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Objective@#To investigate the expression of SnoN in human hypertrophic scar fibroblasts and the mechanism of its participation in hypertrophic scar formation.@*Methods@#Eight patients with hypertrophic scar after burn in need of surgery were admitted in our unit from January to October 2013, and then hypertrophic scar tissue and normal skin tissue of full-thickness skin donor site resected by surgery of the patients were collected. Hypertrophic scar fibroblasts and normal skin fibroblasts of patients were isolated with method of explant culture and then sub-cultured. Cells of the third to fifth passage were used in the following experiments. (1) The protein expressions of SnoN of hypertrophic scar fibroblasts and normal skin fibroblasts were assessed with Western blotting. (2) The mRNA expressions of SnoN of another batch of hypertrophic scar fibroblasts and normal skin fibroblasts were determined with reverse transcription polymerase chain reaction. (3) Another batch of hypertrophic scar fibroblasts and normal skin fibroblasts were treated with 10 ng/mL transforming growth factor beta1 (TGF-β1) for 30 min, 1 h, 2 h, and 6 h, respectively, and then the protein expressions and mRNA expressions of SnoN of untreated cells and treated cells were detected as above. Data were processed with one way analysis of variance and independent sample t test.@*Results@#(1) The protein expression of SnoN of hypertrophic scar fibroblasts was 0.020±0.003, significantly lower than that of normal skin fibroblasts (0.032±0.005, t=7.19, P<0.05). (2) The mRNA expression of SnoN of hypertrophic scar fibroblasts was 0.407±0.157, with no significant difference from that of normal skin fibroblasts (0.339±0.095, t=-1.29, P>0.05). (3) The protein expression of SnoN of normal skin fibroblasts was increased in a time-dependent fashion with the TGF-β1 stimulation, and the protein expressions of SnoN of cells treated with TGF-β1 for 30 min, 1 h, 2 h, and 6 h were significantly higher than those of untreated cells (with t values from 2.27 to 27.89, P values below 0.05). The protein expression of SnoN of hypertrophic scar fibroblasts was decreased in a time-dependent fashion with the TGF-β1 stimulation, and the protein expressions of SnoN of cells treated with TGF-β1 for 30 min, 1 h, 2 h, and 6 h were obviously lower than those of untreated cells (with t values from 10.80 to 13.85, P values below 0.05). (4) The mRNA expressions of SnoN of normal skin fibroblasts and hypertrophic scar fibroblasts were both increased in a time-dependent fashion with the TGF-β1 stimulation, and the mRNA expressions of SnoN of the two types of cells treated with TGF-β1 for 30 min, 1 h, 2 h, and 6 h were both significantly higher than those of untreated cells (with t values from 18.16 to 58.22, P values below 0.05).@*Conclusions@#The protein expression of SnoN in hypertrophic scar fibroblasts is reduced, which weakens its inhibitory effect on TGF-β1 signal, thus amplifying the TGF-β1 signal, and it may participate in the formation of hypertrophic scar.
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Objective To investigate the expression and possible mechanism of miR-21 and Ski-related novel protein N( SnoN) in the renal fibrosis diabetic process.Methods The animal model was established by tail-vein injection of Streptozotocin,and the other group were normal control ( NC) group.After 10 weeks, the rats were sacrificed to measure biochemical parameters and renal index , and to observe the changes of pathomorphology by HE staining as well.Meanwhile, immunohistochemistry and Western blot were employed to examine protein ex-pression of E-cadherin,α-smooth muscle actin(α-SMA), fibronectin(FN), collagen-Ⅰ(Col-Ⅰ), collagen-Ⅲ(Col-Ⅲ), transforming growth factor-β1(TGF-β1), Smad3, p-Smad3(Ser423/425) and SnoN in the renal tissue. In addition, the expression of SonN mRNA and miR-21 were detected by qPCR.Results In DM group,the ex-pressions of Col-Ⅰ, Col-Ⅲ and FN in renal interstitium were increased ( P <0.05 ) , TGF-β1 increased (P<0.05),while E-cadherin decreased(P<0.05).Compared with NC group, the expression of α-SMA,p-Smad3 (Ser423/425) protein increased in DM group(P<0.05),while the protein level of SnoN decreased but the level of SnoN mRNA increased ( P <0.05 ) .Moreover, the level of miR-21 markedly increased in DM group ( P <0.05 ) .Conclusions TGF-β1 may up-regulate the expression of miR-21 but restrain the translational expression of SnoN, aggravating fibrosis.
