Smad7 mRNA Expression Research Articles

Transcription factor Sp2 promotes TGFB-mediated interstitial cell osteogenic differentiation in bicuspid aortic valves through a SMAD-dependent pathway

Calcification of the bicuspid aortic valve (BAV) involves differential expression of various RNA genes, which is achieved through complex regulatory networks that are controlled in part by transcription factors and microRNAs. We previously found that miR-195–5p regulates the osteogenic differentiation of valvular interstitial cells (VICs) by targeting the TGF-β pathway. However, the transcriptional regulation of miR-195–5p in calcified BAV patients is not yet clear. In this study, stenotic aortic valve tissues from patients with BAVs and tricuspid aortic valves (TAVs) were collected. Candidate transcription factors of miR-195–5p were predicted by bioinformatics analysis and tested in diseased valves and in male porcine VICs. SP2 gene expression and the corresponding protein levels in BAV were significantly lower than those in TAV, and a low SP2 expression level environment in VICs resulted in remarkable increases in RNA expression levels of RUNX2, BMP2, collagen 1, MMP2, and MMP9 and the corresponding proteins. ChIP assays revealed that SP2 directly bound to the transcription promoter region of miR-195–5p. Cotransfection of SP2 shRNA and a miR-195–5p mimic in porcine VICs demonstrated that SP2 repressed SMAD7 expression via miR-195–5p, while knockdown of SP2 increased the mRNA expression of SMAD7 and the corresponding protein and attenuated Smad 2/3 expression. Immunofluorescence staining of diseased valves confirmed that the functional proteins of osteogenesis differentiation, including RUNX2, BMP2, collagen 1, and osteocalcin, were overexpressed in BAVs. In Conclusion, the transcription factor Sp2 is expressed at low levels in VICs from BAV patients, which has a negative impact on miR-195–5p expression by binding its promoter region and partially promotes calcification through a SMAD-dependent pathway.

1β-Hydroxyalantolactone from Inulae Flos alleviated the progression of pulmonary fibrosis via inhibiting JNK/FOXO1/NF-κB pathway

Inulae Flos was widely distributed throughout Europe, Africa, and Asia, and was commonly used as a folk medicine in clinic for treating various respiratory diseases, including cough, asthma, bronchitis, pulmonary fibrosis, and pneumonia. However, the ingredients responsible for the pharmacology effects of I. Flos and the underlying mechanisms remain unclear. In this study, the effects of 16 known sesquiterpene lactones and flavonoids from I. Flos on TGF-β1-induced fibroblast activation were assessed by phenotypic high-content screening. Among those sixteen compounds, 1β-hydroxy alantolactone (HAL), the main characteristic sesquiterpene lactone from I. Flos, exhibited remarkable inhibitory activity. The further studies showed that HAL significantly inhibited the proliferation and induced the apoptosis of human fibroblast cell lines HELF and MRC-5 in a concentration-dependent manner. It also reduced intracellular ROS production, suppressed the mRNA expressions of E-cad, TGF-β1, Smad3, Col I, α-SMA and TNF-α, and downregulated protein expressions of α-SMA and F-actin. Furthermore, HAL significantly reduced the levels of HA, LN, PC-III and IV-C in serum, TNF-α and IL-6 in BALF, and TGF-β1, HYP and Col I in lung tissues of bleomycin (BLM)-treated rats. HAL significantly downregulated the expressions of p-JNK, FOXO1, p-p65, α-SMA, p-smad3 and Col I but upregulated p-FOXO1, which could be reversed by JNK agonist anisomycin. These results demonstrated that HAL induced the apoptosis of lung fibroblast cells activated by TGF-β1 and improved BLM-induced lung fibrosis in rats via inhibiting JNK/FOXO1/NF-κB pathway.

Pirfenidone Attenuates Renal Tubulointerstitial Fibrosis through Inhibiting miR-21

