Search

Cell division cycle associated 7 (CDCA7) is a copy number amplification gene that contributes to the metastasis and invasion of tumors, including esophageal squamous cell carcinoma (ESCC). This present study aimed at clarifying whether high expression of CDCA7 promotes the metastasis and invasion of ESCC cell lines and exploring the underlying mechanisms implicated in epithelial-mesenchymal transition (EMT) of ESCC. The role of CDCA7 in the regulation of ESCC metastasis and invasion was evaluated using ESCC cell lines. Expression of EMT-related markers including E-cadherin, N-cadherin, Vimentin, Snail, and Slug, transforming growth factor β (TGF-β) signaling pathway including Smad2/3, p-Smad2/3, Smad4, and Smad7 were detected in CDCA7 knockdown and overexpressed cell lines. Dual-luciferase reporter assay and rescue assay were used to explore the underlying mechanisms that CDCA7 contributed to the metastasis and invasion of ESCC. High CDCA7 expression significantly promoted the metastasis and invasion of ESCC cell lines both in vivo and in vitro. Additionally, the expression of CDCA7 positively correlated with the expression of N-cadherin, Vimentin, Snail, Slug, TGF-β signaling pathway and negatively correlated with the expression of E-cadherin. Furthermore, CDCA7 transcriptionally regulated the expression of Smad4 and Smad7. Knockdown of CDCA7 inhibited the TGF-β signaling pathway and therefore inhibited EMT. Our data indicated that CDCA7 was heavily involved in EMT by regulating the expression of Smad4 and Smad7 in TGF-β signaling pathway. CDCA7 might be a new therapeutic target in the suppression of metastasis and invasion of ESCC.

Glioblastoma (GBM) is the most aggressive primary brain tumor as one of the deadliest cancers. The TGF-β signaling acts as an oncogenic factor in GBM, and plays vital roles in development of GBM. SMAD7 is a major inhibitor of TGF-β signaling, while the deubiquitination of SMAD7 has been poorly studied in GBM. Here, we found USP2 as a new prominent candidate that could regulate SMAD7 stability. USP2 was lost in GBM, leading to the poor prognosis in patients. Moreover, aberrant DNA methylation mediated by DNMT3A induced the low expression of USP2 in GBM. USP2 depletion induced TGF-β signaling and progression of GBM. In contrast, overexpressed USP2 suppressed TGF-β signaling and GBM development. Specifically, USP2 interacted with SMAD7 and prevented SMAD7 ubiquitination. USP2 directly cleaved Lys27- and Lys48-linked poly-ubiquitin chains of SMAD7, and Lys27-linked poly-ubiquitin chains of SMAD7 K185 mediated the recruitment of SMAD7 to HERC3, which regulated Lys63-linked poly-ubiquitination of SMAD7. Moreover, we demonstrated that the DNMT3A inhibitor SGI-1027 induced USP2, suppressed TGF-β signaling and GBM development. Thus, USP2 repressed development of GBM by inhibition TGF-β signaling pathway via the deubiquitination of SMAD7.

Studies over the past decades have implicated lncRNAs in promoting the development, migration and invasion of gastric cancer (GC). However, the role and mechanism of lncRNA MBNL1-AS1 in GC promotion are poorly understood. In this research, qRT-PCR showed that MBNL1-AS1 was down-regulated in GC tissues and cells. Cell experiments and the animal study demonstrated that MBNL1-AS1 knockdown accelerated GC cell proliferation, migration, and invasion, thus restraining cell apoptosis. Meanwhile, overexpression of MBNL1-AS1 repressed GC cell promotion. Bioinformatics analysis confirmed that MBNL1-AS1 binds to miR-424-5p via negative modulation. Rescue experiments showed that decreased miR-424-5p level inhibited GC cell promotion by silencing MBNL1-AS1. Furthermore, Smad7 was identified as a target of miR-424-5p that could reverse the promotion of GC cell growth mediated by miR-424-5p. Western blot results proved that MBNL1-AS1 affected TGF-β/SMAD pathways by regulating the miR-424-5p/Smad7 axis. Collectively, MBNL1-AS1 restrained GC growth via the miR-424-5p/Smad7 axis and thus could be a promising target for GC therapy. These findings illustrate that lncRNA MBNL1-AS1, as a tumor suppressor gene, participates in GC progression by regulating miR-424-5p/Smad7 axis, thus activating TGF-β/EMT pathways. The evidence may provide a potential marker for GC patients.

MicroRNAs (miRNAs) have emerged as important regulators in the development of cardiovascular diseases. miR-410-3p was shown to play a protective or detrimental role in the progression in cardiovascular events. However, the exact role and the underlying mechanism of miR-410-3p in cardiac hypertrophy have not been documented. The current work was aimed to determine the role and underlying mechanism of miR-410-3p on Angiotensin II (Ang II) induced cardiac hypertrophy. FITC-phalloidin staining was used for determination of cardiomyocyte surface area. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed to identify mRNA expression level of hypertrophic markers. Smad7 protein expression level was analyzed using Western blot. Dual-luciferase reporter assay was used to examine the regulatory function of miR-410-3p on Smad7. MiR-410-3p was found significantly up-regulated in Ang II–induced cardiac hypertrophy. MiR-410-3p inhibitor remarkably alleviated cardiomyocyte hypertrophic changes. Dual-luciferase reporter assay result indicated that miR-410-3p directly targeted Smad7 and miR-410-3p inhibitor effectively prevented Ang II triggered down-regulation of Smad7. Moreover, Smad7 overexpression significantly reversed the pro-hypertrophic effect of miR-410-3p. In summary, our findings revealed that miR-410-3p mediated Ang II–induced cardiac hypertrophy via targeting inhibition of Smad7.

