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Background The pathogenesis of beryllium-induced pulmonary fibrosis is unknown and there is no specific treatment for the disease as yet. MicroRNA (miRNA) may play a role in the process of beryllium-induced pulmonary fibrosis. Objective To construct a microRNA-21 (miR-21) interfering cell line, and to investigate the effect of miR-21 on beryllium sulfate (BeSO4)-induced fibrosis in human lung adenocarcinoma alveolar basal epithelial cells (A549 cells) and its potential mechanism. Methods The miR-21 target genes were predicted by the online database miRBase and verified by experiments using dual luciferase reporter gene. After transfecting A549 with miR-21interference lentivirus, puromycin was used to select a stable cell line. An in vitro model of pulmonary fibrosis was established using BeSO4 infecting A549 cells with a concentration of 10 μmol·L−1 and an exposure time of 48 h. Then the treated cells were divided into control group, model group, miR-21 interference group, and miR-21 interference control group. Real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the relative expression level of miR-21 gene. Western blotting was used to detect the relative expression levels of TGF-β1/Smads pathway related proteins [Smad2, Smad3, p-Smad2, p-Smad3, Smad7, and transforming growth factor-β1 (TGF-β1)], myofibrosis cell marker α-smooth muscle actin (α-SMA), andextracellular matrix collagen-I (COL-I) and collagen-Ⅲ (COL-Ⅲ). Results The miRBase predicted that miR-21 had a binding site with Smad7, and the results of the dual luciferase reporter gene experiment showed that the target gene of miR-21 was Smad7. The construction of miR-21 interfered with A549 cell line was successful. Compared with the control group, the relative expression of miR-21 gene in the model group increased by 97.57%; the relative expression of Smad7 protein in the model group decreased by 15.48%; the relative protein expression of Smad2, Smad3, p-Smad2, p-Smad3, TGF-β1, α-SMA, COL-I, and COL-Ⅲ increased by 13.55%, 35.72%, 18.35%, 35.75%, 25.52%, 31.58%, 24.61%, and 11.66% respectively (P<0.05). Compared with the interference control group, the miR-21 gene expression level in the interference group decreased by 28.96%; the relative expression of Smad7 protein increased by 19.07%; the relative protein expression of Smad2, Smad3, p-Smad2, p-Smad3, TGF-β1, α-SMA, COL-I, and COL-Ⅲ decreased by 8.01%, 19.95%, 14.56%, 19.37%, 11.95%, 10.96%, 18.81%, and 31.36% repectively (P<0.05). There was no statistically significant difference in the gene abd protein expression levels of each gene between the model group and the interference control group (P>0.05). Conclusion In an in vitro model of pulmonary fibrosis induced by beryllium compounds, miR-21 may promote fibrosis by targeting Smad7 to regulate the TGF-β1/Smad signaling pathway.
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Objective: To study the therapeutic effect of Kangxianling Decoction on renal fibrosis induced by 5/6 nephrectomization in rats, and discuss its action mechanism. Method: Totally 50 SD rats were randomly divided into normal control group (n=10), sham-operation group (n=10), and 5/6 nephrectomized renal fibrosis model group (n=30). After successful modeling, rats were randomly divided into the model group, Kangxianling group, and Losartan Potassium group. Rats in the Losartan Potassium group were administered with Losartan Potassium by gastrogavage; rats in the Kangxianling group were administered with Kangxianling by gastrogavage. Equal volume of distilled water was administered to rats in the other groups. Rats were sacrificed after 8 weeks of consecutive medication, and then serum creatinine (SCr), urea nitrogen (BUN), 24 hour urine protein (24 h-Pro) were measured in each group. Hematoxylin-eosin (HE) staining was used to observe the renal pathological changes and the score of Kidney damage was made. Masson staining was used to observe the degree of renal fibrosis. Western blot and real-time fluorescent quantitative polymerase chain reaction(Real-time PCR) were used to detect the protein and mRNA expression levels of transforming growth factor (TGF-β1), Smad2, Smad3, and Smad7 in kidneys. Result: As compared with the normal control and sham-operation group, the renal tissue fibrosis, score of kidney damage, SCr, BUN and 24 h-Pro levels were higher in model group (Pβ1, Smad2, and Smad3 (PPPβ1, Smad2, Smad3 were reduced (PPConclusion: Kangxianling decoction could alleviate renal fibrosis by inhibiting TGF-β1/Smad signal pathway.
