Effect of down-regulation of high mobility group box-1 expression on proliferation and migration abilities of human umbilical vein endothelial cells

Abstract

To investigate the effect of down-regulation of high mobility group box-1 (HMGB1) expression on the proliferation and migration abilities of human umbilical vein endothelial cell ( HUVEC) in vitro. The method of stable transfection was used to transfect the pRNA-u6.1/Neo-control and pRNA-u6.1/ Neo-HMGB1 short hairpin RNA (shRNA) plasmid to HUVEC cells, and control and HMGB1 shRNA cell lines were reproduced, and the cells were identified by Western blotting test and real-time fluorescence reverse transcription-polymerase chain reaction (RT-PCR). Then methyl thiazolyl tetrazolium (MTT) was used for the determination of the proliferative ability, flow cytometry was used for determining the changes in cell cycle distribution ability of HUVEC, and the migratory ability was detected by scratch test. Western blotting and RT -PCR detection proved the production of stable transfection cell lines was successful, and the content of HMGB1 protein and mRNA expression in cells were significantly lower after cultivation for 72 hours than those of control cells (protein: 0.436 ± 0.027 vs. 1.017 ± 0.038, I= 12.180, P=0.000; mRNA: 0.436 ± 0.031 vs. 1.020 ± 0.051, T=9.660, P= 0.001 ). The results of MTT showed that the proliferation ability of HMGB1 shRNA cell lines was lower obviously 2, 3, 4, 5 days after transfection than that of control cells ( 2 days: 0.210 ± 0.023 vs. 0.240 ± 0.011, T= 1.050, P=0.351; 3 days: 0.240 ± 0.022 vs. 0.361 ± 0.030, T=3.203, P=0.033; 4 days: 0.373 ± 0.031 vs. 0.531 ± 0.033, T=3.530, P=0.022; 5 days: 0.441 ± 0.031 vs. 0.602 ± 0.030, T=4.180, P=0.106). Flow cytometry results showed that the number of HMGB 1 shRNA cells in S phase was significantly lower than that of the control cell line ( ( 13.10 ± 1.10 )% vs. (21.12 ± 1.20)%, T=4.950, P=0.001). Scratch test showed that the healing ability of HMGB1 shRNA cell line was lowered significantly at 12 hours as compared with that of control ( (20.17 ± 3.33 )% vs. ( 88.53 ± 3.15 )% , T= 14.142, P=0.000). HMGB 1 shRNA can significantly inhibit HUVEC cell proliferation and migration.