Objective To investigate the effect of SMAD family member 3(SMAD3) silenced by small interfering RNA (siRNA) on macrophage polarization and transforming growth factor β1 (TGF-β1)/ SMAD family signaling pathway in rheumatoid arthritis (RA). Methods RA macrophages co-cultured with rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) were used as a cell model. TGF-β1 was used to stimulate macrophages, and SMAD3-specific siRNA (si-SMAD3) and negative control siRNA (si-NC) were transfected into human RA macrophages co-cultured in Transwell^TM chamber. The expression of SMAD3 mRNA was detected by real-time fluorescence quantitative PCR, and the expression of TGF-β1, SMAD3 and SMAD7 protein was detected by Western blot analysis. The contents of TGF-β1 and IL-23 in cell culture supernatant were determined by ELISA. Cell proliferation was detected by CCK-8 assay. Transwell^TM chamber was used to measure cell migration. Results Compared with the model group and the si-NC group, the expression of TGF-β1, SMAD3 mRNA and protein in RA macrophages decreased significantly after silencing SMAD3. In addition, the secretion of IL-23 decreased significantly, and the cell proliferation activity and cell migration were inhibited, with high expression of SMAD7. Conclusion Knockdown of SMAD3 can promote M2 polarization and SMAD7 expression in RA macrophages.
Humans ; Arthritis, Rheumatoid/genetics* ; Interleukin-23 ; Macrophages ; RNA, Messenger ; RNA, Small Interfering/genetics* ; Smad7 Protein/genetics* ; Transforming Growth Factor beta1/genetics* ; Smad3 Protein/genetics* ; Gene Silencing

OBJECTIVETo investigate the relationship between expression of Smad3, Smad7 and ventricular remodeling in rats after myocardial infarction.

METHODSMyocardial infarction was induced by left anterior descending coronary artery ligation in rats (n = 11) and sham-operated rats were used as control (n = 10). The rats were sacrificed 8 weeks later. Heart weight/body weight (HW/BW), mean blood pressure, left ventricular end diastolic pressure (LVEDP), collagen content in the un-infarcted area were examined. The mRNA levels of transforming growth factor (TGF)beta(1), Smad 3, Smad7 were determined by RT-PCR.

RESULTCompared with controls, the level of HW/BW, LVEDP and collagen content were significant increased. The mRNA expression of TGFbeta(1) and Smad3 was significantly increased in areas of myocardial infarction, border of the infarction, interventricular septum and right ventricle. The expression of Smad7 mRNA in these areas was decreased.

CONCLUSIONThese results indicated that TGFbeta(1)-Smads signaling was correlated to the ventricular remodeling after myocardial infarction. Smad3 might promote the process while Smad7 inhibit the process.

Animals ; Male ; Myocardial Infarction ; metabolism ; physiopathology ; RNA, Messenger ; metabolism ; Rats ; Rats, Wistar ; Smad3 Protein ; genetics ; metabolism ; Smad7 Protein ; genetics ; metabolism ; Transforming Growth Factor beta ; metabolism ; Ventricular Remodeling

