Résumé
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.
Résumé
Filtering bleb scarring is the main cause of glaucoma filtration surgery failure. Subconjunctival injection of antimetabolites, such as mitomycin and 5-fluorouracil, is widely used clinically to reduce the incidence of scarring, which improves the success rate of the surgery. However, accompanied side effects such as cytotoxicity should not be ignored. Secreted protein acidic and rich in cysteine (SPARC) as a matricellular protein is widely distributed in the eyes, which plays an important role in the process of wound repairing and tissue remodeling. The expression of SPARC is significantly elevated in the mouse model of subconjunctival scarring. Researches suggest that SPARC participates in and regulate the formation of bleb scarring through multiple pathways, therefore it may become a specific new target in the anti-scarring therapy.
Résumé
Objective:To investigate the effects of vascular endothelial growth factor (VEGF) on the expression of secreted protein acidic and rich in cysteine (SPARC) and the fibrosis in cultured human Tenon's fibroblast (HTF) in vitro. Methods:HTF cells were obtained from Tenon's capsule tissues of patients undergoing strabismus surgery. Immunofluorescence was used to identify the HTF cells. HTF cells were cultured with different concentrations of VEGF, and which were divided into four groups, i.e., 0 ng/mL group, 25 ng/mL group, 50 ng/mL group and 100 ng/mL group. The expression of SPARC, collagen- , and matrix metalloprotein 9 (MMP-9) and the activity of extracellular signal-regulated kinase (ERK) pathway were analyzed by Western blotting and real-time quantitative PCR (qPCR). The abilities of proliferation and migration of HTF cells were detected by MTS assay and scratch test, respectively. Results:HTF cells were observed and identified by inverted phase contrast microscope and immunofluorescence. Under the stimulation of VEGF, the expression of protein and mRNA of SPARC, collagen-I and MMP-9 of HTF cells in other three groups were increased compared with 0 ng/mL group; the phosphorylation activities of ERK pathway were up-regulated, and the proliferation and migration abilities of HTF cells were up-regulated. And the effect was the most obvious in the 50 ng/mL group. Conclusion:VEGF is involved in promoting the fibrosis of HTF cells accompanied by the up-regulation of the SPARC, which suggests SPARC may become a potential regulatory site.
Résumé
Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein highly expressed in bone tissue that acts as a chemoattractant factor promoting the arrival of prostate cancer (PCa) cells to the bone marrow. However, the contribution of SPARC during the early stages of tumor progression remains unclear. In this study, we show that SPARC is highly expressed in PCa tissues with a higher Gleason score. Through stable knockdown and overexpression of SPARC in PC3 and LNCaP cells, respectively, here we demonstrate that endogenous SPARC induces the epithelial-mesenchymal transition (EMT), decreasing E-cadherin and cytokeratin 18 and increasing N-cadherin and vimentin. Moreover, SPARC induces the expression of EMT regulatory transcription factors Snail family transcriptional repressor 1 (Snail), Snail family transcriptional repressor 2 (Slug), and zinc finger E-box binding homeobox 1 (Zeb1). In addition, SPARC knockdown in PC3 cells decreases migration and invasion in vitro, without modifying cell proliferation. Our results indicate that SPARC might facilitate tumor progression by modifying the cellular phenotype in cancer cells.
Résumé
Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein highly expressed in bone tissue that acts as a chemoattractant factor promoting the arrival of prostate cancer (PCa) cells to the bone marrow. However, the contribution of SPARC during the early stages of tumor progression remains unclear. In this study, we show that SPARC is highly expressed in PCa tissues with a higher Gleason score. Through stable knockdown and overexpression of SPARC in PC3 and LNCaP cells, respectively, here we demonstrate that endogenous SPARC induces the epithelial-mesenchymal transition (EMT), decreasing E-cadherin and cytokeratin 18 and increasing N-cadherin and vimentin. Moreover, SPARC induces the expression of EMT regulatory transcription factors Snail family transcriptional repressor 1 (Snail), Snail family transcriptional repressor 2 (Slug), and zinc finger E-box binding homeobox 1 (Zeb1). In addition, SPARC knockdown in PC3 cells decreases migration and invasion in vitro, without modifying cell proliferation. Our results indicate that SPARC might facilitate tumor progression by modifying the cellular phenotype in cancer cells.