Background: Our previous studies had shown pirfenidone (PFD) not only improved tubulointerstitial fibrosis (TIF) but also inhibited the expression of microRNA-21 (miR-21) in the renal tissue of unilateral urethral obstruction (UUO) rats. This study aims to investigate whether PFD can attenuate TIF through inhibiting miR-21 in UUO rats. Methods: Sprague Dawley rats were divided randomly into sham-operated group, UUO group, and PFD and olmesartan (Olm) treatment groups. Samples were collected on day 14. Expression of miR-21, TGF-β1, Smad3, and Smad7 mRNA in the renal tissue was detected using real-time quantitative PCR. Immunohistochemistry was performed to assess the protein expressions of collagen III, E-cadherin, and α-SMA. Automated capillary Western blotting was used to detect the quantitative expression of TGF-β1, Smad3, p-Smad3, Smad7, collagen III, E-cadherin, and α-SMA in renal tissues. The expression of miR-21 and Smad7 mRNA and the protein levels of collagen III and α-SMA were examined in the miR-21-overexpressing cell line, NRK-52E. Results: Compared with the UUO group, both PFD and Olm inhibited renal tubular dilation, diffused epithelial cell degeneration and necrosis, and reduced renal interstitial edema, inflammatory cell infiltration, and collagen fiber deposition, while no significant difference between PFD group and Olm group. Informatics-based approaches identified Smad7 as a likely candidate for regulation by miR-21. Compared with the sham group, miR-21 expression was upregulated in the UUO group resulting in the downregulation of Smad7 expression due to degradation. The overexpression of miR-21 in the in vitro model downregulated Smad7 and promoted EMT and ECM accumulation. Protein levels of TGF-β1, Smad3, p-Smad3, collagen III, and α-SMA were upregulated, while E-cadherin protein was downregulated in the UUO group than in the sham group. PFD rather than Olm decreased the expression of miR-21 and increased the expression level of Smad7 mRNA and then inhibited the TGF-β1/Smad3 signaling pathway. Olm only downregulated the TGF-β1/Smad3 signaling pathway. Conclusions: PFD improves TIF by downregulating the expression of miR-21, then elevating Smad7, and finally inhibiting the activation of the TGF-β1/Smad3 signaling pathway in UUO rats.

Autocross-linked hyaluronic acid gel and adipose-derived mesenchymal stem cell composites for the treatment intrauterine adhesions

ObjectiveTo evaluate the effect of adding adipose-derived mesenchymal stem cells (ASCs) to autocross-linked hyaluronic acid (HA) gel for intrauterine adhesion (IUA) treatment. MethodsA rat IUA model was established by mechanical curettage and infection, and then different treatments were administered to the rats on day 7 after modeling. Ninety-six rats were randomly divided into the following groups: IUA model group, gel therapy group, and combination therapy group (HA gel combined with ASCs). Eight rats per group were sacrificed on days 7, 10, 14 and 21 for the subsequent experiments. Morphological changes were determined by hematoxylin-eosin staining and Masson staining. Smad3 and leukocyte inhibitory factor (LIF) were analyzed by quantitative real-time polymerase chain reaction and immunohistochemistry. ResultsThe endometrial lines in the gel therapy group and the combination therapy group were more complete than those in the model group. Masson staining showed that fibrosis area rates in the gel therapy group and the combination therapy group were significantly lower than those in the model group on day 7(P < 0.05). During the observation period, the fibrosis area rates in the combination therapy group remained lower than those in the model group (P < 0.05). The mRNA expression of Smad3 in the combination therapy group was lower than that in the model group and gel therapy group during the observation period (P < 0.05). The mRNA expression level of LIF in the combination therapy group was higher than that in the model group and the gel therapy group throughout the observation period (P < 0.05). ConclusionsHA gel was effective in preventing the IUA adhesion formation at the early stage of the observation period, while ASC enhanced this effect throughout the observation period. Gel and ASC composites helped to improve endometrial receptivity.

Cold exposure aggravates pulmonary arterial hypertension through increased miR-146a-5p, miR-155-5p and cytokines TNF-α, IL-1β, and IL-6

BackgroundCold temperatures can aggravate pulmonary diseases and promote pulmonary arterial hypertension (PAH); however, the underlying mechanism has not been fully explored. AimTo explore the effect of chronic cold exposure on the production of inflammatory cytokines and microRNAs (miRNAs) in a monocrotaline (MCT)-induced PAH model. MethodsMale Sprague Dawley rats were divided into a Control (23.5 ± 2 °C), Cold (5.0 ± 1 °C for ten days), MCT (60 mg/kg body weight i.p.), and MCT + Cold (ten days of cold exposure after 3 weeks of MCT injection). Hemodynamic parameters, right ventricle (RV) hypertrophy, and pulmonary arterial medial wall thickness were determined. IL-1β, IL-6, and TNF-α levels were determined using western blotting. miR-21-5p and -3p, miR-146a-5p and -3p, and miR-155-5p and -3p and plasma extracellular vesicles (EVs) and mRNA expression of Cd68, Cd163, Bmpr2, Smad5, Tgfbr2, and Smad3 were determined using RT-qPCR. ResultsThe MCT + Cold group had aggravated RV hypertrophy hemodynamic parameters, and pulmonary arterial medial wall thickness. In lungs of the MCT + Cold, group the protein levels of TNF-α, IL-1β, and IL-6 were higher than those in the MCT group. The mRNA expression of Cd68 and Cd163 were higher in the MCT + Cold group. miR-146a-5p and miR-155-5p levels were higher in the plasma EVs and lungs of the MCT + Cold group. Cold exposure promoted a greater decrease in miR-21-5p, Bmpr2, Smad5, Tgfbr2, and Smad3 mRNA expression in lungs of the MCT + Cold group. ConclusionCold exposure aggravates MCT-induced PAH with an increase in inflammatory marker and miRNA levels in the plasma EVs and lungs.