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Objective To investigate the changes in the expression levels of miR-21 and its regulatory proteins in bone tissues of osteoporotic mouse model, and its correlation with bone mineral density. Methods Thirty ovariectomized (ovx) female mice were randomly divided into six groups according to different observation time (0 week, 1 week, 2 weeks, 4 weeks, 6 weeks, and 8 weeks, respectively). Smad7 expression level in the tissues of the distal femur of these mice was immunohistochemically analyzed. Real time PCR was performed at different time points for detecting changes in the mRNA levels of miR-21 and Smad7 target genes in the bone tissue. Dual-energy X-ray absorptiometry (DXA) was used to measure the distal femoral bone density in mice of each group. Results Immunohistochemical study revealed that the expression of Smad7 in the bone tissue of osteoporostic mouse gradually increased over time. Real time PCR analysis showed that the expression of mi R-21 gradually decreased, whereas the expression of Smad7 gradually increased. Over time, the bone density of mice gradually decreased, indicating that miR-21 was positively correlated with bone density, whereas Smad7 was negatively correlated with bone density. Conclusion Bone density of the osteoporotic mice gradually decreases. The expression of miR-21 is positively correlated with bone density, while that of Smad7 is negatively correlated with bone density.
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Objective • To investigate the effects of silencing connective tissue growth factor (Ctgf) gene on the growth, cell cycle and the expression of TGF-β1, Smad3 and Smad7 of rat hepatic stellate cell line HSCT6. Methods • The recombinant lentivirus vector pCDH/Ctgf-shRNA of Ctgf gene was constructed by RNA interference. The recombinant vector was packaged to obtain highly infectious pCDH/Ctgf-shRNA lentivirus particles for HSCT6 infection. The expression of green fluorescent protein (GFP) in the transfected HSCT6 cells was observed under fluorescence microscope. The effects of Ctgf-shRNA lentivirus on the growth of HSCT6 cells were tested by CCK-8. The effects of Ctgf-shRNA lentivirus on the cell cycle of HSCT6 cells were analyzed by flow cytometry (FCM). The effects of Ctgf-shRNA lentivirus on the expression of mRNA of Ctgf, Tgf-β1, Smad3 and Smad7, and their proteins in HSCT6 cells were detected by real-time PCR and Western blotting, respectively. Results • The lentiviral vector pCDH/Ctgf-shRNA has been constructed successfully. The HSCT6 cells transfected by Ctgf-shRNA lentivirus significantly expressed GFP under fluorescence microscope. The results of CCK-8 confirmed that the growth of HSCT6 cells transfected by Ctgf-shRNA lentivirus was slower than that of controls and the differences were statistically significant after being cultured for 72 h (P<0.05). The results of FCM revealed that the growth of HSCT6 cells transfected by Ctgf-shRNA lentivirus was blocked in the S phase of cell cycle. The results of real-time PCR and Western blotting showed that the Ctgf-shRNA lentivirus effectively silenced Ctgf gene, down-regulated the expression of genes and encoding proteins of TGF-β1, and Smad3 of HSCT6 and up-regulated the expression of genes and encoding proteins of Smad7 of HSCT6 cells. The differences between transfected cells and controls were statistically significant (P<0.05). Conclusion • Silencing Ctgf gene can effectively inhibit the growth of HSCT6 cells, down-regulate the expression of TGF-β1 and Smad3 and up-regulate the expression of Smad7. The inhibition of the growth of HSCT6 cells may be closely related to interference of the TGF-β1/Smads (Smad3 and Smad7) signaling pathway.
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Objective: To investigate the effects of Xuanfei Huayu Formula (XFHY) on the expression of TGF-β1/Smad2 in pulmonary fibrosis rats. Methods: Sixty male SPF Wistar rats were randomly divided into six groups: negative control group (group A), pulmonary fibrosis model group (group B), prednisone positive control group (group C, 0.167 mg/kg), the high doses of XFHY groups (group D, 14.38 g/kg), the medium doses of XFHY groups (group E, 7.19 g/kg), and the low doses of XFHY groups (group F, 3.60 g/kg) with ten rats in each group. The pulmonary fibrosis model was established by nasal instillation of bleomycin 7 μg/g (150 μL); In 8 h after the modle establishment, the rats in C, D, E, and F groups were respectively treated with prednisone acetate or XFHY once daily. Negative control group (group A) and model group (group B) were given equal volume physiological saline. The rats in different groups were executed 28 d after modeling for sampling. The HE and sirius red staining were used to observe alveolitis even pulmonary fibrosis changes in lung tissue under the microscope; The alkaline hydrolysis method was adopted to determine the content of hydroxyproline (Hyp) in lung tissue; The immunohistochemical method was used to determine the expression of alpha-SMA, Smad4, and Smad7 in rat lung tissues. The expression levels of TGF-β RII, Smad2, p-Smad2, and Smad7 proteins were detected by Western blotting. Results: The alveolar structure of the model group was severely damaged, and the interstitial hyperplasia, inflammatory cell infiltration, and collagen fibrosis were observed. Compared with the negative control group, the content of hydroxyproline and collagen staining was significantly increased in the model group. Compared with the model group, the expression of collagen fibers in the alveolar interval of three-dose group and prednisolone acetate group was significantly reduced, and the content of hydroxyproline was decreased significantly. Among them, the collagen fiber expression in XFHY high-dose group was less than XFHY low- and medium-dose group, and the hydroxyproline content was much lower. The above results showed that XFHY had a certain dose-effect relationship with the efficacy of pulmonary fibrosis. On this basis, the mechanism of the action of XFHY continues to it should be further explored. It was found that the protein content of TGF-β RII, Smad2, p-Smad2, Smad4, and α-SMA were decreased significantly, while the expression of Smad7 was higher in the XFHY group compared with the model group. Conclusion: XFHY can effectively prevent and treat pulmonary fibrosis, and its mechanism may relate to inhibit the over-expression of the α-SMA by regulating the TGF-β/Smad signaling pathway, thereby reducing the formation of collagen fibers.