Objective: To investigate the clinicopathological features, immunophenotypes and molecular genetics of EWSR1-SMAD3 positive fibroblastic tumor (ESFT) with an emphasis on differential diagnosis. Methods: The clinicopathological data, immunohistochemical profiles and molecular profiles of 3 ESFT cases diagnosed at the Department of Pathology, Fudan University Shanghai Cancer Center from 2018 to 2021were analyzed. The related literature was also reviewed. Results: There were two males and one female. The patients were 24, 12 and 36 years old, respectively. All three tumors occurred in the subcutis of the foot with the disease duration of 6 months to 2 years. The tumors were presented with a slowly growing mass or nodule, accompanied with pain in 1 patient. The tumors ranged in size from 0.1 to 1.6 cm (mean, 1.0 cm). Microscopically, the tumors were located in the subcutaneous tissue with a nodular or plexiform growth pattern. They were composed of cellular fascicles of bland spindle cells with elongated nuclei and fine chromatin. One of the tumors infiltrated into adjacent adipose tissue. There was no nuclear atypia or mitotic activities. All three tumors showed prominent stromal hyalinization with zonal pattern present in one case. Focal punctate calcification was noted in two cases. The immunohistochemical studies showed that tumor cells were diffusely positive for ERG and negative for CD31 and CD34, with Ki-67 index less than 2%. Fluorescence in situ hybridization on the two tested cases identified EWSR1 gene rearrangement. The next generation sequencing analysis demonstrated EWSR1-SMAD3 fusion in all three cases. During the follow up, one patient developed local recurrence 24 months after the surgery. Conclusions: ESFT is a benign fibroblastic neoplasm and has a predilection for the foot, characterized by ERG immunoreactivity and EWSR1-SMAD3 fusion. Local recurrence might occur when incompletely excised. Familiarity with its clinicopathological features is helpful in distinguishing it from other spindle cell neoplasms that tend to occur at acral sites.
Adult ; Child ; Female ; Humans ; Male ; Biomarkers, Tumor/analysis* ; China ; In Situ Hybridization, Fluorescence ; Neoplasms, Fibrous Tissue/pathology* ; RNA-Binding Protein EWS/genetics* ; Smad3 Protein/genetics* ; Soft Tissue Neoplasms/surgery*

OBJECTIVE: To construct the expressing vector of siRNA which can inhibit the Smad3 activity. METHODS: Sixty-four bases of 2 pair oligos for hairp in RNA expression which targeted Smad3 gene were chemically synthesized and annealed. pSUPER vector was linearized with BgL II and Hin d III treated with alkaline phosphatase (CIP). Anneled oligos were inserted into the downstream of the treated pSUPER's pol III H1 promoter to construct RNAi plasmid (pSUPER Smad3). Oligos with a scrambled sequence were used as a negative control. pSUPER Smad3 was transfected into human renal tubular epithelial cells (HKC). RESULTS: Recombinant pSUPER Smad3 vector was identified by the digestion with Eco R I and Hin d III, and confirmed by the sequencing analysis with T3 primer. Sixty-four bases had been inserted into the expected site. Furthermore, the insertion sequence was exactly corrected. The activity evaluation indicated that mRNA and protein of Smad3 but not Smad2 were inhibited by pSUPER Smad3 in HKC. CONCLUSION The pSUPER Smad3 system has been constructed successfully, and has high inhibition and specificity in vitro.
Epithelial Cells ; metabolism ; Humans ; Kidney Tubules ; cytology ; Plasmids ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; Smad3 Protein ; antagonists & inhibitors ; genetics ; Transfection

OBJECTIVETo study the role of Smad3 in transforming growth factor-beta1 (TGF-beta1)-induced bi-directional effects on skin fibroblast proliferation.

METHODSThe Smad3 small interfering (siRNA) plasmid was constructed using a pSUPER vector. The efficiency of cell transfection was detected by fluorescence microscopy, and the inhibitory effect of the plasmid was assessed by real-time quantitative RT-PCR and immunohistochemistry. The effect of the plasmid on the fibroblast proliferation and Smad3 binding activity was analyzed by BRDU ELISA and EMSA, respectively.

RESULTSThe transfection efficiency of the plasmid into the cells was 41.2%. The Smad3 siRNA plasmid produced efficient and specific inhibition of the expression of Smad3, and promoted the cell proliferation in a dose-dependent manner and abrogated the bi-directional effect of TGF-beta1 on the cell proliferation and Smad3 binding activity.

CONCLUSIONThe siRNA targeting Smad3 gene can inhibit the protein expression and RNA transcription of Smad3, and TGF-beta1 exerts bi-directional regulation on fibroblast proliferation by modulating Smad3 activity.

Animals ; Cell Proliferation ; Cells, Cultured ; Fibroblasts ; cytology ; RNA Interference ; RNA, Small Interfering ; genetics ; Rats ; Rats, Wistar ; Skin ; cytology ; Smad3 Protein ; biosynthesis ; genetics ; Transfection ; Transforming Growth Factor beta1 ; pharmacology

Humans ; Nerve Tissue Proteins ; Epithelial-Mesenchymal Transition/genetics* ; Constriction, Pathologic ; Receptors, Immunologic ; Epithelial Cells/metabolism* ; Smad3 Protein/metabolism* ; Transforming Growth Factor beta1/metabolism*

OBJECTIVETo investigate AXIN1-related CSRNP1 gene expression and the mechanism of its transcriptional regulation in TGF-β1-induced tumor cells.