Effects of BiejiaYugan Granule on TGF-β1/Smads pathway in rats with hepatic fibrosis caused by compound factors

Objective: To investigate the therapeutic effects of Biejia Yugan Granule on hepatic fibrosis caused by compound factors in rats and its effect on TGF-β1/Smads signaling pathway. Methods: SD rats were randomly divided into blank control group, model control group, colchicine group, Biejia Yugan Granule low, medium and high dose (1.85, 3.70, 7.40 g/kg) groups (n= 8 in each group). The rat model of hepatic fibrosis was established by treating with 5% alcohol 15 ml/kg (ig) everyday and injecting with 40% carbon tetrachloride (sc) twice a week for 42 days. The effects of Biejia Yugan Granule on liver function, liver index and water content, serum hepatic fibrosis related indicators, key proteins and gene expression of TGF-β1/Smads signaling pathway in rats were observed. Results: Biejia Yugan Granule at the doses of 1.85, 3.70 and 7.40 g/kg could decrease the serum levels of ALT, AST, ALP and HA, PCⅢ, C-Ⅳ, LN significantly, reduce the water content of liver tissue leads to the decrease of liver index, regulate the liver tissue TGF-β1, Smad3 mRNA and Smad7 mRNA expressions. Conclusion: Biejia Yugan Granule has obvious effects of reducing enzyme and protecting liver and inhibiting hepatic fibrosis, and inhibiting TGF-β1/Smads signaling pathway is one of its mechanisms of anti-hepatic fibrosis.

Biological impacts on the lungs in rats internally exposed to radioactive 56MnO2 particle

To understand the radiation effects of the atomic bombing of Hiroshima and Nagasaki among the survivors, radiation from neutron-induced radioisotopes in soil should be considered in addition to the initial radiation directly received from the bombs. 56Mn, which emits both β particles and γ-rays, is one of the dominant radioisotopes created in soil by neutrons from the bomb. Thus we investigated the biological effects of internal exposure to 56MnO2 particle in the lung of male Wistar rats comparing to the effects of external 60Co-γ irradiation. Absorbed doses of internal irradiation of lungs were between 25 and 65 mGy in 56MnO2-exposed animals, while the whole body doses were between 41 and 100 mGy. Animals were examined on days 3 and 61 after the exposure. There were no remarkable pathological changes related to 56MnO2 particle exposure. However, mRNA and protein expressions of aquaporin 5 increased significantly in the lung tissue on day 3 postexposure in 56MnO2 groups (by 1.6 and 2.9 times, respectively, in the highest dose group). Smad7 mRNA expression was also significantly elevated by 30% in the highest dose group of 56MnO2. Our data demonstrated that internal exposure to 56MnO2 induced significant biological responses including gene expression changes in the lungs, while external 60Co-γ irradiation of 2 Gy did not show any changes.

Nuclear Respiratory Factor-1, a Novel SMAD4 Binding Protein, Represses TGF-β/SMAD4 Signaling by Functioning as a Transcriptional Cofactor.

SMAD4, a key regulator of transforming growth factor-β (TGF-β) signaling, plays a major role in cell growth, migration, and apoptosis. In particular, TGF-β/SMAD induces growth arrest, and SMAD4 induces the expression of target genes such as p21WAF1 and p15INK4b through its interaction with several cofactors. Thus, inactivating mutations or the homozygous deletion of SMAD4 could be related to tumorigenesis or malignancy progression. However, in some cancer types, SMAD4 is neither mutated nor deleted. In the current study, we demonstrate that TGF-β signaling with a preserved SMAD4 function can contribute to cancer through associations with negative pathway regulators. We found that nuclear respiratory factor-1 (NRF1) is a novel interaction SMAD4 partner that inhibits TGF-β/SMAD4-induced p15INK4b mRNA expression by binding to SMAD4. Furthermore, we confirmed that NRF1 directly binds to the core region of the SMAD4 promoter, thereby decreasing SMAD4 mRNA expression. On the whole, our data suggest that NRF1 is a negative regulator of SMAD4 and can interfere with TGF-β/SMAD-induced tumor suppression. Our findings provide a novel perception into the molecular basis of TGF-β/SMAD4-signaling suppression in tumorigenesis.