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Objective@#To investigate the effect and regulatory mechanism of Smurf2 on the negative regulator Smad7 of TGF-β1/Smad signaling pathway in hypertrophic scar fibroblasts.@*Methods@#From January to October 2014, 8 patients with hypertrophic scar after burn were admitted. The age of patients ranged from 1 year 8 months to 7 years, and the time of scar hyperplasia ranged from 2 to 12 months. The residual normal skin of the same patient was used as the control. The fibroblasts were isolated from hypertrophic scar and normal skin respectively and cultured. The third to fifth passage cells were used for the experiments. ① The protein expression of Smad7 in the two groups was detected by Western Blot. ② Hypertrophic scar fibroblasts and normal skin fibroblasts were treated with exogenous TGF-β1 at concentration of 10 ng/ml for 0 min, 5 min, 15 min, 30 min, 1 h, 2 h and 12 h. The expression of Smad7 protein and mRNA were detected by Western Blot and RT-PCR, respectively. ③ The cell lysates of the two groups were collected and incubated with the ubiquitin mixture for 1 h, 2 h and 6 h at 37℃, respectively. The degradation level of Smad7 protein was detected by Western Blot. ④ The cell lysate of hypertrophic scar fibroblasts was collected and incubated with ubiquitin mixture with or without proteasome inhibitor (MG132: MG115=1: 1) to study its inhibitory effect on the degradation of Smad7 in vitro. ⑤ Immunoprecipitation (IP) technique was used to detect the interaction between Smad 7 and E3 ubiquitin ligase Smurf2 in hypertrophic scar fibroblasts. ⑥ The expression of Smad7 protein in hypertrophic scar fibroblasts stimulated by TGF-β1 after Smurf2 silencing by small interference RNA (siRNA) technique were detected by Western blot.@*Results@#① There was no significant difference in the expression level of Smad7 protein between hypertrophic scar and normal skin fibroblasts(t=0.76, P=0.46). ② Expressions of Smad7 mRNA and protein in normal skin fibroblasts stimulated by exogenous TGF-β1 gradually increased in a time-dependent manner(P<0.05). The expression of Smad7 mRNA in the hypertrophic scar fibroblasts increased at all-time points except at 5min , (P<0.05), while there was no significant difference in the expression level of Smad7 protein at all-time points with or without TGF-β1 stimulation(P>0.05). ③ Degradation of Smad7 protein was enhanced in hypertrophic scar fibroblasts (the expression level of Smad 7 protein at each time point was compared with that of the control group and the last time point, P<0.05), while there was no significant difference in Smad7 protein degradation in normal skin fibroblasts(P=0.162). ④ Enhanced degradation of Smad7 in hypertrophic scar fibroblasts was blocked by the addition of the proteasome inhibitors MG132/MG115. ⑤ In hypertrophic scar fibroblasts, the Smurf2-Smad7 complex was detected, which indicated the interaction between Smurf2 and Smad7 in hypertrophic scar fibroblasts. ⑥ The expression of Smad7 protein was not increased in the hypertrophic scar fibroblasts stimulated by TGF-β1, whereas the stimulation of TGF-β1 increased the expression of Smad7 protein after silencing of Smurf2 gene expression.@*Conclusions@#In the hypertrophic scar fibroblasts, Smurf 2 attenuates the inhibitory effect of Smad 7 on TGF-β1 signaling pathway through the degradation of Smad7 by ubiquitination, which may be involved in the formation of hypertrophic scar.
