PTTG1 siRNA增加塞来昔布对骨肉瘤细胞生长抑制作用的实验研究

Experimental study on the inhibitory effect of celecoxib on osteosarcoma cell growth by PTTG1 siRNA

目的:研究垂体肿瘤转化基因1(PTTG1)siRNA和塞来昔布对骨肉瘤细胞生长的影响。方法:以不同浓度的塞来昔布处理骨肉瘤MG-63细胞,MTT方法测定细胞增殖和塞来昔布半数抑制浓度。用PTTG1 siRNA慢病毒和阴性对照慢病毒感染MG-63细胞,用Realtime PCR和Western blot检测干扰效果。以半数抑制浓度的塞来昔布处理感染siRNA慢病毒和阴性对照慢病毒的MG-63细胞,MTT法检测增殖,平板克隆实验检测克隆形成,流式细胞术检测细胞凋亡,Western blot检测细胞中活化的caspase-3(c-caspase-3)、活化的caspase-12(c-caspase-12)和CCAAT/增强子结合蛋白同源蛋白(CHOP)蛋白水平。结果:塞来昔布能够抑制MG-63细胞增殖,其半数抑制浓度约为85μmol/L。siRNA慢病毒感染后细胞中PTTG1 mRNA和蛋白水平均低于对照慢病毒感染的MG-63细胞(P<0.05)。半数抑制浓度的塞来昔布或干扰PTTG1后的MG-63细胞增殖能力和克隆形成能力均降低,细胞凋亡增多,细胞中c-caspase-3、c-caspase-12和CHOP蛋白水平升高。干扰PTTG1的MG-63细胞经半数抑制浓度的塞来昔布处理以后,细胞的增殖和克隆形成能力均降低,细胞凋亡率及细胞中c-caspase-3、c-caspase-12、CHOP蛋白水平均升高,与单纯半数抑制浓度塞来昔布或干扰PTTG1的MG-63细胞比较,差异有统计学意义(P<0.05)。结论:PTTG1 siRNA增加塞来昔布对骨肉瘤细胞生长的抑制作用,其作用机制可能与内质网应激诱导的细胞凋亡有关。

Objective:To study the effects of PTTG1 siRNA and celecoxib on the growth of osteosarcoma cells.Methods:Different concentrations of celecoxib were used to treat osteosarcoma MG-63 cells.Cell proliferation and half inhibitory concentration were measured by MTT.MG-63 cells were infected with lentivirus and negative control lentivirus using PTTG1 siRNA,and the interference effects were detected by Realtime PCR and Western blot.Celecoxib was used to treat MG-63 cells infected with siRNA lentiviruses and negative control lentiviruses.MTT assay was used to detect proliferation.Plate cloning was used to detect clone formation.Apoptosis was detected by flow cytometry.Western blot was used to detect the levels of c-caspase-3,c-caspase-12 and CHOP proteins in the cells.Results:Celecoxib can inhibit the proliferation of MG-63 cells,and the median inhibitory concentration was about 85 μmol/L.The levels of PTTG1 mRNA and protein in the cells infected with siRNA lentivirus were lower than those in MG-63 cells infected with negative control lentivirus(P<0.05).The proliferation and cloning ability of MG-63 cells were decreased after half inhibitory concentration of celecoxib or interfering with PTTG1,and apoptosis was increased,and the levels of c-caspase-3,c-caspase-12 and CHOP protein were increased in cells.After interfering with PTTG1,MG-63 cells were treated with half inhibitory concentration of celecoxib,the ability of cell proliferation and colony formation was decreased.The apoptosis rate and the levels of c-caspase-3,c-caspase-12 and CHOP in the cells were all increased.Compared with celecoxib with only half inhibitory concentration or MG-63 cells interfering with PTTG1,the difference was statistically significant(P<0.05).Conclusion:PTTG1 siRNA increased the inhibitory effect of celecoxib on the growth of osteosarcoma cells.The mechanism may be related to endoplasmic reticulum stress induced apoptosis.