The effect of inhibiting exosomes derived from adipose-derived stem cells via the TGF-β1/Smad pathway on the fibrosis of keloid fibroblasts.

The main mechanism of keloid formation is that keloid fibroblasts (KFs) apoptosis is inhibited, leading to excessive proliferation. Transforming growth factor-β1 (TGF-β1) is a key signal molecule in the process of regulating cell fibrosis. This paper discusses the effect of adipose-derived stem cell exosomes (ADSCs-EXO) on the proliferation and apoptosis of KFS and its possible mechanism, in order to provide reference for the clinical intervention of hypertrophic scar. ADSCs were isolated and cultured from human adipose tissue, the supernatant was collected, and the exosomes secreted by ADSCs-EXO were extracted by ultracentrifugation. At the same time, KFs were cultured from human keloid tissue to P3 generation, and then divided into four groups: control group, experimental group A, experimental group B and experimental group C. KFs were then cultured with four concentrations of ADSCs-EXO (0, 1, 10, and 100 µg/mL, respectively). After 24 hours, cells in each group were taken to detect the following: proliferation of cells in each group using the cell counting Kit 8 (CCK-8) method, cell migration ability via the Transwell test, cell apoptosis by flow cytometry, collagen synthesis using the hydroxyproline method, messenger ribonucleic acid (mRNA) expression of fibrosis-related genes in each group by real-time fluorescent polymerase chain amplification, and the expression of fibrosis-related proteins in the cells of each group by western blotting. Compared with the control group, the proliferation rate, migration rate, and collagen synthesis levels in the three experimental groups decreased with the increase of ADSCs-EXO concentration, while the apoptosis rate in the three experimental groups increased with the increase of ADSCs-EXO concentration, and the differences were statistically significant (P<0.05). Also, compared with the control group, the relative mRNA and protein expression of alpha-smooth muscle actin (α-SMA), TGF-β1, and Smad3 in the three groups decreased significantly, while the expression of three kinds of mRNA and protein decreased with the increase of ADSCs-EXO concentration, and the differences were statistically significant (P<0.05). ADSCs-EXO may inhibit the proliferation and migration, and promote the apoptosis of KFs by inhibiting the expression of the TGF-β1/Smad pathway.

Protective Effect of Thymus serrulatus Essential Oil on Cadmium-Induced Nephrotoxicity in Rats, through Suppression of Oxidative Stress and Downregulation of NF-κB, iNOS, and Smad2 mRNA Expression.

The purpose of the research was to examine the protective effect of essential oil from Thymus serrulatus Hochst. ex Benth. (TSA oil) against cadmium (Cd)-induced renal toxicity. The experimental protocol was designed using 30 healthy adult Wistar albino rats allocated into five groups containing six animals in each group. Group 1 was treated as normal control and groups 2, 3, 4, and 5 were treated with cadmium chloride (CdCl2, 3 mg/kg, IP) for 7 days. Group 3 was also treated with silymarin (100 mg/kg, PO) as a standard group, while groups 4 and 5 were administered with TSA oil at doses of 100 and 200 mg/kg PO, respectively. The nephrotoxicity was measured with various parameters such as kidney function markers, oxidative stress markers (glutathione (GSH) and malondialdehyde (MDA)), and messenger ribonucleic acid (mRNA) expression levels of inflammatory factors. The histological studies were also evaluated in the experimental protocol. The CdCl2-treated groups showed a significant increase in the levels of serum kidney function markers along with MDA levels in kidney homogenate. However, renal GSH level was found to be reduced significantly. It was found that CdCl2 significantly upregulated the nuclear factor levels of kappaB (NF-κB p65), inducible nitric oxide synthase (iNOS), and small mothers against decapentaplegic (Smad2) as compared to the normal control group. On the other hand, TSA oil significantly improved the increased levels of serum kidney function markers, non-enzymatic antioxidants, and lipid peroxidation. In addition, TSA oil significantly downregulated the increased expression of NF-κB p65, iNOS, and Smad2 in Cd-intoxicated rats. Moreover, the histological changes in the tissue samples of the kidney of Cd-treated groups were significantly ameliorated in the silymarin- and TSA-oil-treated groups. The present study reveals that TSA oil ameliorates Cd-induced renal injury, and it is also proposed that the observed nephroprotective effect could be due to the antioxidant potential of TSA oil and healing due to its anti-inflammatory action.