Hepatic stellate cells activation (HSCs) plays a crucial role in the pathogenesis of liver fibrosis. Specific microRNAs have been suggested to affect the activation of HSCs via various signalling pathways including TGF-β/smads and Wnt/β-catenin pathways. Dasatinib is a multitarget inhibitor of many tyrosine kinases has recently studied for its anti-fibrotic effects in a variety of fibrous diseases. This study investigated the role of modulation of miRNA-378 and miRNA-17 in the pathogenesis of liver fibrosis through altering Wnt/β-catenin and TGF-β/smads pathways and evaluated the beneficial effect of the tyrosine kinase inhibitor, dasatinib, in thioacetamide-induced liver fibrosis model in mice. Treatment with dasatinib down-regulated miRNA-17 expression, leading to the restoration of WiF-1 and smad-7 which cause the inhibition of both Wnt/β-catenin and TGF-β/smads signalling. In addition, it upregulated miRNA-378 leading to the decrease of Wnt-10 which contributes to the suppression of activated HSCs., Graphical Abstract

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.

Transforming growth factor-β (TGF-β)-mediated accumulation of extracellular matrix proteins such as collagen I is a common feature of fibrosis. Pancreatic stellate cells play an integral role in the pathogenesis of pancreatitis, and their profibrotic ability is mainly mediated by TGF-β signaling. To specifically address the role of fibrogenic cells in experimental pancreatic fibrosis, we deleted Smad7, the main feedback inhibitor of TGF-β signaling in this cell type in mice.A mouse strain harboring a conditional knockout allele of Smad7 (Smad7fl/fl) with the tamoxifen-inducible inducible Col1a2-CreERT allele was generated and compared with wild-type mice challenged with the cerulein-based model of chronic pancreatitis.Pancreatic stellate cells lacking Smad7 had significantly increased collagen I and fibronectin production and showed a higher activation level in vitro. Surprisingly, the fibrotic index in the pancreata of treated conditional knockout mice was only slightly increased, without statistical significance. Except for fibronectin, the expression of different extracellular matrix proteins and the numbers of fibroblasts and inflammatory cells were similar between Smad7-mutant and control mice.There was no clear evidence that the lack of Smad7 in pancreatic stellate cells plays a major role in experimental pancreatitis, at least in the mouse model investigated here.

The transcription factor forkhead box D3 (FOXD3) is an important member of the FOX family, which can maintain the pluripotent properties of cell clusters, neural crest, and trophoblastic progenitor cells in vivo. It has been shown that FOXD3 could affect proliferation, migration, and angiogenesis of various tumors and its deletion and overexpression in organisms will undoubtedly have important influence on the change of cell fate and the occurrence of tumors. However, the underlying functions and molecular mechanisms of FOXD3 in esophageal squamous cell carcinoma (ESCC) have not been fully clarified. According to the present study, the expression levels and functional roles of FOXD3 were investigated, and its prognostic value and molecular mechanisms in tumorigenesis and progression of ESCC were clarified. The expression level of FOXD3 was significantly downregulated in ESCC tissues and cell lines, and correlated with gender, family history of upper gastrointestinal cancer, TNM stage, depth of invasion, lymph node metastasis, and ESCC patients' survival. Moreover, FOXD3 inhibited cells migration and invasion as well as participated in TGF-β1 induced epithelial-mesenchymal transition process. Furthermore, a positive correlation between FOXD3 and SMAD family member 7 (SMAD7) was explored in ESCC. FOXD3 could directly bind to promoter regions of SMAD7 gene, leading to transcriptional promotion of SMAD7 in human esophageal cancer cells. Taken together, FOXD3 may play a tumor suppressor role in ESCC and may be applied as a new therapeutic target and prognostic marker for ESCC.

Background: The extracellular vesicles (EVs) secreted by bone marrow mesenchymal stromal cells (MSCs) have the ability to improve Myocardial infarction (MI). Some microRNAs (miRNAs) including miR-497 and related target genes have been proved to be closely linked with heart diseases. However, EVs could regulate MI process through miR-497, and the mechanisms have not been fully reported. Methods: Ligation of left anterior descending artery was performed to established MI animals model. Hypoxia cell model was established through lowering the level of oxygen. The cell invasion, migration, and proliferation were measured using tanswell, wound heating, and MTT assays. HE, Masson trichrome, and Sirius Red staining were used to investigate the morphological changes. Results: Overexpression of miR-497 reversed the promotion of cell migration, invasion, and proliferation caused by EVs. The improvement of cardiac function induced by EVs could also be reversed by overexpression of miR-497. Direct binding site between Smad7 and miR-497 was identified. Knockdown of Smad7 reversed the improvement of cardiac function induced by EVs. Conclusions: We found that EVs isolated from MSCs might improve the cardiac injury caused by MI through targeting miR497/Smad7. This study provides novel potential therapeutic thought for the prevention and treatment of MI through targeting miR-497/Smad7.