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OBJECTIVE Alcohol is mainly metabolized through liver and excreted by kidney in the body. Kidney damage has been considered as the secondary to liver injury and kidney dysfunction is common in hospitalized patients with severe alcoholic hepatitis. Both acute and chronic alcoholism accumulation can compromise kidney function, although alcoholic kidney disease has drawn much more attention recently,the methodology for establishing the in vivo and in vitro alcoholic renal fibrosis models are still lacking,and the underlying mechanisms are to be determined. METHODS and RESULTS Mice were feed with a liquid diet containing alcohol for 4 weeks, 8 weeks and 12 weeks respectively, results of Masson′s Trichrome staining showed that kidney fibrosis peaked in 8-week model group, which consistent with the results of albumin assay,Western blot,immunostaining and real-time PCR of collagen I and α-SMA.In vitro study also confirmed that ethanol upregulated the level of fibrotic index-es,including collagen I and α-SMA,in tubular epithelial cells(HK2 cells).Additionally,both in vivo and in vitro studies showed that Smad7 was decreased and Smad3 was highly activated. Then we further detected the underlying mechanisms by which alcohol induced the imbalance of Smad7 and Smad3. Results of Genome-wide methylation sequencing found DNA methylation of Smad7 in the alcoholic fibrosis kidney,which may be mainly mediated by DNA methyltransferase 1(DNMT1),because knock-down of DNMT1,but not DNMT2 and 3,largely restored Smad7 level in ethanol-treated HK2 cells.Con-sequently, we found that NADPH Oxidases (nox)-mediated oxidative stress is the major force upregu-lating DNMT1,since knockdown of Nox2 and 4 could both decrease DNMT1 while rebalancing Smad7/Smad3 axis, and thereby relieved ethanol-induced fibrotic response in HK2 cells. More importantly, intraperitoneal injection of apocynin,an inhibitor of NADPH oxidoreductase,attenuated renal fibrosis in alcoholic kidney fibrosis mouse model. CONCLUSION By establishing the novel in vivo and in vitro models,we found that through activating oxidative stress-induced DNA methylation of Smad7,alcohol induces renal fibrosis by breaking the balance between Smad7 and Smad3.Elimination of Nox-mediated oxidative stress may be a potential therapy for treatment of long-term alcohol abuse-induced kidney fibrosis.
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Objective To investigate the effect of Nourishing Yin and Nonifying Yang sequential therapy (NYNYST )com-bined with western medicine on expression levels of Smad2 ,Smad3 ,Smad7 mRNA in rats with diminished ovarian reserve (DOR).Methods Totally 40 female rats were randomly divided into 5 groups ,the normal control group ,the model group ,the western medicine group ,the NYNYST group and the combination group(western medicine combined with NYNYST ) ,8 in each group. The DOR model was established through orally administering tripterygium pill for continuous 2 weeks. The normal con-trol group and the model group were administered with saline for 10 days. The western medicine group was treated with hor-mone replacement therapy(HRT ) and ovarian stimulation. The NYNYST group was administered with Nourishing Yin herbs in proestrus and Nonifying Yang herbs in late estrus and the combination group was administered with combination of Chinese herb and western drugs for 10 days ,with the same dose in the former two groups.Serum levels of FSH ,LH ,E2 ,anti-Müllerian hormone(AMH) ,Transforming growth factor beta 1(TGF-β1)and Inhibin B(INHB)were measured by ELISA.Changes of Smad2 ,Smad3 ,Smad7 mRNA in ovaries were detected by real-time PCR.Results Compared with the model group ,the serum levels of FSH ,LH were decreased significantly in western medicine group ,NYNYST group and combination group(P＜0.01) , the serum levels of E2 ,AMH ,TGF-β1 and INHB increased in the rats of the treatment group(P＜0.05 ,P＜0.01) ,and efficacy of the combination group was significantly superior to that of the western medicine group (P＜0.01 ,P＜0.05);compared with the model group ,Smad2 mRNA increased significantly in NYNYST group and combination group ,Smad3 mRNA increased sig- nificantly in combination group(P＜0.01) ,the efficacy of combination group was superior to that of the western medicine group (P＜0.05);compared with the model group ,Smad7 mRNA of treatment groups was decreased significantly (P＜0.01).Conclu-sion NYTYST combined with western medicine can improve the function of ovaries by up-regulating the expression of Smad2 , Smad3 mRNA and down-regulating the expression of Smad7 mRNA in DOR rats.