Effects and mechanism of lncRNA-27785.1 that regulates TGF-β1 of Sika deer on antler cell proliferation.

Transforming growth factor (TGF-β) plays an important role in the development of deer antlers.The purpose of this study was to investigate the role of long noncoding RNA in the transcriptional regulation of TGF-β1 and its relationship with the proliferation and differentiation of antler chondrocytes. High-throughput sequencing was used to screen lncRNAs related to TGF-β1. Next, the overexpression plasmid and interference sequence of target lncRNA27785.1 were constructed and transfected into chondrocytes. We found that lncRNA27785.1 inhibited the proliferation and migration of chondrocytesand delayed the transition of cells from G1 to S phase. qRT-PCR and Western blot analysis indicated that the overexpression of lncRNA27785.1 may downregulate mRNA and protein expression of TGF-BR2, Smad3, pSmad3, and Smad4. Our findings highlight lncRNA27785.1 as an inhibitor of chondrocytes proliferation and differentiation by negatively regulating the TGF-β/Smad signaling pathway; this implicates an important regulatory role for long noncoding RNA in the regeneration of antler.

Effects of swimming and epigallocatechin gallate on interstitial proteins expression of myocardium from type 2 diabetic rats

To observe the effects of swimming training and/or epigallocatechin gallate(EGCG) on the expression of myocardial interstitial protein and its relationship with transforming growth factor-beta(TGF-β)/Smads signaling pathway in type 2 diabetic rats. Male SD rats(SPF grade, 6 weeks old) were fed with high-fat diet for 6 weeks and intraperitoneal injection of streptozotocin(STZ, 30 mg/kg). The rats were randomly divided into four groups: diabetic model group(D), diabetes EGCG group(DG), diabetes exercise group(DE), and diabetes exercise EGCG group(DEG), with 8 rats in each group, and 6 male SD rats in the same batch were added as the normal C group. The rats in the exercise group were given swimming training without load. The swimming time was 15 min/d in the first week, and the time was increased by 15 min a week until it was extended to 90 min/d. The rats in the exercise group were trained for 5 days a week. Rats in the EGCG group(100 mg/kg BW) were intragastrical administered for 7 days. Swimming training and/or EGCG intervention lasted for 12 weeks. 48 hours after the end of the intervention experiment, the rats were fasted overnight for 12 hours. Myocardial collagen I(Col I), collagen IV(Col IV) and fibronectin(FN) protein expression were detected by immunohistochemistry in some left ventricles, hydroxyproline(Hyp) content was detected by colorimetry, TGF-β_1 protein expression was detected by WB, Smad2 and Smad7 mRNA expression were detected by RT-PCR. The levels of blood glucose, Hyp content, TGF-beta1, Smad2 mRNA, Col I, Col IV, FN of the rats from D group were significantly higher than those of the rats from C group(P&lt;0. 01), but the level of Smad7 mRNA was significantly lower than it of the rats from C group(P&lt;0. 01). Compared with D group, blood glucose, TGF-β_1, Smad2 mRNA expression, Hyp content, Col I, Col IV, FN protein expression in DG group, DE group and DEG group were significantly decreased(P&lt;0. 05 or P&lt;0. 01), and the Smad7 mRNA expression increased significantly(P&lt;0. 05 or P&lt;0. 01). Compared with DG group, blood glucose, Smad2 mRNA expression, Hyp content, Col I, Col IV protein expression in DEG group were significantly decreased(P&lt;0. 05 or P&lt;0. 01), and the myocardial Smad7 mRNA expression was significantly increased(P&lt;0. 05). Compared with DE group, the levels of blood glucose, TGF-β_1, Smad2 mRNA, Hyp content, Col I, Col IV protein expression were significantly decreased in the EGCG group(P&lt;0. 05 or P&lt;0. 01), and the expression of Smad7 mRNA was significantly increased(P&lt;0. 05). Blood glucose, Smad2 mRNA expression, Hyp content, Col I, Col Ⅳ, FN protein expression in(DEG group were significantly higher than those in C group(P&lt;0. 05 or P&lt;0. 01). 12 weeks swimming training and/or EGCG administration can reduce the expression of myocardial interstitial protein in type 2 diabetic rats, and the combined intervention effect is better.