TGF-β1 (transforming growth factor β1) was considered to play a critical role in the forming of hypertrophic scars. Smad, as a kind of signal downstream mediators, can modulate the functions of TGF-β1. Smad7 can regulate TGF-β1/Smad pathway and present negative feedbacks, which prevents fibrosis mediated by TGF-β1. Nonetheless, the mechanisms related to Smad7 activity in regulating hypertrophic scarring are hardly known. The studies have shown that Smad7 decrease induced by the increase of Smurf2 (Smad ubiquitination regulatory factor 2, an E3 ubiquitin ligase of Smad7) ubiquitination degradation plays a part in fibrosis. We thus made a hypothesis that Smad7 could not inhibit TGF-β1 because Smurf2 ubiquitin degradation was increased in hypertrophic scar fibroblasts. In our research, it was discovered that there was an increase in Smad7 mRNA levels but no increase in Smad7 protein levels in the fibroblasts of hypertrophic scars after TGF-β1 treatment. The ubiquitination activity and degradation of Smad7 protein were increased in the fibroblasts of hypertrophic scars compared with the fibroblasts of normal skin. Enhanced degradation of Smad7 protein in the fibroblasts of hypertrophic scars was prevented by proteasome inhibitors MG132 / MG115. Furthermore, it was found that TGF-β1 stimulation increased Smad7 protein expression after silencing Smurf2 gene in hypertrophic scar fibroblasts, and enhanced Smad7 degradation was prevented in hypertrophic scar fibroblasts after Smurf2 was silenced. It was implied that ubiquitin degradation mediated by Smurf2 might contribute to decreased Smad7 protein levels following TGF-β1 stimulation in the fibroblasts of hypertrophic scars.

Transforming growth factor β (TGF-β) superfamily proteins are potent regulators of cellular development and differentiation. Nodal/Activin/TGF-β and BMP ligands are both present in the intra- and extracellular milieu during early development, and cross-talk between these two branches of developmental signaling is currently the subject of intense research focus. Here, we show that the Nodal induced lncRNA-Smad7 regulates cell fate determination via repression of BMP signaling in mouse embryonic stem cells (mESCs). Depletion of lncRNA-Smad7 dramatically impairs cardiomyocyte differentiation in mESCs. Moreover, lncRNA-Smad7 represses Bmp2 expression through binding with the Bmp2 promoter region via (CA)12-repeats that forms an R-loop. Importantly, Bmp2 knockdown rescues defects in cardiomyocyte differentiation induced by lncRNA-Smad7 knockdown. Hence, lncRNA-Smad7 antagonizes BMP signaling in mESCs, and similarly regulates cell fate determination between osteocyte and myocyte formation in C2C12 mouse myoblasts. Moreover, lncRNA-Smad7 associates with hnRNPK in mESCs and hnRNPK binds at the Bmp2 promoter, potentially contributing to Bmp2 expression repression. The antagonistic effects between Nodal/TGF-β and BMP signaling via lncRNA-Smad7 described in this work provides a framework for understanding cell fate determination in early development.

Purpose Tumor metastasis is the main cause of death from breast cancer patients and cell migration plays a critical role in cancer metastasis. Recent studies have shown long non-coding RNAs (lncRNAs) play an essential role in the initiation and progression of cancer. In the present study, the role of an LncRNA, Rho GTPase Activating Protein 5- Antisense 1 (ARHGAP5-AS1) in breast cancer was investigated. Methods RNA sequencing was performed to find out dysregulated LncRNAs in MDA-MB-231-LM2 cells. Transwell migration assays and F-actin staining were utilized to estimate cell migration ability. RNA pulldown assays and RNA immunoprecipitation were used to prove the interaction between ARHGAP5-AS1 and SMAD7. Western blot and immunofluorescence imaging were used to examine the protein levels. Dual luciferase reporter assays were performed to evaluate the activation of TGF-β signaling. Results We analyzed the RNA-seq data of MDA-MB-231 and its highly metastatic derivative MDA-MB-231-LM2 cell lines (referred to as LM2) and identified a novel lncRNA (NR_027263) named as ARHGAP5-AS1, which expression was significantly downregulated in LM2 cells. Further functional investigation showed ARHGAP5-AS1 could inhibit cell migration via suppression of stress fibers in breast cancer cell lines. Afterwards, SMAD7 was further identified to interact with ARHGAP5-AS1 by its PY motif and thus its ubiquitination and degradation was blocked due to reduced interaction with E3 ligase SMURF1 and SMURF2. Moreover, ARHGAP5-AS1 could inhibit TGF-β signaling pathway due to its inhibitory role on SMAD7. Conclusion ARHGAP5-AS1 inhibits breast cancer cell migration via stabilization of SMAD7 protein and could serve as a novel biomarker and a potential target for breast cancer in the future.