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Objective To explore the effects and possible mechanism of histone deacetylase inhibitor SAHA on the interstitial fibrosis induced by diabetes.Methods The SD rats were divided into three groups:control group (Con,n=9),diabetes mellitus (DM) group (n=9) and SAHA treatment group (n=9).The diabetic rat model was established by injecting streptozotocin (STZ) through tail vein.After 8 weeks,the SAHA treatment group rats were fed with a SAHA solution (25 mg· kg-1 · d-1) by gastric gavage.After 16 weeks,all rats were sacrificed to detect relevant biochemical parameters,and observe the changes of pathomorphology in kidney.In addition,immunohistochemistry staining and Western blotting were employed to detect the protein expressions of transforming growth factor-β1 (TGF-β1),Smad2,Smad3,p-Smad2,p-Smad3,Smad7,collagen-Ⅰ and collagen-Ⅲ,respectively.Results Compared with Con group,the levels of blood glucose (BG),urinary trace albumin/urinary creatinine (ACR),triglyceride (TG) and total cholesterol (TC) in the diabetic group were all increased significantly (all P ＜ 0.05),the protein expressions of TGF-β1,p-Smad2,p-Smad3,collagen-Ⅰ and collagen-Ⅲ in kidney were all increased in diabetic group (all P ＜ 0.05),and the expression of Smad7 was significantly reduced (P ＜ 0.05).Compared with DM group,the levels of ACR was reduced,the renal fibrosis was alleviated,the protein expressions of TGF-β1,p-Smad2,p-Smad3,collagen-Ⅰ and collagen-Ⅲ in SAHA group were all decreased (all P ＜ 0.05),and the expression of Smad7 was increased significantly (P ＜ 0.05).Conclusion SAHA may restore the protein level of Smad7 by enhancing protein stability,then promote the moderate transduction of TGF-β1/Smads signaling pathway,which reduce the fibrosis of renal tubules in diabetic rats.
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AIM:To investigate the effect of rhein on bleomycin-induced pulmonary fibrosis and the expression of microRNA-21 (miR-21) and transforming growth factor-β1 (TGF-β1)/Smad signaling molecules in rats.METHODS:A single dose of bleomycin was intratracheal injected into the SD rats to induce pulmonary fibrosis .After injection of bleo-mycin, the rats were randomly divided into low-, medium-and high-dose rhein treatment groups and model group .The rats that were instilled with normal saline intratracheally served as control group .After the treatment for 28 d, the pulmonary pathologic changes were observed under microscope with hematoxylin-eosin staining .The lung coefficient and hydroxypro-line content were also measured .The expression of miR-21 and the mRNA levels of TGF-β1 and Smad7 in the lung tissues were detected by real-time PCR.The protein levels of TGF-β1 and Smad7 were determined by Western blot .RESULTS:Rhein significantly attenuated the experimental alveolitis , pulmonary fibrosis , lung coefficient and hydroxyproline contents in the rats.Rhein obviously decreased the expression of miR-21,and the mRNA and protein levels of TGF-β1, but signifi-cantly increased the mRNA and protein levels of Smad 7 in the lung tissues .CONCLUSION: Rhein effectively prevents bleomycin-induced pulmonary fibrosis by inhibiting the expression of miR-21 and promoting the expression of Smad 7, thus regulating the TGF/Smad signaling pathway to decrease extracellular matrix deposition .
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Objective To observe the effect of curcumin on the expression of smad7 and TGF-β1 in rats renal tubular cells of with Ang II, and discuss the mechanism of curcumin to improve renal interstitial fibrosis.Methods Cultured rat renal tubular epithelial cells were divided into the blank group, the Ang II control group and the low, medium and high dose curcumin group. The rest of the groups were intervened by 10-8 mol/L Ang II except the blank group; the low, medium and high dose groups of curcumin were intervened by 2.5, 5.0, 10.0μmol/L curcumin. Then Western blot was used to detect the expression of TGF-β1 and smad7 protein, RT-PCR was used to detect the TGF-β1 and smad7 mRNA expression.Results Compared with the blank group, the expression of TGF-β1 protein (0.23 ± 0.03vs. 0.16 ± 0.01), and TGF-β1 mRNA (1.89 ± 0.20vs. 1.00 ± 0.00) significantly increasedin AngⅡ control group (P<0.05), and the expression of smad7 protein (0.19 ± 0.03vs. 0.24 ± 0.02), and smad7 mRNA (0.48 ± 0.05vs. 1.00 ± 0.00) significantly reduced in AngⅡcontrol group (P<0.05). Compared with the AngⅡ control group, the expression of TGF-β1 protein in low, medium and high dose curcumingroup (0.18 ± 0.02, 0.17 ± 0.02, 0.16 ± 0.03vs. 0.23 ± 0.03) and TGF-β1 mRNA (1.58 ± 0.11, 1.34 ± 0.16, 0.97 ± 0.19vs. 1.89 ± 0.20) significantly decreased (P<0.05), and the expression of smad7 protein (0.28 ± 0.04, 0.31 ± 0.03, 0.34 ± 0.04vs. 0.19 ± 0.03) and smad7 mRNA (0.68 ± 0.07, 0.80 ± 0.06, 0.98 ± 0.09vs.0.48 ± 0.05) increased significantly (P<0.05).Conclusions Curcumin can thus play its role in renal protection by counteract the AngⅡ mediated renal interstitial fibrosis. Its mechanism may be related to the reduction of TGF-β1 protein and its mRNA expression, up regulation of smad7 protein and its mRNA expression.