Long non-coding RNA growth arrest specific transcript 5 acting as a sponge of MicroRNA-188-5p to regulate SMAD family member 2 expression promotes myocardial ischemia-reperfusion injury

ABSTRACT The purpose of this work is to probe into the potential role of long non-coding RNA growth arrest specific transcript 5 (lncGAS5)/ microRNA (miR)-188-5p/SMAD2 axis in MIRI. Through ligating the left anterior descending (LAD) coronary artery, MIRI animal model and hypoxia/reoxygenation (H/R) myocardial injury model in vitro were established. Via adenovirus or plasmid transfection, lncGAS5/MiR-188-5p/SMAD2 expression was up-regulated or down-regulated in the study. RT-qPCR was applied to check LncGAS5/MiR-188-5p/SMAD2 mRNA expression, HE staining for histopathological staining, TUNEL staining and flow cytometry to examine cardiomyocyte apoptotic rate, CCK-8 to check cell viability, ELISA to detect inflammatory factor levels, Western blot to examine Bax, Bcl-2, cleaved caspase-3, NF-κB and SMAD2 expression, and dual luciferase reporter experiment to examine the targeting relationship of miR-188-5p with LncGAS5 and SMAD2. The results indicated that LncGAS5 and SMAD2 were highly expressed in MIRI and miR-188-5p was under-expressed. Silencing LncGAS5 and SMAD2 or overexpressing miR-188-5p could reduce MIRI in myocardial tissue, cardiomyocyte apoptosis, inhibit Bax, cleaved caspase-3 and NF-κB expressions and promote Bcl-2 expression, while reducing inflammatory factors TNF -α, IL-1β and IL-6 levels. Overexpressing LncGAS5 promoted MIRI. Additionally, the impact of silencing LncGAS5 on MIRI could be reversed through inhibiting miR-188-5p. LncGAS5 acted as a sponge of miR-188-5p to target SMAD2 expression. In conclusion, Silencing LncGAS5 is available to improve MIRI through regulating miR-188-5p/SMAD2 axis, and may be used as a potential target for treating MIRI in the future.

MicroRNA-216a Promotes Endothelial Inflammation by Smad7/IκBα Pathway in Atherosclerosis.

Background The endothelium is the first line of defence against harmful microenvironment risks, and microRNAs (miRNAs) involved in vascular inflammation may be promising therapeutic targets to modulate atherosclerosis progression. In this study, we aimed to investigate the mechanism by which microRNA-216a (miR-216a) modulated inflammation activation of endothelial cells. Methods. A replicative senescence model of human umbilical vein endothelial cells (HUVECs) was established, and population-doubling levels (PDLs) were defined during passages. PDL8 HUVECs were transfected with miR-216a mimics/inhibitor or small interfering RNA (siRNA) of SMAD family member 7 (Smad7). Real-time PCR and Western blot assays were performed to detect the regulatory role of miR-216a on Smad7 and NF-κB inhibitor alpha (IκBα) expression. The effect of miR-216a on adhesive capability of HUVECs to THP-1 cells was examined. MiR-216a and Smad7 expression in vivo were measured using human carotid atherosclerotic plaques of the patients who underwent carotid endarterectomy (n = 41). Results Luciferase assays showed that Smad7 was a direct target of miR-216a. Smad7 mRNA expression, negatively correlated with miR-216a during endothelial aging, was downregulated in senescent PDL44 cells, compared with young PDL8 HUVECs. MiR-216a markedly increased endothelial inflammation and adhesive capability to monocytes in PDL8 cells by promoting the phosphorylation and degradation of IκBα and then activating NF-κB signalling pathway. The effect of miR-216a on endothelial cells was consistent with that blocked Smad7 by siRNAs. When inhibiting endogenous miR-216a, the Smad7/IκBα expression was rescued, which led to decreased endothelial inflammation and monocytes recruitment. In human carotid atherosclerotic plaques, Smad7 level was remarkably decreased in high miR-216a group compared with low miR-216a group. Moreover, miR-216a was negatively correlated with Smad7 and IκBα levels and positively correlated with interleukin 1 beta (IL1β) expression in vivo. Conclusion In summary, our findings suggest a new mechanism of vascular endothelial inflammation involving Smad7/IκBα signalling pathway in atherosclerosis.

