沉默SPARC基因抑制高氟介导的甲状腺细胞凋亡
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摘要
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-1 R were measured by Western blot.In addition,si-SPARC and si-IGF-1 R 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-1 R(1.95±0.24 vs.0.93±0.08,P<0.05),and inhibition of IGF-1 R 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-1 R.
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-1 R were measured by Western blot.In addition,si-SPARC and si-IGF-1 R 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-1 R(1.95±0.24 vs.0.93±0.08,P<0.05),and inhibition of IGF-1 R 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-1 R.
引文
[1] 曾强,崔玉山,张磊,等. 氟对甲状腺细胞凋亡的影响及其机制研究[J]. 中华预防医学杂志, 2012, 46(3):233-236.
[2] BOBEK S, KAHL S, EWY Z. Effect of long-term fluoride administration on thyroid hormones level blood in rats[J]. Endocrinol Exp, 1976, 10(10):289-295.
[3] WANG H, YANG Z, ZHOU B, et al. Fluoride-induced thyroid dysfunction in rats:Roles of dietary protein and calcium level[J]. Toxicol Ind Health, 2009, 25(1):49-57.
[4] 崔玉山,赵亮,曾强,等. 高碘和/或高氟致人甲状腺细胞凋亡与内质网应激的关系[J]. 中国预防医学杂志, 2015, 16(10):751-756.
[5] ASEER KR, SILVESTER AJ, KUMAR A, et al. SPARC paucity alleviates superoxide-mediated oxidative stress, apoptosis, and autophagy in diabetogenic hepatocytes[J]. Free Radic Biol Med, 2017, 108:874-895.
[6] LIU H, ZENG Q, CUI Y, et al. The role of the IRE1 pathway in excessive iodide- and/or fluoride-induced apoptosis in Nthy-ori 3-1 cells in vitro[J]. Toxicol Lett, 2014, 224(3):341-348.
[7] CHLENSKI A, LIU S, GUERRERO LJ, et al. SPARC expression is associated with impaired tumor growth, inhibited angiogenesis and changes in the extracellular matrix[J]. Int J Cancer, 2006, 118(2):310-316.
[8] BREKKEN RA, PUOLAKKAINEN P, GRAVES DC, et al. Enhanced growth of tumors in SPARC null mice is associated with changes in the ECM[J]. J Clin Invest, 2003, 111(4):487-495.
[9] PRASAD NB, SOMERVELL H, TUFANO RP, et al. Identification of genes differentially expressed in benign versus malignant thyroid tumors[J]. Clin Cancer Res, 2008, 14(11):3327-3337.
[10] PAN L, SHI X, LIU S, et al. Fluoride promotes osteoblastic differentiation through canonical Wnt/β-catenin signaling pathway[J]. Toxicol Lett, 2014, 225(1):34-42.
[11] 贾志红,于燕妮,杨小蓉,等. 氟对大鼠成骨细胞SPARC mRNA及其蛋白表达的影响[J]. 中国预防医学杂志, 2013, 14(7):485-489.
[12] BARBIER O, ARREOLA-MENDOZA L, RAZO LMD. Molecular mechanisms of fluoride toxicity[J]. Chem Biol Interact, 2010, 188(2):319-333.
[13] LIU H, ZENG Q, CUI Y, et al. The role of the IRE1 pathway in excessive iodide- and/or fluoride-induced apoptosis in Nthy-ori 3-1 cells in vitro[J]. Toxicol Lett, 2014, 224(3):341-348.
[14] CHANG W, WEI K, JACOBS SS, et al. SPARC suppresses apoptosis of idiopathic pulmonary fibrosis fibroblasts through constitutive activation ofbeta-catenin[J]. J Biol Chem, 2010, 285(11):8196-8206.
[15] YIU GK, CHAN WY, NG SW, et al. SPARC (secreted protein acidic and rich in cysteine) induces apoptosis in ovarian cancer cells[J]. Am J Pathol, 2001, 159(2):609-622.
[16] AOI W, NAITO Y, TAKAGI T, et al. A novel myokine, secreted protein acidic and rich in cysteine (SPARC), suppresses colon tumorigenesis via regular exercise[J]. Gut, 2013, 62(6):882-889.
[17] MITSIADES CS, MITSIADES N, POULAKI V, et al. Activation of NF-kappaB and upregulation of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells: Therapeutic implications[J]. Oncogene, 2002, 21(37):5673-5683.
