Down-regulation of miR-4443/TIMP2 signaling pathway enhances sorafenib sensitivity in HepG2 cells

Abstract

Objective To investigate the significance of down-regulating the miR-4443/TIMP2 pathway on sorafenib sensitivity in HepG2 cells. Methods HepG2 cells were divided into control group (DMSO) and sorafenib treatment group (10 μmol/L). After treatment for 24 h, the mRNA level of miR-4443 was measured by RT-qPCR. CCK-8 assay was used to study the effects of transfection with miR-4443 mimics or pretreatment of its inhibitor on the cell viability of HepG2 cells treated with sorafenib for 24 h. Bioinformatics analysis was used to predict the downstream target gene of miR-4443. The expression of TIMP2 at mRNA and protein levels was assessed by RT-qPCR and Western blotting in the HepG2 cells treated with sorafenib for 24 h, transfected with miR-4443 mimics or pretreatment of its inhibitor followed by sorafenib treatment. Then TIMP2 siRNA was transfected into HepG2 cells by liposome transfection, the content of TIMP2 protein was detected by Western blotting, and the effect of TIMP2 knockdown combined with sorafenib treatment on the viability of HepG2 cells was measured with CCK-8 assay. Results Sorafenib treatment decreased the expression of miR-4443 in HepG2 cells (P < 0.05). Overexpression of miR-4443 (transfection of miR-4443 mimics) significantly decreased the inhibitory effect of sorafenib on cell viability (P < 0.05), while pretreatment of miR-4443 inhibitor could enhance the effect of the drug (P < 0.05). Bioinformatics analysis indicated that the downstream target gene of miR-4443 was TIMP2. The mRNA and protein levels of TIMP2 were obviously increased in HepG2 cells after sorafenib treatment (P < 0.05), but the transfection of miR-4443 mimics or pretreatment of its inhibitor would down- or up-regulate the mRNA level of TIMP2, and the transfection of miR-4443 mimics decreased the protein level of TIMP2, indicating the regulative relationship between the 2 molecules. The transfection of TIMP2 siRNA decreased the expression of TIMP2 and then enhanced the inhibitory effect of sorafenib on HepG2 viability (P < 0.05). Conclusion Down-regulation of the miR-4443/TIMP2 pathway enhances sorafenib sensitivity in HepG2 cells.

Keywords

• sorafenib
• hepg2 cells
• mir-4443
• timp2
• drug resistance