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何静宇, 周雪琴, 王文涛. miRNA-3679抑制下游ZADH2-靶基因促进肝癌细胞增殖的机制研究[J]. 四川大学学报(医学版), 2022, 53(5): 744-751. DOI: 10.12182/20220960505
引用本文: 何静宇, 周雪琴, 王文涛. miRNA-3679抑制下游ZADH2-靶基因促进肝癌细胞增殖的机制研究[J]. 四川大学学报(医学版), 2022, 53(5): 744-751. DOI: 10.12182/20220960505
HE Jing-yu, ZHOU Xue-qin, WANG Wen-tao. Mechanism of miRNA-3679 Inhibiting Downstream ZADH2-Target Genes to Promote Hepatocellular Carcinoma Cell Proliferation[J]. Journal of Sichuan University (Medical Sciences), 2022, 53(5): 744-751. DOI: 10.12182/20220960505
Citation: HE Jing-yu, ZHOU Xue-qin, WANG Wen-tao. Mechanism of miRNA-3679 Inhibiting Downstream ZADH2-Target Genes to Promote Hepatocellular Carcinoma Cell Proliferation[J]. Journal of Sichuan University (Medical Sciences), 2022, 53(5): 744-751. DOI: 10.12182/20220960505

miRNA-3679抑制下游ZADH2-靶基因促进肝癌细胞增殖的机制研究

Mechanism of miRNA-3679 Inhibiting Downstream ZADH2-Target Genes to Promote Hepatocellular Carcinoma Cell Proliferation

  • 摘要:
      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。

     

    Abstract:
      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.

     

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