Effects of danshensu on the transforming growth factor-β1/smads signaling pathway in rats with lung injury

Observe the therapeutic effect of Danshensu on lung injury for rats, as well as explore the mechanism of Danshensu in TGF-β1/Smads signaling. Thirty Sprague-Dawley (SD) rats were intratracheally instilled with bleomycin to induce lung injury and interstitial fibrosis. Divided Thirty rats into three groups. DA group (η = 10): Inject 15 mg/kg Danshensu into the abdominal cavity; DXM group (η = 10): Inject 1 mg/kg dexamethasone into the abdominal cavity; BLM group (η = 10): Inject 2 mL physiological saline into the abdominal cavity. Then ten SD rats were intratracheally instilled with physiological saline as normal control group, NC group: Inject 2 mL physiological saline into the abdominal cavity. After a period of 28 days, the degree of pulmonary alveolitis was evaluated using hematoxylin-eosin (HE) staining, and the degree of lung fibrosis was evaluated using Masson?s trichrome (MT) staining. The immunohistochemistry was used to determine the expression of α-SMA. Magnetic nanoparticles+rtQ-PCR was used to determine the mRNA expressions for TGF-β1, Smad3, and Smad7. The alveolitis and pulmonary fibrosis in DA rats were obviously less than those in BLM rats and DXM rats. The expression of α-SMA in DA rats was obviously less than that of in BLM rats and DXM rats; the mRNA expression of TGF-β1 and Smad3 in DA rats were obviously reduced; the Smad7 mRNA expression was obviously up-regulated. DA can alleviate rat lung injury caused by bleomycin. Inhibiting the TGF-β1 and Smad3 mRNA expression, as well as boosting the Smad7 mRNA expression is one of the mechanisms by which Danshensu reduces lung injury.

SMAD3 Hypomethylation as a Biomarker for Early Prediction of Colorectal Cancer.

The incidence and mortality rates of colorectal cancer (CRC) have been high in recent years. Prevention and early detection are crucial for decreasing the death rate. Therefore, this study aims to characterize the alteration patterns of mothers against decapentaplegic homolog 3 (SMAD3) in patients with CRC and its applications in early detection by using a genome-wide methylation array to identify an aberrant hypomethylation site in the intron position of the SMAD3 gene. Quantitative methylation-specific polymerase chain reaction showed that hypomethylated SMAD3 occurred in 91.4% (501/548) of Taiwanese CRC tissues and 66.6% of benign tubular adenoma polyps. In addition, SMAD3 hypomethylation was observed in 94.7% of patients with CRC from The Cancer Genome Atlas dataset. A decrease in circulating cell-free methylation SMAD3 was detected in 70% of CRC patients but in only 20% of healthy individuals. SMAD3 mRNA expression was low in 42.9% of Taiwanese CRC tumor tissues but high in 29.4% of tumors compared with paired adjacent normal tissues. Hypomethylated SMAD3 was found in cancers of the digestive system, such as liver cancer, gastric cancer, and colorectal cancer, but not in breast cancer, endometrial cancer, and lung cancer. In conclusion, SMAD3 hypomethylation is a potential diagnostic marker for CRC in Western and Asian populations.

MiR-18a Contributes to Preeclampsia by Downregulating Smad2 (Full Length) and Reducing TGF-β Signaling

The study investigated the regulation of Smad2 by miR-18a and its role in preeclampsia (PE). Bioinformatics analysis showed that both Smad2 and Smad3 were the predicted targets for miR-18a. Mass spectrum analysis showed that two mature Smad2 isoforms existed in human placenta: full length, Smad2(FL), and that lacking exon3, Smad2(Δexon3). The protein level of Smad2(FL), but not Smad2(Δexon3) or Smad3, was significantly increased in severe PE (sPE) placenta, which was inversely correlated with the level of miR-18a. Elevated Smad2(FL) phosphorylation level appeared in sPE placenta, and Smad2 was colocalized with miR-18a in various subtypes of trophoblasts in human placenta. Smad2(FL) was validated as the direct target of miR-18a in HTR8/SVneo cells. miR-18a enhanced trophoblast cell invasion, which was blocked by the overexpression of Smad2(FL). Furthermore, overexpression of miR-18a repressed Smad2 activation and the inhibition of trophoblast cell invasion by transforming growth factor-β (TGF-β). In conclusion, our results suggest that miR-18a inhibits the expression of Smad2(FL), but not Smad2(Δexon3) or Smad3, which can reduce TGF-β signaling, leading to the enhancement of trophoblast cell invasion. A lack of miR-18a, which results in the upregulation of Smad2(FL), contributes to the development of PE.

