[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 GLUD 1, B 3 GAT 1, SLC 46 A 3, MAP 2 K 3, ATF 5, and ZADH 2, were found to be down-regulated in HCC. qPCR showed that ZADH 2 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- ZADH 2 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- ZADH 2 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 ZADH 2 gene and affect its expression. Moreover, miRNA-3679 promotes the proliferation of HCC cells and inhibits their apoptosis by suppressing ZADH2.
(Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).)