Table_1_Downregulation of miR-654-3p in Colorectal Cancer Indicates Poor Prognosis and Promotes Cell Proliferation and Invasion by Targeting SRC.docx

BackgroundMicroRNAs (miRNAs), such as miR-654-3p, regulate gene expression at the post-transcriptional level affecting malignant tumor behavior. However, the expression levels, function, and mechanism of miR-654-3p in colorectal cancer (CRC) are unknown.
MethodsThe expression levels of miR-654-3p and SRC in 103 CRC tissues and matched normal colorectal tissues were detected by a quantitative real-time polymerase chain reaction (qRT-PCR). miR-654-3p was overexpressed by RNA mimics and SRC knockdown by siRNA. Function-based experiments were carried out to detect the proliferation and migration abilities in CRC cell lines. Flow cytometry assay was performed to evaluate the effect of miR-654-3p on cell apoptosis and cycle distribution. Xenograft tumor models in nude mice were utilized to evaluate miR-654-3p functions in vivo. Dual-fluorescence reporter assay was used to verify the direct binding between miR-654-3p and SRC.
ResultsmiR-654-3p was downregulated in CRC tissues as compared to matched normal colorectal tissues. The expression levels of miR-654-3p were closely associated with distant metastasis. In addition, elevated expression of miR-654-3p in CRC patients prolonged the overall survival. Upregulated miR-654-3p significantly suppressed the proliferation and migration capacity of CRC cells by enhancing apoptosis and promoting G0/G1 phase arrest. The direct binding between miR-654-3p and SRC was verified by the dual-luciferase reporter gene. Furthermore, the suppression of proliferation and migration capacity by elevated miR-654-3p level could be reversed by overexpressing SRC.
ConclusionmiR-654-3p acts as a tumor suppressor through regulating SRC. It might also serve as a diagnostic and prognostic indicator and a novel molecular target for CRC therapy.

背景：微小RNA（MicroRNAs, miRNAs）如miR-654-3p可在转录后水平调控基因表达，进而影响恶性肿瘤的生物学行为。然而，miR-654-3p在结直肠癌（colorectal cancer, CRC）中的表达水平、功能及作用机制尚未明确。
方法：采用实时定量聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）检测103例结直肠癌组织及配对正常结直肠组织中miR-654-3p与SRC的表达水平。通过RNA模拟物过表达miR-654-3p，利用小干扰RNA（small interfering RNA, siRNA）敲低SRC的表达。开展功能实验以检测结直肠癌细胞系的增殖与迁移能力；采用流式细胞术评估miR-654-3p对细胞凋亡及细胞周期分布的影响；构建裸鼠异种移植瘤模型，在体评价miR-654-3p的生物学功能；采用双荧光素酶报告基因实验验证miR-654-3p与SRC的直接靶向结合。
结果：相较于配对正常结直肠组织，miR-654-3p在结直肠癌组织中表达下调。miR-654-3p的表达水平与结直肠癌患者的远处转移密切相关。此外，结直肠癌患者中miR-654-3p高表达可延长总生存期。过表达miR-654-3p可显著抑制结直肠癌细胞的增殖与迁移能力，具体机制为增强细胞凋亡并诱导G0/G1期细胞周期阻滞。双荧光素酶报告基因实验证实miR-654-3p可直接结合SRC。进一步研究发现，过表达SRC可逆转miR-654-3p高表达所介导的增殖与迁移抑制效应。
结论：miR-654-3p通过靶向调控SRC发挥肿瘤抑制因子的作用，其有望成为结直肠癌诊断与预后评估的生物标志物，以及结直肠癌治疗的新型分子靶点。