Background Lung cancer is one of the most common malignant tumors threatening human health. The aim of this study was to investigate the function of miR‐21‐5p in lung cancer progression. Methods We analyzed the expression levels of miR‐21‐5p in lung cancer tissues and cell lines. The qRT‐PCR and MTT assays were performed after transfection with miR‐21‐5p mimic, inhibitor and negative control into lung cancer cells. Results Luciferase reporter assays showed miR‐21‐5p directly target SMAD7. The miR‐21‐5p inhibitor significantly suppressed lung cancer cell proliferation, invasion and migration. We found that SMAD7 was upregulated in lung cancer tissue. In addition, we found that SMAD7 inhibited lung cancer cell proliferation and miR‐21‐5p mimic damaged the inhibitory effect of SMAD7. Conclusions miRNA‐21‐5p may promote cell proliferation, migration and invasion by spoiling SMAD7 expression in lung cancer cells., miR‐21‐5p directly targets SMAD7. miR‐21‐5p/SMAD7 signal controls lung cancer progression.

We aimed to investigate the role of cMet agonistic antibody (cMet Ab) in preventing kidney fibrosis during acute kidney injury (AKI) to chronic kidney disease (CKD) transition. Additionally, we explored the effect of cMet Ab on TGF-β1/Smad pathway during the pathogenesis of kidney fibrosis. A unilateral ischemia–reperfusion injury (UIRI) mouse model was established to induce AKI-to-CKD transition. Furthermore, we incubated human proximal tubular epithelial cells (hPTECs) under hypoxic conditions as in vitro model of kidney fibrosis. We analyzed the soluble plasma cMet level in patients with AKI requiring dialysis. Patients who did not recover kidney function and progressed to CKD presented a higher increase in the cMet level. The kidneys of mice treated with cMet Ab showed fewer contractions and weighed more than the controls. The mice in the cMet Ab-treated group showed reduced fibrosis and significantly decreased expression of fibronectin and α-smooth muscle actin. cMet Ab treatment decreased inflammatory markers (MCP-1, TNF-α, and IL-1β) expression, reduced Smurf1 and Smad2/3 level, and increased Smad7 expressions. cMet Ab treatment increased cMet expression and reduced the hypoxia-induced increase in collagen-1 and ICAM-1 expression, thereby reducing apoptosis in the in vitro cell model. After cMet Ab treatment, hypoxia-induced expression of Smurf1, Smad2/3, and TGF-β1 was reduced, and suppressed Smad7 was activated. Down-regulation of Smurf1 resulted in suppression of hypoxia-induced fibronectin expression, whereas treatment with cMet Ab showed synergistic effects. cMet Ab can successfully prevent fibrosis response in UIRI models of kidney fibrosis by decreasing inflammatory response and inhibiting the TGF-β1/Smad pathway.

Cavernous nerve injury is the main cause of erectile dysfunction (ED) after radical prostatectomy (RP). In our previous study, injection of adipose-derived stem cells (ADSCs) into the cavernosum can repair damaged cavernosum nerves and ED can be restored to a certain extent. In order to improve these therapeutic effects, we evaluated the efficacy of ADSCs co-modified with VEGF and Smad7 in a rat model. SD rats were randomly divided into six groups: a sham surgery group, and the five bilateral cavernous nerve injury (BCNI) groups were injected with ADSC or ADSCs genetically modified by VEGF (ADSC-V), Smad7 (ADSC-S), or VEGFSmad7 (ADSC-VS) or phosphate-buffered saline (PBS). The results indicated that the erectile function of the ADSC-V, ADSC-S, and ADSC-VS groups was significantly recovered, and the erectile function of the ADSC-VS group was more distinctly recovered as compared to the other groups. The same results are shown in the expression of neuronal nitric oxide synthase and the smooth muscle/collagen ratio of penile tissue comparing the ADSC-VS group to the ADSC-V and ADSC-S group. These experimental data suggest that ADSCs co-overexpressed with VEGF and Smad7 can significantly improve erectile function after BCNI. This study provides new therapeutic thoughts for ED following RP.

Objective Peyronie’s disease (PD) is a fibrotic disorder of the penis, but effective treatments are lacking. Here, we observed the effects of rat-derived bone marrow mesenchymal stem cells (BMSCs) injection in the active phase and chronic phase in a rat model of PD, and the possible mechanism was analysed with fibroblasts derived from rat penile tunica albuginea (TA). Methods Thirty-two male Sprague-Dawley rats were divided into four groups. In sham group, the rats were injected with 50 µL of vehicle. In the PD group, the rats were injected with 50 µg TGF-β1. In the PD + BMSCs early treatment group, the rats were injected with 50 µg TGF-β1 and injected with 1 × 106 BMSCs after 1 day. In the PD + BMSCs late treatment group, the rats were injected with 50 µg TGF-β1 and injected with 1 × 106 BMSCs after 28 days. Twenty-seven days after the last injection, the erectile function of the rats was measured, and then, penile fibrosis was analysed by histology and western blot. In vitro, fibroblasts derived from rat penile TA were used to identify a possible antifibrotic mechanism of BMSCs, and a Smad7 expression vector was used as a positive control. Fibroblasts were pretreated with the Smad7 expression vector or BMSCs for 48 h and then activated with 10 ng/mL TGF-β1 for 24 h. Cells viability was assessed, and Smad7, collagen 3, elastase-2B and osteopontin expression levels were analysed by immunofluorescence and western blot. Furthermore, fibroblasts were transfected with Smad7 siRNA or scramble control to observe whether the effects of BMSCs could be offset. Results Erectile function obviously improved, and fibrosis of penile TA was prevented after BMSCs treatment compared with that in the rats with PD. Furthermore, the effects of BMSCs treatment in the active phase were better than those in the chronic phase. After cocultured with BMSCs, cell viability was not affected, Smad7 expression was upregulated, and collagen 3, elastase-2B and osteopontin levels were decreased in the TGF-β1-treated fibroblasts. After transfection with Smad7 siRNA, the antifibrotic effects of BMSCs were offset. Conclusions The antifibrotic effects of BMSCs treatment in the active phase of the PD rat model were better than those in the chronic phase. A possible mechanism of BMSCs treatment was related to increased Smad7 expression, suggesting a possible effective and safe procedure for the treatment of PD.

