The effects of up-regulating the expression of microRNA-1224-5p on hepatocellular carcinoma tumor endothelial cells

Abstract

Objective To investigate the effect of microRNA-1224-5p (miR-1224-5p) on tumor endothelial cells (TECs) of human hepatocellular carcinoma (HCC). Methods Oligonucleotides were chemically synthesized and transfected into TECs using Lipofectamine 2000. TECs were divided into 3 groups: a control (CON) group consisted of TECs without transfection, a negative control (NC) group consisted of TECs transfected with negative control oligonucleotides and Green Fluorescent Protein (GFP), and a micro-up (MU) group consisted of TECs transfected with miR-1224-5p mimic and GFP. The expression of miR-1224-5p was quantified via quantitative reverse transcription polymerase chain reaction (RT-qPCR). The proliferation of TECs was detected using MTT (Thiazolyl Blue Tetrazolium Bromide) assay. Apoptosis and cell cycle were detected via flow cytometry. The migration and invasion of TECs were detected using Ttranswell assay. The ability of tube fromation of TECs was detected using tube formation assay. RESULTS Oligonucleotides were successfully transduced into TECs, and specifically up-regulated the expression of miR-1224-5p. Results The results of RT-qPCR showed that the expression of miR-1224-5p was significantly increased in the MU group (2-ΔΔCt=3.27±0.15) compared with the CON group (2-ΔΔCt=1.00) and the NC group (2-ΔΔCt=1.08±0.11) (P<0.01). The results of MTT assay showed that the cell proliferation was all significantly inhibited in the MU group for the 4 time points compared with the CON group and the NC group (P<0.01). The results of flow cytometry showed that the apoptosis was significantly increased in the MU group (19.29±0.95)% compared with the CON group (8.73±0.64)% and the NC group (9.51±0.56)% (P<0.01). The results of cell cycle detection showed that percentage of G1 phase cells in the MU group (73.3±2.0)% was significantly higher than the CON group (50.7±1.3)% and the NC group (51.7±1.9)% and the percentage of S phase cells in the MU group (23.0±3.0)% was significantly decreased compared with the CON group (31.4±2.7)%, and the NC group (29.1±1.6)%. (P<0.05 or P<0.01). The ability of migration was significantly inhibited in the MU group (51.0±3.6) compared with the CON group (77.7±2.5), and the NC group (79.2±3.5) (P<0.01). And the ability of invasion of TECs was also significantly inhibited in the MU group (9.8±1.3) compared with the CON group (15.8±0.8) and the NC group (15.4±0.9) (P<0.01). The ability of tube formation of TECs in the MU group was inhibited absolutely, but TECs in the CON group and the NC group were not affected. Conclusion Up-regulation of miR-1224-5p could decrease proliferation, induce apoptosis, inhibit migration and invasion, and supress tube formation of TECs of human HCC. Key words: Hepatocellular carcinoma; Vascular endothelial cells of human hepatocellular carcinoma; MicroRNA-1224-5p