GSTO1基因沉默对氟致成骨细胞自噬与凋亡的影响

Effects of GSTO1 Gene Silencing on Autophagy and Apoptosis of Fluoride-induced Osteoblasts

为探讨氟致成骨细胞自噬和凋亡中GSTO1的作用机制,利用RNA干扰(RNAi)技术,抑制GSTO1基因在小鼠成骨细胞中表达,检测染氟小鼠成骨细胞中自噬和凋亡相关基因表达。针对小鼠成骨细胞GSTO1mRNA序列设计、合成3对21核苷酸序列siRNA(siRNA1、siRNA2、siRNA3);从3对序列中筛选最佳有效片段,然后检测不同质量浓度氟化钠(10~4、10~3、10~2、10、1mg·L^(-1))下GSTO1的mRNA转录,确定最佳氟浓度。试验分为空白对照组(control)、阴性对照组(NC-siRNA)、氟化钠组(1mg·L^(-1))、沉默目的基因组(siRNA-GSTO1)和氟化钠加沉默目的基因组(NaF/siRNA-GSTO1),用RT-PCR技术检测成骨细胞自噬相关基因LC3、p62、Beclin1、Atg3、Atg5和凋亡相关基因BCL2、Caspase-3的转录,同时应用Western blot技术检测LC3、p62、Beclin1、Atg3和Atg5蛋白表达。结果显示:用HE、吉姆萨、碱性磷酸酶、茜素红四种染色法鉴定试验所用细胞为成骨细胞。确定试验模型中50nmol·L^(-1)为siRNA的最佳转染浓度,siRNA2为最佳沉默转染片段,NaF的最佳浓度为1mg·L^(-1)。成骨细胞染氟与GSTO1基因抑制均可使LC3、Beclin1、Atg5和BCL2基因转录显著升高,p62和Caspase-3基因转录显著下降;但与NaF组相比较,NaF/siRNA-GSTO1组LC3、Beclin1、和Atg5基因mRNA转录水平显著降低(P<0.05),而p62和Caspase-3基因转录显著升高(P<0.05)。Western blot检测成骨细胞自噬相关蛋白LC3、Beclin1、Atg5和p62的表达与基因转录趋势完全一致。结果表明,低剂量氟与沉默GSTO1表达均可致成骨细胞自噬增强的同时凋亡减弱,GSTO1对低剂量氟致成骨细胞自噬增强与抑制凋亡有一定的协同效应。

To investigate the role of GSTO1 in the fluoride-induced autophagy and apoptosis in the osteoblasts,RNA interference(RNAi)technology was used to inhibit the expression of GSTO 1 gene in mouse osteoblasts,and the expressions of autophagy and apoptosis related genes were detected.Three pairs of 21 nucleotide sequence siRNA(siRNA1,siRNA2,siRNA3)were designed and synthesized according to the mouse GSTO 1 mRNA sequence,and the best effective fragment was screened.Then,the mRNA expression levels of GSTO 1 under different concentrations of sodium fluoride(NaF)(10^4,10^3,10^2,10,and 1 mg·L^-1)were evaluated to determine the optimal fluoride concentration.This experiment included control group,negative control group(NC-siRNA),NaF group(1 mg·L^-1),silencing target gene group(siRNA-GSTO1)and NaF+silencing target gene group(NaF/siRNA-GSTO1).RT-PCR was used to detect the transcription of autophagy-related genes(LC3,p62,Beclin 1,Atg3,Atg5)and apoptosis-related genes(BCL2,Caspase-3)in osteoblasts.The protein expression of LC3,p62,Beclin1,Atg3 and Atg5 were detected by Western blot.Results were as follows:Osteoblasts were identified by HE,Giemsa,alkaline phosphatase,and alizarin red staining.In the experimental model,50 nmol·L^-1 was found to be the optimal transfection concentration of siRNA,and siRNA2 was the best silent transfection fragment,at 1 mg·L^-1 NaF optimal concentration.The NaF treatment group and siRNA-GSTO1 group inhibited the transcription of LC3,Beclin1,Atg5 and BCL2,and decreased the transcription of p62 and Caspase-3.However,compared with the NaF group,the transcription of LC3,Beclin 1 and Atg5 mRNA were significantly decreased(P<0.05),while the transcription of p62 and Caspase-3 were significantly increased in the NaF/siRNA-GSTO1 group(P<0.05).The expression of autophagy-related proteins LC3,Beclin1,Atg5 and p62 in osteoblasts were completely consistent with the gene transcription.In summary,both low-dose fluoride and silencing of GSTO1 expression increased autophagy and decreased apoptosis in the osteoblasts.GSTO1 had synergistic effect on low-dose fluoride-induced autophagy enhancement and apoptosis inhibition in the osteoblasts.