Smad7 restrains TGF-β responses, and has been suggested to exert both pro- and anti-inflammatory actions that may involve effects on macrophages. Myocardial infarction triggers a macrophage-driven inflammatory response that not only plays a central role in cardiac repair, but also contributes to adverse remodeling and fibrosis. We hypothesized that macrophage Smad7 expression may regulate inflammation and fibrosis in the infarcted heart through suppression of TGF-β responses, or via TGF-independent actions. In a mouse model of myocardial infarction, infiltration with Smad7+ macrophages peaked 7 days after coronary occlusion. Myeloid cell-specific Smad7 loss in mice had no effects on homeostatic functions and did not affect baseline macrophage gene expression. RNA-seq predicted that Smad7 may promote TREM1-mediated inflammation in infarct macrophages. However, these alterations in the transcriptional profile of macrophages were associated with a modest and transient reduction in infarct myofibroblast infiltration, and did not affect dysfunction, chamber dilation, scar remodeling, collagen deposition, and macrophage recruitment. In vitro, RNA-seq and PCR arrays showed that TGF-β has profound effects on macrophage profile, attenuating pro-inflammatory cytokine/chemokine expression, modulating synthesis of matrix remodeling genes, inducing genes associated with sphingosine-1 phosphate activation and integrin signaling, and inhibiting cholesterol biosynthesis genes. However, Smad7 loss did not significantly affect TGF-β-mediated macrophage responses, modulating synthesis of only a small fraction of TGF-β-induced genes, including Itga5, Olfml3, and Fabp7. Our findings suggest a limited role for macrophage Smad7 in regulation of post-infarction inflammation and repair, and demonstrate that the anti-inflammatory effects of TGF-β in macrophages are not restrained by endogenous Smad7 induction.

Abundant preclinical work showed that in Crohn's disease (CD), the defective activity of the immunosuppressive cytokine tumor necrosis factor (TGF)-β1 due to high levels of the intracellular inhibitor Smad7 contributes to amplify the tissue-damaging inflammatory response. Consistently, phase I and II studies documented clinical and endoscopic benefit in active CD patients treated with mongersen, an oral antisense oligonucleotide targeting Smad7. However, a multicenter, randomized, double-blind, placebo-controlled, phase III study was prematurely discontinued as a futility analysis showed that mongersen was not effective in CD patients. The reasons why the phase III study failed despite the fact that previous clinical trials documented the efficacy of the drug remain unknown. The primary objective of this Viewpoint was to provide clues about the factors explaining discrepancies among the clinical trials. We illustrate the recent data indicating that the various batches of mongersen, used during the phase III program, are chemically different, with some of them being unable to downregulate Smad7 expression. Overall, these findings suggest the necessity of new clinical studies to further evaluate the efficacy of chemically homogenous batches of mongersen in patients with inflammatory bowel diseases (IBDs), and, at the same time, they can help understand the failure of other clinical trials with antisense oligonucleotides in IBD (i.e. alicaforsen).

Transforming growth factor-β (TGF-β) signaling plays a key role in excessive fibrosis. As a class IIa family histone deacetylase (HDAC), HDAC5 shows a close relationship with TGF-β signaling and fibrosis. However, the effect and regulatory mechanism of HDAC5 in hypertrophic scar (HS) formation remain elusive. We show that HDAC5 was overexpressed in HS tissues and depletion of HDAC5 attenuated HS formation

