GSTO1基因沉默对氟致成骨细胞自噬与凋亡的影响
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摘要
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 GSTO1 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 GSTO1 mRNA sequence,and the best effective fragment was screened.Then,the mRNA expression levels of GSTO1 under different concentrations of sodium fluoride (NaF) (10~4,10~3,10~2,10,and 1mg·L~(-1))were evaluated to determinethe optimal fluoride concentration.This experiment included control group,negative control group (NC-siRNA),NaF group (1mg·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,Beclin1,Atg3,Atg5)and apoptosis-related genes (BCL2,Caspase-3)in osteoblasts.The protein expression of LC3,p62,Beclin1,Atg3 and Atg5were detected by Western blot.Results were as follows:Osteoblasts were identified by HE,Giemsa,alkaline phosphatase,and alizarin red staining.In the experimental model,50nmol·L~(-1) was found to be the optimal transfection concentration of siRNA,and siRNA2 was the best silent transfection fragment,at 1mg·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,Beclin1 and Atg5mRNA 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.
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 GSTO1 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 GSTO1 mRNA sequence,and the best effective fragment was screened.Then,the mRNA expression levels of GSTO1 under different concentrations of sodium fluoride (NaF) (10~4,10~3,10~2,10,and 1mg·L~(-1))were evaluated to determinethe optimal fluoride concentration.This experiment included control group,negative control group (NC-siRNA),NaF group (1mg·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,Beclin1,Atg3,Atg5)and apoptosis-related genes (BCL2,Caspase-3)in osteoblasts.The protein expression of LC3,p62,Beclin1,Atg3 and Atg5were detected by Western blot.Results were as follows:Osteoblasts were identified by HE,Giemsa,alkaline phosphatase,and alizarin red staining.In the experimental model,50nmol·L~(-1) was found to be the optimal transfection concentration of siRNA,and siRNA2 was the best silent transfection fragment,at 1mg·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,Beclin1 and Atg5mRNA 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.
引文
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[20]PAUL S,JAKHAR R,BHARDWAJ M,et al.Glutathione-S-transferase omega 1(GSTO1-1)acts as mediator of signaling pathways involved in aflatoxin B1-induced apoptosis-autophagy crosstalk in macrophages[J].Free Rad Biol Med,2015,89:1218-1230.
[21]陈科,程汉华,周荣家.自噬与泛素化蛋白降解途径的分子机制及其功能[J].遗传,2012,34(1):5-18.CHEN K,CHENG H H,ZHOU R J.Molecular mechanisms and functions of autophagy and the ubiquitin-proteasome pathway[J].Hereditas,2012,34(1):5-18.(in Chinese)
[22]陈金东.细胞自噬与细胞凋亡在细胞中的作用[J].遵义医学院学报,2016,39(3):217-222.CHEN J D.The role of autophagy and apoptosis in cells[J].Journal of Zunyi Medical University,2016,39(3):217-222.(in Chinese)
[23]PANKIV S,CLAUSEN T H,LAMARK T,et al.p62/SQSTM1binds directly to Atg8/LC3to facilitate degradation of ubiquitinated protein aggregates by autophagy[J].J Biol Chem,2007,282(33):24131-24145.
[2]刘永林,马培,雒昆利,等.1991-2012年中国地方性氟中毒病情动态变化[J].重庆师范大学学报:自然科学版,2016,33(2):142-151.LIU Y L,MA P,LUO K L,et al.The state and trend of endemic fluorosis from 1991to 2012in China[J].Journal of Chongqing Normal University:Natural Science Edition,2016,33(2):142-151.(in Chinese)
[3]李彤,张秀云,徐辉,等.氟对体外培养成骨细胞氧化应激和增殖活性的影响[J].中国实验诊断学,2007,11(2):194-197.LI T,ZHANG X Y,XU H,et al.The effects of fluoride on oxidative stress and proliferative activity of osteoblasts[J].Chinese Journal of Laboratory Diagnosis,2007,11(2):194-197.(in Chinese)
[4]LEVINE B,KROEMER G.Autophagy in the pathogenesis of disease[J].Cell,2008,132(1):27-42.
[5]GANESAN S,PEARCE S C,GABLER N K,et al.Short-term heat stress results in increased apoptotic signaling and autophagy in oxidative skeletal muscle in Sus scrofa[J].J Therm Biol,2018,72:73-80.
[6]YANG H L,MEI J,CHANG K K,et al.Autophagy in endometriosis[J].Am J Transl Res,2017,9(11):4707-4725.
[7]MONTICOLO F,COLANTUONO C,CHIUSANOM L.Shaping the evolutionary tree of green plants:evidence from the GST family[J].Sci Rep,2017,7:14363.