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Objective:To investigate the role of Smad7 in the Hepatocellular carcinoma (HCC) migration and proliferation and its clinical significance.Methods:Through transfecting pcDNA3.1 (+)-Smad7 or siRNA Smad7 to overexpress or knockdown the Smad7 expression in HCC cell lines HepG2 and Huh7.The MTT assays were used to test the role of Smad7 in proliferation of HCC cells.Transwell and wound-healing assays were used to detect the effect of Smad7 on migratory ability in both tow cell lines.RT-PCR was used to test the Smad7 expression in 9 clinical HCC patients' specimens.Results:As the results,overexpression of Smad7 significantly inhibited the proliferation of cells compared with the control group,while knockdown Smad7 promoted the proliferation.At the same time,overexpression of Smad7 could inhibit the migratory ability of HCC cells compared with the control group,while knockdown smad7 could accelerate this ability.The expression of Smad7 in cancer tissue was significantly lower compared with normal mucosa tissue adjacent to cancer.Conclusions:Smad7 is a kind of anti-progressive molecule in HCC.
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Many cancer researchers use gene expression analysis for differentiation between tumor and normal cells for diagnostic, prognostic and therapeutic purposes. Most of studies compare either tumor cell lines by normal cell lines or tumor tissue of affected individuals by normal healthy control tissue. But expression of each special gene is unique in different individuals and also in different tissue of same individual. For this reason, here we compare the gene expression levels of SMAD7 and KLF10 in tumor cells and its adjacent normal tissue of breast cancer patients and compared them. For this purpose, a total of 40 tumor and matched tumor-free margin samples were obtained during surgery. The SMAD7 and KLF10 mRNA expression levels in tumor and marginal samples were examined by real-time quantitative PCR. Results are not concordant with previous studies and comparison of only SMAD7 or KLF10 is not useful for differentiating between tumor and margin cells, but ratio analysis of these two genes, SMAD7/ KLF10, can be indicative than study of one gene alone. We concluded that gene expression analysis of tumor cells with adjacent normal tissue are essential for precise identification and interpretation of cancer alterations and have important implications for the diagnostic and therapeutic management of cancer patients.
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Objective:To investigate the mechanism of Smad7 gene modified bone marrow mesenchymal stem cells ( Smad7-BMSCs) to prevent hepatic fibrosis in vitro. Methods:Smad7-EGFP-BMSCs were established by isolating and purifying BMSCs of rats, and transfecting Ad-Smad7-EGFP. HSC-T6 were divided into Group A, Group B, Group C and Group D, which were respectively incubated with Smad7-EGFP-BMSCs, BMSCs, Smad7 plasmid and PBS for 72 hours. The level of Smad7and TGF-β1protein in the culture solution was determined by ELISA. The expression of mRNA and protein of Smad7,TGF-β1,Col Ⅰ and α-SMA in the hepatic stellate cells were respectively determined by Western blot and RT-PCR. Cellular apoptosis was determined by flow cytometry. Results:(1)The results of ELISA showed that the level of TGF-β1 protein decreased(P<0. 01) but the level of Smad7 protein increased (P<0. 01) in Group B,Group C and Group D compared with Group A;the level of TGF-β1 protein decreased(P<0. 01) but the level of Smad7 protein increased (P<0. 01) in Group D compared with Group B and Group C. (2)The results of Western blot and RT-PCR showed that the level of mRNA and protein of Smad7,TGF-β1,Col Ⅰ and α-SMA decreased(P<0. 01) but the level of mRNA and protein of Smad7 protein increased (P<0. 01) in Group B,Group C and Group D compared with Group A;the level of mRNA and protein of Smad7,TGF-β1,ColⅠandα-SMA decreased(P<0. 01) but the level of mRNA and protein of Smad7 protein increased (P<0. 01) in Group D compared with Group B and Group C. (3)The results of flow cytometry showed that the rate of cellular apoptosis de-creased(P<0. 01),but the level of Smad7 protein increased (P<0. 01) in Group B,Group C and Group D compared with Group A;the rate of cellular apoptosis decreased(P<0. 01)in Group D compared with Group B and Group C. Conclusion:Smad7-BMSCs can have the effect of anti-hepatic fibrosis by affecting TGF-β1 signal pathway and promoting cellular apoptosis in hepatic stellate cells.