BACKGROUND: Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) and mothers against decapentaplegic homolog 7 (SMAD7) are important transforming growth factor-β (TGF-β) signaling antagonists, however their roles in acute myeloid leukemia (AML) remains unclear. Telomerase reverse transcriptase (TERT) may be involved in regulating BAMBI and SMAD7 expressions; a role beyond telomeres that is not clinically validated yet. OBJECTIVE: In this study, we examined the expression levels and prognostic values of BAMBI, SMAD7 and TERT and their association with AML patients’ outcomes. METHODS: Blood samples were collected from 74 de-novo AML patients and 16 controls. Real-time quantitative PCR (qRT-PCR) was performed to analyze BAMBI, SMAD7 and TERT expressions. RESULTS: BAMBI and SMAD7 expression in AML were significantly upregulated versus controls (p< 0.05). BAMBI, SMAD7 and TERT levels were significantly correlated together (p< 0.001). Kaplan-Meier analysis indicated that patients with high BAMBI, SMAD7 and TERT expression levels had markedly shorter event free survival (EFS) and overall survival (OS) time (p< 0.01). Furthermore, multivariate analysis revealed that only high BAMBI expression was an independent risk factor for OS (p= 0.001). CONCLUSIONS: BAMBI is a novel biomarker in predicting prognosis in AML patients. Moreover, a potential interplay is found between BAMBI, SMAD7 and TERT in AML pathogenies.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive fatal interstitial lung disease characterized by abnormal transition and proliferation of fibroblasts. The uncontrolled transition of fibroblasts, commonly known as myofibroblasts, are the principal source of the enormous extracellular matrix (ECM) depositing in lung parenchyma, leading to gradual failure of gas exchange and mortality of the patients. However, up to now, rare effective therapeutic strategies have been developed to blockade fibroblast-to-myofibroblast transition (FMT) in IPF. Method: We illustrated that the lungs originated from IPF patients and mice with pulmonary fibrosis are characterized by the overexpression of sushi-repeat-containing protein, X-linked 2 (SRPX2). Further functionality studies identified the pivotal role of SRPX2 in FMT. Mechanistically, SRPX2 was involved in a TGFβR1/SMAD3/SRPX2/AP1/SMAD7 positive feedback loop. Specifically, SRPX2 was upregulated by TGF-β1 in a TGFβR1/SMAD3-dependent manner, after which SRPX2 in turn repressed the expression of AP1, subsequently minimized SMAD7 expression, through which it reduced the formation of inhibitory complex with TGFβR1 and enhanced SMAD signaling pathway to promote FMT and exacerbate pulmonary fibrosis. Notably, intratracheal administration of siRNA-loaded liposomes could effectively suppress the expression of Srpx2 in the lung and remarkably protect mice against BLM-induced pulmonary fibrosis, concomitant with a significant reduction of FMT. Results: Accordingly, these data indicate that Srpx2 plays an essential role in the pathogenesis of pulmonary fibrosis and suggests the strategy aiming at silencing Srpx2 could be a promising therapeutic approach against pulmonary fibrosis in clinical settings.

Background/Aims: Current asthma therapies remain unsatisfactory for con trolling airway remodelling in asthma. MicroRNA-21 is a key player in asthma pathogenesis, but the molecular mechanisms underlying its effects on airway remodelling are not completely understood. We investigated the effects of inhibi tion of microRNA-21 on allergic airway inflammation and remodelling. Methods: Female BALB/c mice were divided into four groups: control, ovalbu min-sensitized and -challenged for 3 months, microRNA-negative control-treat ed ovalbumin-treated, and microRNA-21 inhibitor-treated ovalbumin-treated groups. Parameters related to airway remodelling, cytokine production, airway inflammation, and airway hyperresponsiveness were compared between groups. Human bronchial smooth muscle cells were used in a mechanism study. Results: In this asthma model, ovalbumin-sensitized and -challenged mice ex hibited allergic airway inf lammation and airway remodelling. MicroRNA-21 inhibitor-treated mice had fewer inflammatory cells, lower TH2 cytokine produc tion, and suppressed parameters related to remodelling such as goblet cell hyper plasia, collagen deposition, hydroxyproline content, and expression of smooth muscle actin. Inhibition of microRNA-21 decreased transforming growth factor β1 expression and induced Smad7 expression in lung tissue. In human bronchial smooth muscle cells stimulated with transforming growth factor β1, microR NA-21 inhibition upregulated Smad7 expression and decreased markers of airway remodelling. Conclusions: Inhibition of microRNA-21 had both anti-inflammatory and anti-re modelling effects in this model of ovalbumin-induced chronic asthma. Our data suggest that the microRNA-21-transforming growth factor β1-Smad7 axis modu lates the pathogenesis of ovalbumin-induced chronic asthma and in human bron chial smooth muscle cells. MicroRNA-21 inhibitors may be a novel therapeutic target in patients with allergic asthma, especially those with airway remodelling.

TGFβ signaling is a known pathway to be involved in colorectal cancer (CRC) progression and miRNAs play crucial roles by regulating different components of this pathway. Hence, finding the link between miRNAs and the pathway could be beneficial for CRC therapy. Array data indicated that miR-186-5p is a differentially expressed miRNA in colorectal Tumor/Normal tissues and bioinformatics tools predicted SMAD6/7 (inhibitory SMADs) as bona fide targets of this miRNA. Here, we intended to investigate the regulatory effect of the miR-186-5p expression on TGFβ signaling in CRC. Firstly, the miR-186-5p overexpression in HCT116 cells resulted in a significant reduction of SMAD6/7 expression, measured through RT-qPCR. Then, the direct interactions of miR-186-5p with SMAD6/7 3′UTRs were supported through dual luciferase assay. Furthermore, miR-186-5p overexpression suppressed proliferation, cell viability, and migration while, it increased apoptosis in CRC cells, assessed by cell cycle, MTT, scratch and Annexin V/PI apoptosis assays. Consistently, miR-186-5p overexpression resulted in reduced CyclinD1 protein using western blot, and also resulted in increased P21 and decreased c-Myc expression. Overall, these results introduced miR-186-5p as a cell cycle suppressor through downregulation of SMAD6/7 expression. Thus, miR-186-5p might be served as a novel tumor suppressive biomarker and therapeutic target in CRC treatment.