METHODSHuman lung carcinoma A549 cells or human prostate cancer PC3 cells were treated with TGF-β1 at different doses (0, 20, 40, and 80 ng/ml) or at 20 ng/ml for 0, 8, 12, or 24 h, and the dose and time effect of TGF-β1 on CSRNP1 mRNA expression in the tumor cells were evaluated with real-time RT-PCR. A549 cells were also treated with TGF-β1 and cycloheximide to clarify whether CSRNP1 expression induced by TGF-β1 required de novo protein synthesis. A549 cells transfected with pcDNA3.1, flag-SMAD3, or flag-SMAD3-mu, after serum starvation, were treated with or without TGF-β1 (20 ng/mL) for 24 h, and the overexpression of wild-type SMAD3 and dominant negative SMAD3-mu mutant were confirmed by Western blotting. The effect of SMAD3 or SMAD3-mu overexpression on CSRNP1 mRNA expression was also measured by real-time RT-PCR.

RESULTSIn both A549 and PC3 cells, TGF-β1 dose- and time-dependently stimulated CSRNP1 expression, which required de novo protein synthesis in A549 cells. Overexpression of wild-type SMAD3 significantly increased the expression of CSRNP1 mRNA induced by TGF-β1, while overexpression of dominant negative SMAD3 mutant remarkably reduced CSRNP1 mRNA expression in response to TGF-β1 in A549 cells.

CONCLUSIONTGF-β1 may contribute to CSRNP1 expression through SMAD3 activation and downstream signaling in tumor cells.

Apoptosis Regulatory Proteins ; genetics ; metabolism ; Axin Protein ; genetics ; metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Humans ; RNA, Messenger ; genetics ; Signal Transduction ; Smad3 Protein ; genetics ; metabolism ; Transfection ; Transforming Growth Factor beta1 ; pharmacology

OBJECTIVEIn order to explore the roles of Huoxueruanjian compound on liver fibrogenesis and its molecular mechanism, this paper has investigated the Influence of blood serum with such traditional Chinese medicine compound on the expression of Smad3, Smad7 and procollagen alpha2(I) gene in hepatic stellate cell (HSC).

METHODSHSC-T6 was deal with different Concentration of blood serum medicine with Heluoshugan which was made by routine way. Then expression change of Smad3, Smad7 and procollagen alpha2(I) mRNA among each groups were observed by RT-PCR. Furthermore, the expression change of Smad3 protein were examined by Western blot.

RESULTSExpression of Smad3 and procollagen alpha2(I) mRNA as well as Smad3 protein had been downregulated after treating with blood serum medicine of Heluoshugan (P<0.01, P<0.05, respectively). The expression of procollagen alpha2(I) mRNA changed at the same tendency as those of Smad3. The role of blood serum medicine was significant difference between different concentration, P<0.05. And the expression of procollagen alpha2(I) mRNA changed in concentration-dependent manner. Blood serum medicine has no effects on the Smad7 mRNA.

CONCLUSIONThe anti-fibrosis roles of HuoXueruanjian Compound maybe influence the function of TGF-beta and Smad by nonspecific action, thereby inhibit the transcription of procollegan alpha2(I) mRNA and decrease the production of ECM. As regards Smad3, it may be facilitating the development of liver fibrosis when its expression increases. Otherwise, it manifest with anti-fibrosis role.

Animals ; Collagen Type I ; genetics ; DNA-Binding Proteins ; genetics ; Liver Cirrhosis ; drug therapy ; etiology ; pathology ; Medicine, Chinese Traditional ; RNA, Messenger ; analysis ; Rats ; Smad3 Protein ; Smad7 Protein ; Trans-Activators ; genetics

OBJECTIVETo explore the regulatory mechanisms of transforming growth factor-beta1 (TGF-beta1) and two transcriptional factors Smad 2, 3 on hypertrophic scar formation and fetal scarless healing.