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Transforming growth factor (TGF)-β signaling plays an important role in the pathogenesis of psoriasis. CD109, a novel TGF-β co-receptor, which inhibits TGF-β signaling by enhancing Smad7-dependent degradation of TGF-β type I receptor (TGF-β RI), is abnormally expressed in psoriasis. To date, the expression of Smad7 and the correlation between CD109 and Smad7 expression in psoriasis have not been fully elucidated. This study was designed to investigate the expression and the correlation of CD109 and TGF-β signaling associated proteins in psoriasis and their roles in the pathogenesis of psoriasis. Thirty-two psoriasis specimens were subjected to immunohistochemical staining for CD109, Smad7, TGF-β RI and Ki67. Ten normal skin (NS) specimens served as controls. The positive expression rate (% positive cells) of Smad7 and Ki67 in psoriasis was significantly higher than in NS (62.6%±19.9% vs. 17.2%±4.4%, and 50.7%±14.3% vs. 19.5%±3.2%, respectively, P<0.001), and the expression levels of CD109 and TGF-β RI were reduced significantly in psoriasis as compared with NS (8.1%±6.7% vs. 35.8%±6.7% and 27.3%±3.4% vs. 3.0%±3.4%, respectively, P<0.001). There were significantly negative correlations between CD109 and Smad7 (r=-0.831, P<0.01). These findings indicated that CD109 might play a certain role in the pathogenesis of psoriasis. Lower expression of CD109 and TGF-β RI was highly correlated with higher expression of Smad7 and Ki67, suggesting that CD109 may induce the pathogenesis of psoriasis through Smad7-mediated degradation of TGF-β RI, and lead to the termination of TGF-β signaling.
Subject(s)
Adolescent , Adult , Female , Humans , Male , Middle Aged , Antigens, CD , Genetics , Metabolism , Case-Control Studies , Down-Regulation , GPI-Linked Proteins , Genetics , Metabolism , Neoplasm Proteins , Genetics , Metabolism , Psoriasis , Metabolism , Pathology , Signal Transduction , Smad7 Protein , Genetics , Metabolism , Transforming Growth Factor beta , Metabolism , Up-RegulationABSTRACT
BACKGROUND/AIMS: Transforming growth factor-beta (TGF-β) is a cytokine implicated in the susceptibility, development, and progression of gastrointestinal cancer and certain other neoplasms. In the later stages of cancer, TGF-β not only acts as a bystander of host-immune response, but also contributes to cell growth, invasion, and metastasis. In the current study, we generated gastric mucosal cells that stably express Smad7, and explored the Helicobacter pylori-associated biological changes between mock-transfected and Smad7-transfected RGM1 cells. METHODS: RGM1 cells stably transfected with Smad7 were infected with H. pylori, and molecular changes in apoptotic markers and inflammatory mediators were examined. Several candidate genes were explored in Smad7-overexpressing cells after H. pylori infection. RESULTS: Overexpression of Smad7 in RGM1 cells significantly increased the H. pylori-induced cytotoxicity compared to mock-transfected cells. Exaggerated increases in inflammatory mediators, cyclooxygenase 2, inducible NO synthase, and augmented apoptosis were noted in Smad7-overexpressing cells, whereas mitigated heme oxygenase 1 was noted in Smad7- overexpressing cells. These phenomena were reversed in cells transfected with Smad7 siRNA. CONCLUSIONS: These data suggest that inhibition of Smad7 is a possible target for mitigating H. pylori-associated inflammation.
