关键词: 骨肉瘤, FHL2, U2OS细胞株, 增殖, 凋亡

Abstract: ObjectiveTo explore the effect of silencing the expression of four and a half LIM domains protein 2 （FHL2） on the proliferation and apoptosis of osteosarcoma U2OS cells and its mechanism by shRNA. MethodsFHL2 was knocked down in U2OS cells by constructing the shRNA interference carrier PLKO.1 of FHL2. The lentivirus was prepared and infected with U2OS cells， which were divided into shFHL2 target gene interference group （shFHL2） and unrelated sequence control group （SCR）. At 96 h after transfection， realtime quantitative PCR （QPCR） and Western blotting were used to detect the mRNA and protein levels of FHL2 in two groups. MTT assay and flow cytometry were performed to detect the proliferation， cell cycle distribution and apoptotic rate of U2OS cells. Western blotting was used to analyze the level of cell cycle and apoptoticrelated proteins. Dual luciferase assay was conducted to investigate the transcriptional activity of p53 on Bax when FHL2 was overexpressed or was knocked down. 
ResultsAt 96 h after transfection， the mRNA and protein levels of FHL2 in shFHL2 group were significantly lower than those in SCR group （P＜0.05）. The proliferative ratios of shFHL2 group at 24， 48， 72 and 96 h after transfection were 173±008， 249±038， 326±045 and 428±029， and the data of 96 h was lower than SCR group （P＜0.05）. The apoptotic rate of shFHL2 group at 96 h after transfection was （17.55±1.05）％， higher than （7.73±1.12）％ of SCR group （P＜0.05）. The proportion of shFHL2 group cells in G0／G1 phase at 96 h after transfection was （68.18±0.78）％， higher than （59.73±2.28）％ of SCR group， and the difference was statistically significant （P＜0.05）. Western blotting showed that the level of cell cyclerelated protein p21 and proapoptotic protein p53 and Bax increased. Dual luciferase assay suggested that p53 could increase the transcriptional activity of Bax， furthermore， when FHL2 was knocked down at the same time， the transcriptional activity of p53 was enhanced. ConclusionCell proliferation reducing， cell cycle arresting at G0／G1 phase， and cell apoptosis increasing occurred in FHL2 knockdown U2OS cells. Moreover， FHL2 participated in the regulation of the transcriptional activation function of p53. These results indicated that FHL2 might be a novel potential target for osteosarcoma treatment.

Key words: Osteosarcoma, Four and a half LIM domains protein 2（FHL2）, U2OS cell line, Proliferation, Apoptosis