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

  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.