-3p was upregulated in CRC tissue and cell lines. The high expression of miR-200a-3p was significantly associated with tumor size，tumor metastasis，and TNM stage. The expression of miR-200a-3p could be regarded as an independent prognostic biomarker for overall survival（hazard ratio ［HR］=2.077，95%CI=1.079-3.998，P=0.029） and disease-free survival（HR=2.927，95%CI=1.179-7.270，P=0.021）. Functional study revealed that miR-200a-3p could bind to the 3’-UTR region of PDCD4 and reduced the expression of PDCD4 in CRC cells. Overexpression of PDCD4 inhibited the promoting effect of miR-200a-3p on the proliferation，colony formation，migration，and invasion of CRC cells. In the mouse model of metastasis and caudal vein injection，the miR-200a-3p-OE+PDCD4 group had significant reductions in lung colonization and lung metastatic lesions compared with the miR-200a-3p-OE group（P<0.05），which was consistent with the in vitro results. Conclusion There is a significant increase in the expression of miR-200a-3p in CRC cells，tissue，and serum，and it can be used as a biomarker for diagnosis and prognosis. Overexpression of miR-200a-3p promotes the progression of CRC by inhibiting PDCD4 expression.

关键词

miR-200a-3p / 程序性细胞死亡4 / 结直肠癌 / 诊断 / 预后

Key words

miR-200a-3p / programmed cell death 4 / colorectal cancer / diagnosis / prognosis

基金资助

湖南省卫生健康委科研计划资助项目(202203102964)