[18] GRUBER HE, NORTON HJ, HANLEY EN. Anti-apoptotic effects of IGF-1 and PDGF on human intervertebral disc cells in vitro[J]. Spine, 2000, 25(17):2153-2157.
[19] OCK S, AHN J, LEE SH, et al. IGF-1 receptor deficiency in thyrocytes impairs thyroid hormone secretion and completely inhibits TSH-stimulated goiter[J]. FASEB J, 2013, 27(12):4899-4908.
[2] BOBEK S, KAHL S, EWY Z. Effect of long-term fluoride administration on thyroid hormones level blood in rats[J]. Endocrinol Exp, 1976, 10(10):289-295.
[3] WANG H, YANG Z, ZHOU B, et al. Fluoride-induced thyroid dysfunction in rats:Roles of dietary protein and calcium level[J]. Toxicol Ind Health, 2009, 25(1):49-57.
[4] 崔玉山,赵亮,曾强,等. 高碘和/或高氟致人甲状腺细胞凋亡与内质网应激的关系[J]. 中国预防医学杂志, 2015, 16(10):751-756.
[5] ASEER KR, SILVESTER AJ, KUMAR A, et al. SPARC paucity alleviates superoxide-mediated oxidative stress, apoptosis, and autophagy in diabetogenic hepatocytes[J]. Free Radic Biol Med, 2017, 108:874-895.
[6] LIU H, ZENG Q, CUI Y, et al. The role of the IRE1 pathway in excessive iodide- and/or fluoride-induced apoptosis in Nthy-ori 3-1 cells in vitro[J]. Toxicol Lett, 2014, 224(3):341-348.
[7] CHLENSKI A, LIU S, GUERRERO LJ, et al. SPARC expression is associated with impaired tumor growth, inhibited angiogenesis and changes in the extracellular matrix[J]. Int J Cancer, 2006, 118(2):310-316.
[8] BREKKEN RA, PUOLAKKAINEN P, GRAVES DC, et al. Enhanced growth of tumors in SPARC null mice is associated with changes in the ECM[J]. J Clin Invest, 2003, 111(4):487-495.
[9] PRASAD NB, SOMERVELL H, TUFANO RP, et al. Identification of genes differentially expressed in benign versus malignant thyroid tumors[J]. Clin Cancer Res, 2008, 14(11):3327-3337.
[10] PAN L, SHI X, LIU S, et al. Fluoride promotes osteoblastic differentiation through canonical Wnt/β-catenin signaling pathway[J]. Toxicol Lett, 2014, 225(1):34-42.
[11] 贾志红,于燕妮,杨小蓉,等. 氟对大鼠成骨细胞SPARC mRNA及其蛋白表达的影响[J]. 中国预防医学杂志, 2013, 14(7):485-489.
[12] BARBIER O, ARREOLA-MENDOZA L, RAZO LMD. Molecular mechanisms of fluoride toxicity[J]. Chem Biol Interact, 2010, 188(2):319-333.
[13] LIU H, ZENG Q, CUI Y, et al. The role of the IRE1 pathway in excessive iodide- and/or fluoride-induced apoptosis in Nthy-ori 3-1 cells in vitro[J]. Toxicol Lett, 2014, 224(3):341-348.
[14] CHANG W, WEI K, JACOBS SS, et al. SPARC suppresses apoptosis of idiopathic pulmonary fibrosis fibroblasts through constitutive activation ofbeta-catenin[J]. J Biol Chem, 2010, 285(11):8196-8206.
[15] YIU GK, CHAN WY, NG SW, et al. SPARC (secreted protein acidic and rich in cysteine) induces apoptosis in ovarian cancer cells[J]. Am J Pathol, 2001, 159(2):609-622.
[16] AOI W, NAITO Y, TAKAGI T, et al. A novel myokine, secreted protein acidic and rich in cysteine (SPARC), suppresses colon tumorigenesis via regular exercise[J]. Gut, 2013, 62(6):882-889.
[17] MITSIADES CS, MITSIADES N, POULAKI V, et al. Activation of NF-kappaB and upregulation of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells: Therapeutic implications[J]. Oncogene, 2002, 21(37):5673-5683.
[18] GRUBER HE, NORTON HJ, HANLEY EN. Anti-apoptotic effects of IGF-1 and PDGF on human intervertebral disc cells in vitro[J]. Spine, 2000, 25(17):2153-2157.
[19] OCK S, AHN J, LEE SH, et al. IGF-1 receptor deficiency in thyrocytes impairs thyroid hormone secretion and completely inhibits TSH-stimulated goiter[J]. FASEB J, 2013, 27(12):4899-4908.
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