TGF-β1 has long been considered as a key mediator in diabetic kidney disease (DKD) but anti-TGF-β1 treatment fails clinically, suggesting a diverse role for TGF-β1 in DKD. In the present study, we examined a novel hypothesis that latent TGF-β1 may be protective in DKD mice overexpressing human latent TGF-β1. Streptozotocin-induced Type 1 diabetes was induced in latent TGF-β1 transgenic (Tg) and wild-type (WT) mice. Surprisingly, compared to WT diabetic mice, mice overexpressing latent TGF-β1 were protected from the development of DKD as demonstrated by lowing microalbuminuria and inhibiting renal fibrosis and inflammation, although blood glucose levels were not altered. Mechanistically, the renal protective effects of latent TGF-β1 on DKD were associated with inactivation of both TGF-β/Smad and nuclear factor-κB (NF-κB) signaling pathways. These protective effects were associated with the prevention of renal Smad7 from the Arkadia-induced ubiquitin proteasomal degradation in the diabetic kidney, suggesting protection of renal Smad7 from Arkadia-mediated degradation may be a key mechanism through which latent TGF-β1 inhibits DKD. This was further confirmed in vitro in mesangial cells that knockdown of Arkadia failed but overexpression of Arkadia reversed the protective effects of latent TGF-β1 on high glucose-treated mesangial cells. Latent TGF-β1 may protect kidneys from TGF-β1/Smad3-mediated renal fibrosis and NF-κB-driven renal inflammation in diabetes through inhibiting Arkadia-mediated Smad7 ubiquitin degradation.

OBJECTIVES/HYPOTHESIS Vocal fold fibrosis remains a significant clinical challenge. Estrogens, steroid hormones predominantly responsible for secondary sexual characteristics in women, have been shown to alter wound healing and limit fibrosis, but the effects on vocal fold fibrosis are unknown. We sought to elucidate the expression of estrogen receptors and the effects of estrogens on TGF-β1 signaling in rat vocal fold fibroblasts (VFFs). STUDY DESIGN In vitro. METHODS VFFs were isolated from 10-week-old, male Sprague-Dawley rats, and estrogen receptor alpha (ERα) and G protein-coupled receptor 30 (GPR30) were examined via immunostaining and quantitative polymerase chain reaction (qPCR). VFFs were treated with estradiol (E2, 10-7 , 10-8 or 10-9 M) ± transforming growth factor beta 1 (TGF-β1, 10 ng/mL). ICI 182,780 (ICI, 10-7 M) or G36 (10-7 M) were employed as antagonists of ERα or GPR30, respectively. qPCR was employed to determine estrogen receptor-mediated effects of E2 on genes related to fibrosis. RESULTS ERα and GPR30 were expressed in VFFs at both the protein and the mRNA levels. E2 (10-7 M) did not alter Smad3, Smad7, Acta2 mRNA, or extracellular matrix related genes. However, the combination of E2 (10-8 M) and TGF-β1 significantly increased Smad7 (P = .03) and decreased Col1a1 (P = .04) compared to TGF-β1 alone; this response was negated by the combination of ICI and G36 (P = .009). CONCLUSIONS E2 regulated TGF-β1/Smad signaling via estrogen receptors in VFFs. These findings provide insight into potential mechanisms of estrogens on vocal fold injury with the goal of enhanced therapeutics for vocal fold fibrosis. LEVEL OF EVIDENCE NA Laryngoscope, 131:2285-2291, 2021.

Oxidative stress is a major cause of aging related skin injuries. Hydrogen peroxide related ROS accumulation triggers increase in matrix metalloproteinases and elevated collagen degradation, which is a characteristic of skin aging. In this study, we investigated the protective effect of Poria cocos, used widely in the treatment of inflammatory diseases, against H2 O2 induced oxidative stress. The aqueous extract of dried P. cocos was obtained by heating 10 g in 500 ml of distilled water. The mixture was evaporated up to 400 ml and the remaining 100 ml was filtered through muslin cloth repeatedly to obtain a clear aqueous extract of the P. cocos. Hs68 human dermal fibroblast cells were challenged with 100 μM of H2 O2 for 24 h. Following H2 O2 challenge, the cells were treated with increasing concentration of P. cocos extract (100-400 μg/ml) for 24 h. P. cocos extract hindered the H2 O2 induced cell death significantly that was correlated with reduction in ROS accumulation. Western blot analysis show that P. cocos extract suppressed the expression of metallomatrix proteinases, inflammatory markers and skin aging markers, but increased TGF-β1 levels and antioxidant related proteins. These data suggest that P. cocos is effective in attenuating oxidative stress associated skin aging effects and may be a potential agent in cosmetics products.

