Objective: To examine the relationship between miRNA-3679 and hepatocellular carcinoma (HCC) cell lines, and to verify the downstream target genes of miRNA-3679.
Methods: PCR was used to determine the expression of miRNA-3679 in liver cancer cell lines, and databases, including ENCORI, miRDB and TargetScan, were used to predict the downstream target genes of miRNA-3679. qPCR of the normal control group (or NC group), miR-3679 inhibitor group and transfection negative control group (or inhibitor NC group) was done to determine the transfection efficiency of the target gene, thereby identifying zinc-binding alcohol dehydrogenase domain containing 2 (ZADH2) as the target gene. Western blot was used to determine the ZADH2 protein expression after miRNA-3679 inhibitor transfection. 5-Ethynyl-2'-deoxyuridine (EdU) staining was done to determine the effect of transfection of miRNA-3679 inhibitor and simultaneous transfection of miRNA-3679 and ZADH2 inhibitors on cell proliferation. Clone formation assay was done to determine the ability of cell clone formation. Flow cytometry was done to examine cell apoptosis.
Results: The expression level of miRNA-3679 in HCC cell lines was higher than that in normal human liver cell lines (P<0.05). Through screening conducted with the databases, six genes, including GLUD1, B3GAT1, SLC46A3, MAP2K3, ATF5, and ZADH2, were found to be down-regulated in HCC. qPCR showed that ZADH2 expression increased significantly after transfection with miRNA-3679 inhibitor (P<0.01) and luciferase activity increased after transfection with miR-3679 inhibitor (P<0.01). Western blot results showed that ZADH2 protein expression of the miR-3679 inhibitor group was higher than that of the NC group (P<0.01). EdU analysis showed that the number of positive cells in the miRNA-3679 inhibitor group was lower than that in the NC group and the Inhibitor NC group (P<0.05). The clone count of the miR-3679 inhibitor+si-ZADH2 group was significantly higher than that of the miR-3679 inhibitor group (P<0.01). Flow cytometry showed that the number of apoptotic cells of the miR-3679 inhibitor+si-ZADH2 group was significantly lower than that of the miR-3679 inhibitor group (P<0.01).
Conclusion: miRNA-3679 is significantly highly expressed in HCC cells and miRNA-3679 can directly interact with ZADH2 gene and affect its expression. Moreover, miRNA-3679 promotes the proliferation of HCC cells and inhibits their apoptosis by suppressing ZADH2.
目的: 寻找miRNA-3679与肝癌细胞系之间的关系，并验证其下游靶基因。
方法: 通过PCR检测miRNA-3679在肝癌细胞系中的表达，使用ENCORI、miRDB、TargetScan数据库预测miRNA-3679的下游靶基因；通过qPCR检测（空白对照组、转染组及转染阴性对照组）转染靶基因表达水平确定靶基因为含锌结合醇脱氢酶结构域-2（ZADH2）；Western blot检测转染miRNA-3679抑制剂后ZADH2蛋白表达；EdU染色检测转染miRNA-3679抑制剂及同时转染miRNA-3679、ZADH2抑制剂后对细胞增殖的影响；克隆形成实验检测细胞克隆形成能力；流式细胞术检测细胞凋亡。
结果: miRNA-3679在肝癌细胞系中的表达水平均高于正常人肝细胞株（P<0.05），数据库中共筛选出6个在肝癌中下调的基因：GLUD1、B3GAT1、SLC46A3、MAP2K3、ATF5、ZADH2；qPCR检测转染miRNA-3679抑制剂后ZADH2表达升高（P<0.01）；转染miR-3679抑制剂后荧光素酶活性升高（P<0.01）；Western blot检测miR-3679 inhibitor组中的ZADH2蛋白表达高于NC组（P<0.01）；EdU分析检测miRNA-3679 inhibitor组阳性细胞数低于NC组及Inhibitor NC组（P<0.05）；miR-3679 inhibitor+si-ZADH2组克隆计数多于miR-3679 inhibitor组（P<0.01）；流式细胞术检测提示miR-3679 inhibitor+si-ZADH2组细胞凋亡数目低于miR-3679 inhibitor组（P<0.01）。
结论: miRNA-3679在肝癌细胞中显著高表达，可直接作用于ZADH2基因并影响其表达，且通过抑制ZADH2发挥促进HCC细胞的增殖及抑制其凋亡。
Keywords: HCC; Micro-ribonucleic acid; Micro-ribonucleic acid-3679; Plasticity-related gene 3; Zinc-binding alcohol dehydrogenase domain containing 2.
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