Effect of tissue inhibitor of metalloproteinase-1 and 2 siRNA on the expression of smad2/3/4 protein in hepatic stellate cells

Objective: To study the effect of tissue inhibitor of metalloproteinases (TIMP)-1 siRNA and TIMP-2 siRNA on the expression of smad2/3/4 protein in CCl4-induced liver fibrosis rat hepatic stellate cells (HSC). Methods: Rat's liver tissues with liver fibrosis after treatment with pre-built TIMP-1siRNA and TIMP-2 siRNA were used as the research subjects. Immunohistochemistry, Western blotting and real-time PCR were used to detect the protein and corresponding mRNA expression levels on smad2/3/4. TUNEL and α-smooth muscle actin (α-SMA) positive cells were quantified by double-labeled immunofluorescence. Analysis of variance (ANOVA) was used to compare the means between multiple groups, and the SNK test was used for the pairwise comparison of means. Results: The results of immunohistochemistry showed that the protein expressions of smad2, smad3, and smad4 in the TIMP-1 siRNA group and TIMP-2 siRNA group were significantly reduced than those of the model and the negative control group (P < 0.05). In addition, Western blotting results had also shown the same trend. The protein expression of smad2, smad3, and smad4 in the TIMP-1siRNA group and TIMP-2siRNA group were significantly reduced than those of the model and the negative control group (P < 0.01). The mRNA expression of smad2, smad3, and smad4 in TIMP-1siRNA group and TIMP-2siRNA group was significantly reduced than those of the model and negative control group (P < 0.05). Immunofluorescence showed that the apoptosis of activated HSC in the TIMP-1 siRNA group(0.014 3 ± 0.002 4) and TIMP-2 siRNA group(0.010 7 ± 0.004 4) was increased than those of the model(0) and the negative control group (0.002 4 ± 0.002 4, P < 0.05). Conclusion: TIMP-1 siRNA and TIMP-2 siRNA promote the apoptosis of activated HSCs. In addition, it also has a significant inhibitory effect on the expression of smad protein.

Smad7 Enhances TGF-β-Induced Transcription of c-Jun and HDAC6 Promoting Invasion of Prostate Cancer Cells.

SummaryTransforming growth factor β (TGF-β) enhances migration and invasion of cancer cells, causing life-threatening metastasis. Smad7 expression is induced by TGF-β to control TGF-β signaling in a negative feedback manner. Here we report an additional function of Smad7, i.e., to enhance TGF-β induction of c-Jun and HDAC6 via binding to their regulatory regions, promoting migration and invasion of prostate cancer cells. Lysine 102 in Smad7 is crucial for binding to specific consensus sites in c-Jun and HDAC6, even when endogenous Smad2, 3, and 4 were silenced by siRNA. A correlation between the mRNA expression of Smad7 and HDAC6, Smad7 and c-Jun, and c-Jun and HDAC6 was found in public databases from analyses of prostate cancer tissues. High expression of Smad7, HDAC6, and c-Jun correlated with poor prognosis for patients with prostate cancer. The knowledge that Smad7 can activate transcription of proinvasive genes leading to prostate cancer progression provides clinically relevant information.

Renoprotective Effect of Formononetin by Suppressing Smad3 Expression in Db/Db Mice.

PurposeGlomerular sclerosis and renal interstitial fibrosis are the most important pathologies in the development of kidney damage under diabetic conditions. Smad3 plays antagonistic roles in high glucose-induced renal tubular fibrosis, which is an important treatment target for diabetic nephropathy (DN). Formononetin (FMN) has multiple effects on diabetic vascular complications including DN. However, whether it plays an anti-fibrosis role by regulating smad3 is unclear. The purpose of this study was to evaluate the renoprotective effect of FMN by suppressing smad3 expression in db/db mice.MethodsFMN was orally administered to db/db mice with a dose of 25 or 50 mg/kg/day for 8 weeks. At the end of the study, serum, urine, and kidney samples were collected for biochemical and pathological examinations. The expressions of proteins and mRNA associated with renal fibrosis were determined by biochemical, histological, immunofluorescence, and real-time PCR analysis.ResultsThe results showed that FMN substantially improved the glucolipid metabolism, reduced the oxidative stress, and protected renal function in db/db mice. Meanwhile, protein and mRNA expression of smad3 and related regulatory factor of extracellular matrix deposition were significantly suppressed.ConclusionThe present study suggested that FMN has a good renoprotective effect in DN, which plays an anti-fibrosis role in db/db mice by suppressing the expression of smad3.