Background and purpose Tooth eruption is a complicated process regulated by the dental follicles (DF). Our recent study discovered that tooth eruption was inhibited upon injection of bleomycin into DF. However, the mechanisms were unknown. Experimental approach Human dental follicle cells (hDFCs) were treated by bleomycin or exogenous TGF-β1 or transfected by plasmids loading SMAD7 or shRNA targeting SMAD7, followed by osteogenesis induction assay and signalling analysis. Human fresh DF tissues and Wistar rats were used to further confirm bleomycin function. Key results Bleomycin decreased expression of RUNX2 and osteogenic genes in hDFCs, reducing osteogenic capacity. TGF-β1 expression was up-regulated in bleomycin-treated hDFCs. The effects of exogenous TGF-β1 were similar to those of bleomycin in hDFCs. Additionally, compared to SMAD2/3, SMAD7 expression increased more in bleomycin- or TGF-β1-treated hDFCs. Overexpression of SMAD7 likewise significantly decreased RUNX2 expression and osteogenic capacity of hDFCs. Knockdown of SMAD7 markedly attenuated the inhibitory effects of bleomycin and TGF-β1 on osteogenic capacity and RUNX2 expression of hDFCs. Most importantly, changes in TGF-β1, SMAD7, and RUNX2 expressions were similar in the DF of rats and humans treated with bleomycin. Conclusion and implications SMAD7 was a negative regulator of osteogenic differentiation in DFCs through suppressing RUNX2 expression. Bleomycin or TGF-β1 inhibited osteogenic differentiation of DFCs via a TGF-β1/SMAD7/RUNX2 pathway. Our findings might be beneficial for enhancing the osteogenic activity of DFCs or inhibiting the eruption of undesirable teeth.

Myocardial infarction has been recognized globally as a serious problem featured with high mortality and morbidity. In addition, hypoxia represents the central feature of myocardial infarction. Recently, it has been reported that circular RNAs can exert critical functions in the biological processes of diseases. However, the functions of most circular RNAs remain unclear in cells cultured under hypoxic conditions. In this study, we focused on exploring the role of circ_SMAD7 (namely hsa_circ_0000848 in this study) in cardiomyocyte cells cultured under hypoxic conditions to provide a novel insight for future myocardial infarction studies.Firstly, a real-time quantitative polymerase chain reaction assay was adopted to analyze hsa_circ_0000848 expression. Functional assays were performed to detect the functions of hsa_circ_0000848 in cardiomyocyte cells cultured under hypoxic conditions. Furthermore, mechanism assays were implemented to explore the regulatory mechanism of hsa_circ_0000848.Hsa_circ_0000848 was notably downregulated in hypoxia-induced cardiomyocytes. The silencing of hsa_circ_0000848 hindered the proliferation while accelerating the apoptosis of hypoxia-induced cardiomyocytes cells. Moreover, hsa_circ_0000848 interacted with ELAV-like RNA-binding protein 1 protein to stabilize SMAD family member 7 mRNA. Moreover, SMAD family member 7 overexpression could reverse the suppressive effect of hsa_circ_0000848 knockdown on myocardial infarction progression.Our research was the first in the field to confirm that the hsa_circ_0000848/ ELAV-like RNA-binding protein 1/SMAD family member 7 axis could affect the development of cardiomyocyte cells cultured under hypoxia, indicating that hsa_circ_0000848 might function as a novel biomarker in cells under hypoxia thus laying the groundwork for future study on myocardial infarction.

Anti-cancer effects of curcumol on various cancers have been reported previously. This study focused on investigating the role of curcumol in pancreatic cancer from the molecular perspective. The survival of pancreatic cancer patients with high or low expression of miR-21-5pand the target gene of miR-21-5pwere analyzed by bioinformatics. MiR-21-5p expression in cancer tissues was analyzed by RT-qPCR. Anxenograft-tumor BALB/c nude mice model was established and pancreatic cancer cells were cultured. Later, the mice and cells were further treated with curcumol. The tumor size and weightas well as mice body weight were recorded. The viability, proliferation, migration, and invasion of the cells were evaluated by MTT, colony formation, and transwell assays, respectively. The expressions of molecules in the xenograft-tumor tissues or cells were detected by immunohistochemical assay, Western blot, or RT-qPCR. MiR-21-5p was high-expressed in pancreatic cancer tissues and patients with high expression of miR-21-5p had poor survival. Curcumol inhibited the xenograft-tumor size, tumor weight, and PCNA and miR-21-5p expressions while promoting Cleaved caspase-3 expression in xenograft-tumor tissues. Curcumol inhibited the viability, proliferation, migration, invasion, and miR-21-5p expression, but increased SMAD7 expression in cancer cells. MiR-21-5p overexpression reversed the effect of curcumol on cancer cells, and decreased the E-cadherin expression while elevating the expressions of PCNA, N-cadherin, Vimentin, p-SMAD2, and p-SMAD3 in curcumol-treated cells. The overexpression of SMAD7, a target gene of miR-21-5p, reversed the effect of miR-21-5p on curcumol-treated cells. Curcumol inhibited growth of xenograft-tumors and the biological activities of pancreatic cancer cells by regulating the miR-21-5p/SMAD7 axis.