[8]BOARD P G,MENON D.Structure,function and disease relevance of Omega-class glutathione transferases[J].Arch Toxicol,2016,90(5):1049-1067.
[9]SIMON M J K,BEIL F T,RTHER W,et al.High fluoride and low calcium levels in drinking water is associated with low bone mass reduced bone quality and fragility fractures in sheep[J].Osteop Int,2014,25(7):1891-1903.
[10]YAN X Y,YAN X T,MORRISON A,et al.Fluoride induces apoptosis and alters collagen I expression in rat osteoblasts[J].Toxicol Lett,2011,200(3):133-138.
[11]郭晓东,杨茂伟,梁单,等.氟诱导成骨细胞MC3T3-E1自噬与凋亡及其相互作用分析[J].中国地方病防治杂志,2012,27(3):165-168.GUO X D,YANG M W,LIANG D,et al.An analysis on fluorine-induced autophagy and apoptosis of MC3T3-E1and their interaction[J].Chinese Journal of Control of Endemic Diseases,2012,27(3):165-168.(in Chinese)
[12]XU H,ZHOU Y L,ZHANG X Y,et al.Activation of PERK signaling through fluoride-mediated endoplasmic reticulum stress in OS732cells[J].Toxicology,2010,277(1-3):1-5.
[13]ZHAO Y F,LI Y Y,GAO Y F,et al.TGF-β1acts as mediator in fluoride-induced autophagy in the mouse osteoblast cells[J].Food Chem Toxicol,2018,115:26-33,doi:10.1016/j.fct.2018.02.065.
[14]张颖,孙贵范,金亚平,等.氟对成骨细胞增殖分化的影响及维生素C的拮抗作用[J].中华劳动卫生职业病杂志,2003,21(4):250-252.ZHANG Y,SUN G F,JIN Y P,et al.Effects of fluoride on proliferation and differentiation of rat osteoblasts in vitro and the antagonistic action of vitamin C[J].Chinese Journal of Industrial Hygiene and Occupational Diseases,2003,21(4):250-252.(in Chinese)
[15]GUNEY M,ORAL B,TAKE G,et al.Effect of fluoride intoxication on endometrial apoptosis and lipid peroxidation in rats:role of vitamins E and C[J].Toxicology,2007,231(2-3):215-223.
[16]于倩,左楠,贾海燕,等.硒对氟中毒雏鸡肝脏细胞色素P450酶系主要亚型的影响[J].畜牧兽医学报,2009,40(6):922-927.YU Q,ZUO N,JIA H Y,et al.The effect of selenium on main subtypes of hepatic microsomal-cytochrome P450sin chickens with exposure of fluoride[J].Acta Veterinaria et Zootechnica Sinica,2009,40(6):922-927.(in Chinese)
[17]PIAGGI S,RAGGI C,CORTI A,et al.Glutathione transferase omega 1-1(GSTO1-1)plays an anti-apoptotic role in cell resistance to cisplatin toxicity[J].Carcinogenesis,2010,31(5):804-811.
[18]MENG F,ZHANG Y Y,LIU F,et al.Characterization and mutational analysis of omega-class GST(GSTO1)from Apis cerana cerana,agene involved in response to oxidative stress[J].PLoS One,2014,9(3):e93100.
[19]ZHANG S,NIU Q,GAO H,et al.Excessive apoptosis and defective autophagy contribute to developmental testicular toxicity induced by fluoride[J].Environ Poll,2016,212:97-104.
[20]PAUL S,JAKHAR R,BHARDWAJ M,et al.Glutathione-S-transferase omega 1(GSTO1-1)acts as mediator of signaling pathways involved in aflatoxin B1-induced apoptosis-autophagy crosstalk in macrophages[J].Free Rad Biol Med,2015,89:1218-1230.
[21]陈科,程汉华,周荣家.自噬与泛素化蛋白降解途径的分子机制及其功能[J].遗传,2012,34(1):5-18.CHEN K,CHENG H H,ZHOU R J.Molecular mechanisms and functions of autophagy and the ubiquitin-proteasome pathway[J].Hereditas,2012,34(1):5-18.(in Chinese)
[22]陈金东.细胞自噬与细胞凋亡在细胞中的作用[J].遵义医学院学报,2016,39(3):217-222.CHEN J D.The role of autophagy and apoptosis in cells[J].Journal of Zunyi Medical University,2016,39(3):217-222.(in Chinese)
[23]PANKIV S,CLAUSEN T H,LAMARK T,et al.p62/SQSTM1binds directly to Atg8/LC3to facilitate degradation of ubiquitinated protein aggregates by autophagy[J].J Biol Chem,2007,282(33):24131-24145.