沉默SPARC基因抑制高氟介导的甲状腺细胞凋亡

Silencing SPARC inhibits high fluoride-induced apoptosis of thyrocytes

目的研究富含半胱氨酸的酸性分泌蛋白(SPARC)对高氟介导的甲状腺细胞凋亡的作用及可能机制。方法培养人甲状腺细胞Nthy-ori 3-1,给予不同浓度的NaF(0.1、1、10 mmol/L)处理24 h,通过实时定量PCR和蛋白印迹法评价SPARC的表达,确定后续实验NaF作用浓度。另将细胞分组进行实验:对照组、NaF组(1 mmol/L孵育24 h)、si-SPARC组(转染SPARC siRNA 48 h用NaF处理)和si-NC组(转染Negative control siRNA 48 h用NaF处理)。CCK-8法和乳酸脱氢酶(LDH)试剂盒检测细胞毒性;细胞凋亡试剂盒检测细胞凋亡率,蛋白印迹法检测活化caspase3(c-caspase3)和IGF-1R的蛋白表达。此外,将siSPARC和siIGF-1R共同转染至甲状腺细胞,通过评价细胞凋亡进一步探讨SPARC的作用机制。结果随着NaF的浓度增大,SPARC mRNA和蛋白表达均逐渐升高(P<0.05)。si-SPARC组细胞活性较si-NC组增高[(84.02±9.51)%vs.(58.31±6.86)%,P<0.05],且LDH释放率减少[(134.25±18.98)%vs.(195.18±23.50)%,P<0.05]。si-SPARC组较si-NC组细胞凋亡率减少[(124.67±19.44)%vs.(175.24±16.46)%,P<0.05]。此外,si-SPARC组(1.95±0.24 vs. 0.93±0.08,P<0.05)IGF-1R蛋白表达上调,抑制IGF-1R逆转SPARC对细胞凋亡的作用。结论沉默SPARC基因降低高氟介导的细胞毒性、抑制细胞凋亡,其机制可能是通过负调控IGF-1R实现的。

Objective To investigate the effects and mechanisms of secreted protein,acidic and rich in cysteine(SPARC)on high fluoride-induced apoptosis of thyrocytes.Methods Human thyroid cells(Nthy-ori 3-1)were cultured and treated with various concentrations(0.1,1 and 10 mmol/L)of NaF for 24 h,and the expression of SPARC was evaluated using Real-time PCR and Western blot,respectively.The cells were divided into four groups:control group,NaF group,si-SPARC group(cells were transfected with SPARC siRNA for 48 h and then exposed to NaF for 24 h),and si-NC group(cells were transfected with negative control siRNA for 48 h and then exposed to NaF for 24 h).Cytotoxicity was assayed using CCK-8 and LDH;cell apoptosis rate was detected with ELISA.The expressions of cleaved caspase3(c-caspase3)and IGF-1R were measured by Western blot.In addition,si-SPARC and si-IGF-1R were co-transfected into thyrocytes to further explore mechanisms of SAPRC by evaluating apoptosis.Results The mRNA and protein levels of SPARC were augmented with the increase of NaF(P<0.05).Cell viability was significantly higher in si-NC group than that in si-SPARC group[(84.02±9.51)%vs.(58.31±6.86)%,P<0.05],and the release rate of LDH was lower[(134.25±18.98)%vs.(195.18±23.50)%,P<0.05].Cell apoptosis rate was lower in si-SPARC group than that in si-NC group[(124.67±19.44)%vs.(175.24±16.46)%,P<0.05].In addition,silencing SPARC upregulated the expression of IGF-1R(1.95±0.24 vs.0.93±0.08,P<0.05),and inhibition of IGF-1R reversed the effect of SPARC on apoptosis.Conclusion Inhibition of SPARC reduces high fluoride-induced cytotoxicity and blocks cell apoptosis.The possible mechanism is through the negative regulation of IGF-1R.