METHODSThirty-two cases were detected to compare the gene expression of TGF-beta1, Smad 2 and Smad 3 with RT-PCR. Among those cases, there were 8 cases of hypertrophic scars, 8 cases of control skins, 8 cases of fetal skins and 8 cases of adult skins.

RESULTSTGF-beta1, Smad 2 and Smad 3 gene expression could all be detected in hypertrophic scars, fetal and adult skins. Among 8 groups examinated in this experiment (each group comprised a hypertrophic scar and its corresponding normal skin), there were 5, 8 and 5 groups in which TGF-beta1, Smad 2 and Smad 3 gene expression were higher in hypertrophic scars than in normal skins respectively. The fetal skins showed significantly lower level of TGF-beta1 and Smad 3 gene expression compared with adult skins (t = 2.204, P < 0.05 and t = 4.269, P < 0.01 respectively), while mRNA contents of Smad 2 were obviously higher in fetal skins than in adult skins (t = 6.685, P < 0.01).

CONCLUSIONTGF-beta1 and its downstream signal molecules Smad 2, Smad 3 might be involved in hypertrophic scar formation. Higher gene expression of TGF-beta1, Smad 2 and Smad 3 in hypertrophic scars might lead to stimulating extracellular matrix deposition, inducing fibroblast proliferation and accelerating fibrogenesis. Lower mRNA contents of TGF-beta1 and Smad 3 in fetal skins compared with adult skins might be associated with fetal scarless healing.

Cicatrix, Hypertrophic ; metabolism ; DNA-Binding Proteins ; genetics ; Gene Expression Regulation ; Reverse Transcriptase Polymerase Chain Reaction ; Skin ; metabolism ; Smad2 Protein ; Smad3 Protein ; Trans-Activators ; genetics ; Transforming Growth Factor beta ; genetics ; Transforming Growth Factor beta1

To explore the influence for combination of nourishing yin and tonifying yang sequential therapy (NYTYST) with Western medicine in treating anovulatory infertility rats with diminished ovarian reserve (DOR) based on TGF-β1/Smads signaling pathway. 
 Methods: A total of 40 female rats were randomly divided into 5 groups, a normal control group, a model group, a Western medicine group, a NYTYST group and a combination group (n=8 in each group). The DOR model was established through orally taking tripterygium pill for continuous 2 weeks. The normal control group and the model group were treated with saline for 10 days. The Western medicine group was treated with hormone replacement therapy (HRT) and ovarian stimulation. The NYTYST group was treated with nourishing yin herbs in proestrus and tonifying yang herbs in late estrus and the combination group was treated with Chinese herb and Western drugs for 10 days. HE staining was used to observe histopathologic changes in ovary. Expression levels of transforming growth factor β1 receptor (TGF-β1R) in rats ovarian were detected by immunohistochemistry. Expression levels of Smad2, Smad3 and Smad7 protein in rat ovarian were detected by Western blot.
 Results: Compared with the control group, the numbers of developing follicles, mature follicles and corpus luteum were decreased , while atrefic follicles were increased significantly in the model group (P<0.01); the levels of TGF-β1R, Smad2 and Smad3 were decreased significantly, while Smad7 was increased significantly (P<0.01). Compared with the model group, the numbers of developing follicles, mature follicles and corpus luteum, Smad2 and Smad3 expression were increased, while atrefic follicles and Smad7 were decreased significantly in the treatment group (P<0.05 or P<0.01). The numbers of developing follicles and corpus luteum in the combination group was superior to the Western medicine group (P<0.05). Compared with the Western medicine group, the levels of TGF-β1R, Smad2 and Smad3 were increased significantly, while Smad7 was decreased significantly in the combination group (P<0.05 or P<0.01). 
 Conclusion: NYTYST combined with Western medicine can improve the function of ovaries reserve by up-regulation of TGF-β1R, Smad2 and Smad3 while down-regulation of Smad7 in DOR rats.
Animals ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Gene Expression Regulation ; drug effects ; Infertility ; therapy ; Medicine, Chinese Traditional ; Ovarian Reserve ; drug effects ; Rats ; Signal Transduction ; drug effects ; Smad2 Protein ; genetics ; metabolism ; Smad3 Protein ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism