目的： 分析miR-224在肝癌患者肿瘤组织和血浆中的表达水平及其与临床病理特征、诊断和预后之间的相关性，并进一步通过生物信息学方法和体外实验分析其在肝癌发生发展中的作用机制。方法： 利用基因表达数据库（Gene Expression Omnibus，GEO）中大样本数据分析miR-224在肝癌患者肿瘤组织及正常组织中的表达水平。采用qPCR法检测2017年1月至2020年1月间于河北省人民医院手术切除的80例临床肝癌患者肿瘤组织和对应癌旁组织中miR-224的表达水平，检测miR-224在30例肝癌患者血浆中的表达水平。利用Kaplan-Meier plotter数据库分析miR-224的表达水平与肝癌患者的总生存时间之间的相关性。利用生物信息学手段分析miR-224所参与的生物学过程和信号通路。向肝癌HepG2细胞转染miR-224 inhibitor，采用克隆形成实验、Transwell小室实验、qPCR和WB法分别检测敲低miR-224表达对肝癌HepG2细胞增殖、侵袭能力以及EMT相关分子表达水平的影响。结果： GEO数据库分析结果显示，miR-224在肝癌组织中的表达水平显著高于正常组织。临床标本验证结果表明，miR-224在肝癌患者肿瘤组织和血浆中的表达水平显著高于对应的癌旁组织和健康人血浆（均P<0.01）。miR-224的表达水平与肝癌患者的TNM分期、淋巴结转移状态和肿瘤大小显著相关（P<0.05或P<0.01）。ROC分析发现，miR-224在肝癌诊断方面具有较强的能力，且miR-224的表达水平升高与肝癌患者的预后不良显著相关（P<0.05）。功能富集分析发现，miR-224主要参与mTOR信号通路、AGE-RAGE信号通路、Rap1信号通路、Ras信号通路、ErbB信号通路、HIF-1信号通路以及p53信号通路等肿瘤发生发展相关的信号通路。敲低miR-224可显著抑制HepG2细胞的克隆形成和侵袭，影响EMT相关标志物的表达（P<0.05或P<0.01）。结论： miR-224在肝癌组织和血浆中显著高表达，且与肝癌患者的预后不良显著相关，敲低miR-224表达可抑制肝癌HepG2细胞的克隆形成、侵袭和EMT进程。

Objective: To analyze the expression level of miR-224 in cancer tissues and plasma of hepatocellular carcinoma (HCC) patients, and its correlation with clinicopathological characteristics, diagnosis and prognosis of HCC patients, and to further analyze its mechanism of action in the occurrence and development of liver cancer through bioinformatics analysis and in vitro experiments.Methods: The expression level of miR-224 in HCC tissues and normal tissues was analyzed using large sample data from Gene Expression Omnibus (GEO). qPCR method was used to verify the expression level of miR-224 in the tumor tissues and corresponding adjacent tissues that surgically resected from 80 HCC patients in Hebei Provincial People’s Hospital from January 2017 to January 2020; in addition, the miR-224 level was also examined in plasma samples from 30 HCC patients. The Kaplan-Meier plotter database was used to analyze the correlation between the miR-224 expression and the overall survival time of HCC patients. The biological processes and signal pathways involving miR-224 were analyzed using bioinformatics tools. Hepatocellular carcinoma HepG2 cells were transfected with miR-224 inhibitor, and then Clone formation experiment, Transwell chamber experiment, qPCR and WB methods were used to detect the effect of miR-224 knockdown on the proliferation and invasion of HepG2 cells and the expression level of EMT-related molecules.Results: The results of GEO database analysis showed that the expression level of miR-224 in HCC tissues was significantly higher than that in normal tissues. The results of clinical specimen verification showed that the expression level of miR-224 in the tumor tissues and plasma of HCC patients was significantly higher than that in the corresponding adjacent tissues and plasma from healthy controls (all P<0.01). The expression level of miR-224 was significantly correlated with the TNM stage, lymph node metastasis status and tumor size of HCC patients (P<0.05 or P<0.01). ROC analysis indicated that miR-224 showed a prominent diagnostic value in liver cancer, and the increased expression level of miR-224 was significantly related to the poor prognosis of HCC patients (P<0.05). Functional enrichment analysis revealed that miR-224 was mainly involved in the mTOR signaling pathway, AGE-RAGE signaling pathway, Rap1 signaling pathway, Ras signaling pathway, ErbB signaling pathway, HIF-1 signaling pathway and p53 signaling pathway and other signaling pathways related to tumor occurrence and development. Knockdown of miR-224 could significantly inhibit the colony formation and invasion of HepG2 cells and affect the expression of EMT-related markers (P<0.05 or P<0.01).Conclusion: miR-224 is highly expressed in HCC tissues and plasma and is significantly related to the poor prognosis of HCC patients. Knockdown of miR-224 expression can inhibit the colony formation, invasion and EMT process of liver cancer HepG2 cells.

R735.7；R730.7

河北省医学科学研究青年课题计划资助项目（No. 20170297）；河北省教育厅2020年度河北省高等学校人文社会科学研究重点项目资助（No. SD201076）