miR-1229-3p抑制结直肠癌疾病进展及作为潜在生物标志物的研究

miR-1229-3p inhibits the malignant progression of colorectal cancer and serves as a potential biomarker

目的:探讨微小RNA(microRNA, miRNA)在结直肠癌(colorectal cancer, CRC)疾病进展中的作用及临床应用价值。方法:基于TCGA数据库,筛选分析人类结肠癌及癌旁组织的转录组差异表达miRNA,以miRNA-1229-3p作为研究对象,在GEO数据库与Starbase数据库中验证其表达水平。采用生物信息学方法对miR-1229-3p的生物功能特征进行分析,构建miR-1229-3p过表达细胞系及miR-1229-3p敲降细胞系,通过CCK-8实验、克隆形成实验、EdU实验、Transwell实验、流式细胞术实验在体外探究miR-1229-3p对CRC细胞增殖、转移、凋亡的影响,并通过裸鼠移植瘤试验探究miR-1229-3p对CRC细胞体内增殖的影响。通过DIANA、TargetScan、miRDB数据库筛选miR-1229-3p的靶基因,并通过KEGG富集分析、GO分析靶基因的生物学功能。检测60例CRC患者及60例健康体检者血浆外泌体中的miR-1229-3p含量,采用受试者工作特征曲线(receiver operating characteristic curve, ROC)分析其对CRC患者的诊断效能。结果:数据分析显示,miR-1229-3p在CRC细胞和组织中表达下调。生物信息学方法预测结果显示,miR-1229-3p与肿瘤的进展途径(如上皮间质转化、血管形成等)呈显著负相关(R<0,P<0.05)。体内、外研究表明,过表达miR-1229-3p后,CRC细胞的增殖、迁移和侵袭能力受到抑制,且其潜在靶基因SETD7在细胞中的表达下调;而敲低miR-1229-3p后可抑制CRC细胞的凋亡。CRC患者血浆外泌体中的miR-1229-3p含量较健康体检者降低,ROC曲线分析显示,miR-1229-3p诊断CRC的约登指数最大值为0.586 8,曲线下面积为0.863 2(P<0.01)。结论:miR-1229-3p在CRC中表达下调,SETD7是其潜在的靶基因,miR-1229-3p可抑制CRC细胞增殖、迁移与侵袭,促进CRC细胞凋亡,有望作为诊断CRC的潜在生物标志物。

Objective:To investigate the role and clinical application of microRNA in the malignant progression of colorectal cancer(CRC).Methods:The transcriptome data of human colorectal cancer and adjacent tissues from TCGA database were analyzed to screen out the differentially expressed microRNAs(miRNAs),among which miR-1229-3p with the most significant difference was selected as the target miRNA,and furthermore the expression of miR-1229-3p was verified through GEO and Starbase databases.The biological functional characteristics of miR-1229-3p was analyzed through Bioinformatics.The miR-1229-3p overexpression cell line and miR-1229-3p knockdown cell line were constructed.The effect of miR-1229-3p on the proliferation,metastasis,and apoptosis of CRC cells was explored in vitro by CCK-8 experiment,cloning formation experiment,EdU experiment,transwell,and flow cytometry experiment.The effect of miR-1229-3p on the proliferative capacity of CRC in vivo was explored by nude mouse transplantation tumor experiment.The target genes of miR-1229-3p were screened out by DIANA,TargetScan,and miRDB databases.KEGG and GO were used to analyze the biological function of target genes.The expression of miR-1229-3p in plasma exosomes of 60 CRC patients and 60 healthy examiners obtained from Nanjing First Hospital were analyzed,and receiver operating characteristic curve(ROC)was used to distinguish its diagnostic efficacy for CRC patients.Results:The expression of miR-1229-3p was downregulated in CRC cells and tissues.Bioinformatics predicted a significant negative correlation between miR-1229-3p and tumor progression pathways,such as epithelial-mesenchymal transition and angiogenesis(R<0,P<0.05).In vitro and in vivo studies showed that after the overexpression of miR-1229-3p,the proliferation,migration and invasion of CRC cells were inhibited,and the potential target gene SETD7 was downregulated,while the apoptosis of CRC cells could be inhibited after downregulating miR-1229-3p.miR-1229-3p from plasma exosomes of CRC was downregulated,which could distinguish CRC patients from normal people.ROC curve showed that the optimal critical value of miR-1229-3p for diagnosing CRC was 0.5868,and the area under the curve was 0.8632(P<0.01).Conclusions:The expression of miR-1229-3p is downregulated in CRC,and SETD7 is a potential target gene for miR-1229-3p.miR-1229-3p can inhibit the proliferation,migration and invasion of CRC cells,and promote apoptosis of CRC cells.miR-1229-3p can be served as a potential biomarker for CRC diagnosis.