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This study was aimed to demonstrate preliminarily the effects and mechanisms of uremic clearance granule (UCG) ameliorating renal interstitial fibrosis (RIF) by regulating transforming growth factor (TGF)-β1/SnoN/Smads signaling pathway in vivo. Fifteen rats were randomly divided into 3 groups：the normal group,the model group and the UCG group. The rats with renal failure were induced by intragastric administration of adenine and unilateral ureteral obstruction (UUO). After modeling,the rats in the UCG group and in the other groups were intervened by intragastric administration of UCG and distilled water respectively during 3 weeks. The body weight and 24 h urinary protein excretion (Upro) in all rats were tested after drug administration. All rats were killed after drug administration for 3 weeks,blood and kidneys were collected and weighted,kidney appearance and renal morphological characteristics were observed. In addition,serum biochemical indices and the protein expressions of TGF-β1,SnoN,phosphorylated Smad2/3 (p-Smad2/3) and Smad7 in the kidney were evaluated respectively. The results indicated that,after the intervention of UCG,the general state of health,kidney appearance,serum creatinine (Scr),blood urea nitrogen (BUN),uric acid (UA),albumin (Alb),Upro and renal morphological change in model rats were improved in different degrees,respectively. Moreover,UCG down-regulated the protein expressions of TGF-β1 and p-Smad2/3,and up-regulated the protein expressions of SnoN and Smad7 in the kidney. In conclusion,UCG reduces extracellular matrix (ECM) synthesis and delays the progression of renal failure via possibly multi-targeting at regulating TGF-β1/SnoN/Smads signaling pathway in vivo.
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AIM:To investigate the effects of proteasome inhibitor MG 132 on the expression of SnoN in renal tubule epithelial cells incubated in high glucose , and to explore the possible mechanism and function that MG 132 reduces or slows down renal tubular interstitial injury after incubated in high glucose .METHODS:The NRK-52E cells were divid-ed into normal control group (NG), high glucose group (HG) and high glucose plus pretreatment with different doses of MG132 group (HG+MG132).The immunofluorescence staining was used to detect the protein expression of E-cadherin and α-smooth muscle actin (α-SMA) in NRK-52E cells under different conditions .The relative protein expression levels of SnoN, Smad ubiquitination regulatory factor 2 (Smurf2), Arkadia, E-cadherin, α-SMA and collagen type Ⅰ(Col-Ⅰ) were detected by Western blotting .RESULTS:Compared with NG group , the expression of E-cadherin and SnoN was de-creased (P<0.05), while the expression of α-SMA, Col-Ⅰ, Smurf2 and Arkadia was increased (P<0.05).Compared with HG group, the protein expression of SnoN and E-cadherin was significantly up-regulated in HG+MG132 group ( P<0.05 ) , and the protein expression of α-SMA and Col-Ⅰwas significantly down-regulated in a dose-depended manner ( P<0.05).However, no effect on the protein expression of Smurf2 and Arkadia was observed.CONCLUSION: MG132 in-hibits the degradation of SnoN protein induced by high glucose , thus reducing the renal fibrosis .
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OBJECTIVE: TGF-beta signaling is dependent on the heterodimerization of the type II TGF-beta receptor (TbetaR-II) with the type I TGF-beta receptor (TbetaR-I). which mediate intracellular signals through Smad proteins. Whereas physiologic concentrations of SnoN and Ski allow a feedback regulation of TGF-beta signaling, deregulation of SnoN or Ski expression leads to total inhibition of TGF-beta signaling and of the tumor suppressors Smad2 and Smad4, which can explain the role of SnoN and Ski as oncogenes. In order to identify possible molecular mechanisms responsible for TGF-beta resistance, the author investigated the mutation and expression of TGF-beta1, its receptors, Ski/SnoN in cervical carcinomas. METHODS: From December 1995 to December 1999, 45 carcinomas and 7 normal cervical tissue specimens were obtained by surgical resection in the Kyung Hee University Medical Center. Tissue specimens were snap-forzen in liquid N2 and stored at -70 degrees C until used. Total RNA was extracted from specimens and evaluated the expression levels using densitometric analysis of quantitative RT-PCR products (TGF-beta1, Tbeta1R-I, Tbeta1R-II, Ski/SnoN), and the mutations were investigated by quantitative genomic-PCR followed by nonisotopic RT-PCR-SSCP analysis (Tbeta1R-II, Tbeta1R-I, Ski/SnoN). The abnorally expressed levels of RT-PCR products (TGF-beta1, Tbeta1R-II) were analysed for the clinicopathologic characteristics. RESULTS: Quantitative RT-PCR analysis demonstrated variable expression of TGF-beta1 mRNA (0.05-0.89) in tumors and significantly increased TGF-beta1 expression level (>0.48) in 15 of 45 samples (33.3%). There is no significant reduction of Tbeta1R-I expression (1.36) in 2 of 45 samples (4.4%), and there is no amplification of Ski/SnoN gene by quantitative genomic-PCR analysis. CONCLUSIONS: The overexpression of TGF-beta1 mRNA and the reduced or absent expression of Tbeta1R-II may be an important contributing factors, and the abnormally low genomic levels and no mutational alterations of Tbeta1R-II is caused by monoallelic deletion suggesting that Tbeta1R-II might play as a tumor suppressor of haloinsufficiency in cervical carcinomas. We could not show that high levels of Ski/SnoN expression could produce a disruption of TGF-beta signaling in cervical carcinomas.