Subject(s)
Apoptosis , Cyclooxygenase 2 , Gastritis , Gastrointestinal Neoplasms , Helicobacter pylori , Helicobacter , Heme Oxygenase-1 , Inflammation , Neoplasm Metastasis , Nitric Oxide Synthase , RNA, Small Interfering , Transforming Growth Factor betaABSTRACT
The endothelial-mesenchymal transition (EndMT) is known to be involved in the transformation of vascular endothelial cells to mesenchymal cells. EndMT has been confirmed that occur in various pathologic conditions. Transforming growth factor β1 (TGF-β1) is a potent stimulator of the vascular endothelial to mesenchymal transition (EMT). Aspirin-triggered resolvin D1 (ATRvD1) has been known to be involved in the resolution of inflammation, but whether it has effects on TGF-β1-induced EndMT is not yet clear. Therefore, we investigated the effects of AT-RvD1 on the EndMT of human umbilical vein vascular endothelial cells line (HUVECs). Treatment with TGF-β1 reduced the expression of Nrf2 and enhanced the level of F-actin, which is associated with paracellular permeability. The expression of endothelial marker VE-cadherin in HUVEC cells was reduced, and the expression of mesenchymal marker vimentin was enhanced. AT-RvD1 restored the expression of Nrf2 and vimentin and enhanced the expression of VE-cadherin. AT-RvD1 did also affect the migration of HUVEC cells. Inhibitory κB kinase 16 (IKK 16), which is known to inhibit the NF-κB pathway, had an ability to increase the expression of Nrf2 and was associated with the inhibition effect of AT-RvD1 on TGF-β1-induced EndMT, but it had no effect on TGF-β1-induced EndMT alone. Smad7, which is a key regulator of TGF-β/Smads signaling by negative feedback loops, was significantly increased with the treatment of AT-RvD1. These results suggest the possibility that AT-RvD1 suppresses the TGF-β1-induced EndMT through increasing the expression of Smad7 and is closely related to oxidative stress.
Subject(s)
Humans , Actins , Endothelial Cells , Human Umbilical Vein Endothelial Cells , Inflammation , Oxidative Stress , Permeability , Phosphotransferases , Transforming Growth Factors , Umbilical Veins , VimentinABSTRACT
Objective Human bone marrow mesenchymal stem cells were transfected by Smad7 and labeled with green fluores-cent mark.BMMSCs were implanted into rabbit glaucoma operational model and observed surviving condition.Methods Through BP and LR reaction,Smad7 with green fluorescent mark was inserted into human bone marrow mesenchymal stem cells by bacterio-phage,filtered positive colony and picked out cell line.15 New Zealand white rabbits were enforced trabeculectomy with BMMSC, then following up cell survival condition.Results Smad7 expressed stable in human bone marrow mesenchymal stem cells with sat-isfactory green fluorescent mark.BMMSC survived in rabbit trabecula with stable green fluorescent and effective ocular press.Con-clusion Smad7 with green fluorescent mark could be inserted into human bone marrow mesenchymal stem cells stably,and has ef-fective results in rabbit model.
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Objective:To investigate the inhibitory effects of Sedum Sarmentosum total flavonoids ( SSTF) on hepatic fibrosis in-duced by CCl4 ,and examine the effects on the expression of TGF-β1 and Smad7. Methods:Sixty male SD rats were randomly divided into the normal control group, the model group, 100, 200 and 400 mg·kg-1 SSTF groups and colchicine positive control group. The experimental model of hepatic fibrosis in rats was established by the injection of CCL4 . The liver histopathology was examined by Mas-son stain, and the protein expression and mRNA of TGF-β 1 and Smad7 were assessed by RT-PCR and Western blotting. Results:Compared with the model group, every SSTF group could significantly reduce the degree of liver fibrosis induced by CCL4(P<0. 05). The middle and high dose SSTF gouprs could significantly reduce the protein expression and mRNA of TGF-β1 (P<0. 05), and sig-nificantly increase the protein expression and mRNA of Smad 7 (P<0. 05). Conclusion:SSTF exhibits anti-hepatic fibrosis effects in rats through down-regulating the expression of TGF-β1 and up-regulating the expression of Smad7 in fibrotic liver tissue.
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Objective To explore miR-21 regulation of Smad7 in lung cancer A549 cell line as well as its impact on the proliferation of lung cancer A549 cell line. Methods miR-21 mimic and inhibitors in lung cancer A549 cell line were transfected by using Lipofectamine 2000 Reagent. After 48 h, Western blot and qRT-PCR were applied to assess the expression of protein and mRNA of Smad7 . MTT assay was used to determine the proliferation influence of the transfected lung cancer A549 cell line. Results Western blot and qRT-PCR showed that A549 cell trans-fected miR-21 mimic exhibited down-regulated Smad7 protein and mRNA expression, and A549 cell transfected miR-21 inhibitor exhibited up-regulated Smad7 protein and mRNA expression. The A549 cell proliferation activity decreased significantly after transfected miR-21 inhibitors. Conclusion miR-21 inhibitors can increase Smad7 pro-tein and mRNA expression, and suppress the proliferation activity of lung cancer A549 cell significantly.