Long noncoding RNA NEAT1 promotes cardiac fibrosis in heart failure through increased recruitment of EZH2 to the Smad7 promoter region

Cardiac fibrosis, a well-known major pathological process that ultimately leads to heart failure, has attracted increasing attention and focus in recent years. A large amount of research indicates that long noncoding RNAs (lncRNAs) play an important role in cardiac fibrosis, but little is known about the specific function and mechanism of the lncRNA NEAT1 in the progression of cardiac fibrosis to heart failure. In the present study, we have demonstrated that the lncRNA NEAT1 is upregulated in patients with heart failure. Similarly, the expression of Neat1 was also increased in the left ventricular tissue of transverse aortic constriction (TAC) surgery mice and cardiac fibroblasts treated with TGF-β1. Further, gain-of-function and loss-of-function experiments showed that silencing of Neat1 attenuated cardiac fibrosis, while overexpression of Neat1 with adenovirus significantly aggravated the in vitro progression of fibrosis. With regard to the underlying mechanism, our experiments showed that Neat1 recruited EZH2 to the promoter region of Smad7 through physical binding of EZH2 to the promoter region, as a result of which Smad7 expression was inhibited and the progression of cardiac fibrosis was ultimately exacerbated. We found that the introduction of shNeat1 carried by adeno-associated virus-9 significantly ameliorated cardiac fibrosis and dysfunction caused by TAC surgery in mice. Overall, our study findings demonstrate that the lncRNA Neat1 accelerates the progression of cardiac fibrosis and dysfunction by recruiting EZH2 to suppress Smad7 expression. Thus, NEAT1 may serve as a target for the treatment of cardiac fibrosis.

Sox9-Increased miR-322-5p Facilitates BMP2-Induced Chondrogenic Differentiation by Targeting Smad7 in Mesenchymal Stem Cells

Bone morphogenetic protein 2 (BMP2) induces effective chondrogenesis of mesenchymal stem cells (MSCs) by promoting Sox9 expression. However, BMP2 also induces chondrocyte hypertrophy and endochondral ossification by upregulating Smad7 expression, which leads to the disruption of chondrogenesis. In addition, Smad7 can be inhibited by Sox9. Therefore, the underlying mechanism is not clear. Currently, an increasing number of studies have shown that microRNAs play a pivotal role in chondrogenic and pathophysiological processes of cartilage. The purpose of this study was to determine which microRNA is increased by Sox9 and targets Smad7, thus assisting BMP2 in maintaining stable chondrogenesis. We found that miR-322-5p meets the requirement through next-generation sequencing (NGS) and bioinformatic analysis. The targeting relationship between miR-322-5p and Smad7 was confirmed by dual-luciferase reporter assays, qPCR, and western blotting (WB). The in vitro study indicated that overexpression of miR-322-5p significantly inhibited Smad7 expression, thus causing increased chondrogenic differentiation and decreased hypertrophic differentiation, while silencing of miR-322-5p led to the opposite results. Flow cytometry (FCM) analysis indicated that overexpression of miR-322-5p significantly decreased the rate of early apoptosis in BMP2-stimulated MSCs, while silencing of miR-322-5p increased the rate. A mouse limb explant assay revealed that the expression of miR-322-5p was negatively correlated with the length of the BMP2-stimulated hypertrophic zone of the growth plate. An in vivo study also confirmed that miR-322-5p assisted BMP2 in chondrogenic differentiation. Taken together, our results suggested that Sox9-increased miR-322-5p expression can promote BMP2-induced chondrogenesis by targeting Smad7, which can be exploited for effective tissue engineering of cartilage.

Smad7 Deficiency in Myeloid Cells Does Not Affect Liver Injury, Inflammation or Fibrosis after Chronic CCl4 Exposure in Mice

Myeloid cells play an essential role in the maintenance of liver homeostasis, as well as the initiation and termination of innate and adaptive immune responses. In chronic hepatic inflammation, the production of transforming growth factor beta (TGF-β) is pivotal for scarring and fibrosis induction and progression. TGF-β signalling is tightly regulated via the Smad protein family. Smad7 acts as an inhibitor of the TGF-β-signalling pathway, rendering cells that express high levels of it resistant to TGF-β-dependent signal transduction. In hepatocytes, the absence of Smad7 promotes liver fibrosis. Here, we examine whether Smad7 expression in myeloid cells affects the extent of liver inflammation, injury and fibrosis induction during chronic liver inflammation. Using the well-established model of chronic carbon tetrachloride (CCl4)-mediated liver injury, we investigated the role of Smad7 in myeloid cells in LysM-Cre Smadfl/fl mice that harbour a myeloid-specific knock-down of Smad7. We found that the chronic application of CCl4 induces severe liver injury, with elevated serum alanine transaminase (ALT)/aspartate transaminase (AST) levels, centrilobular and periportal necrosis and immune-cell infiltration. However, the myeloid-specific knock-down of Smad7 did not influence these and other parameters in the CCl4-treated animals. In summary, our results suggest that, during long-term application of CCl4, Smad7 expression in myeloid cells and its potential effects on the TGF-β-signalling pathway are dispensable for regulating the extent of chronic liver injury and inflammation.

FOXO3 regulates Smad3 and Smad7 through SPON1 circular RNA to inhibit idiopathic pulmonary fibrosis

Forkhead box protein O3 (FOXO3) has good inhibition ability toward fibroblast activation and extracellular matrix, especially for the treatment of idiopathic pulmonary fibrosis. How FOXO3 regulates pulmonary fibrosis remains unclear. In this study, we reported that FOXO3 had binding sequences with F-spondin 1 (SPON1) promoter, which can activate its transcription and selectively promote the expression of SPON1 circRNA (circSPON1) but not mRNA expression. We further demonstrated that circSPON1 was involved in the extracellular matrix deposition of HFL1. In the cytoplasm, circSPON1 directly interacted with TGF-β-induced Smad3 and inhibited the activation of fibroblasts by inhibiting nuclear translocation. Moreover, circSPON1 bound to miR-942-5p and miR-520f-3p that interfered with Smad7 mRNA and promoted Smad7 expression. This study revealed the mechanism of FOXO3 in the occurrence and development of pulmonary fibrosis. Potential therapeutic targets and new insights into the diagnosis and treatment of idiopathic pulmonary fibrosis based on circRNA were also provided.

SMAD7 is differentially expressed in brain metastatic human breast cancer.

Metastasis to the brain is a clinical problem in patients with breast cancer (1-3). We mined published microarray data (4, 5) to compare primary and metastatic tumor transcriptomes for the discovery of genes associated with brain metastasis in humans with metastatic breast cancer. We found that mothers against decapentaplegic homolog 7, encoded by SMAD7, was among the genes whose expression was most different in the brain metastases of patients with metastatic breast cancer as compared to primary tumors of the breast. SMAD7 mRNA was present at decreased quantities in brain metastatic tissues as compared to primary tumors of the breast. Importantly, expression of SMAD7 in primary tumors was significantly correlated with patient recurrence-free survival. Modulation of SMAD7 expression may be relevant to the biology by which tumor cells metastasize from the breast to the brain in humans with metastatic breast cancer.

Triptolide Improves Renal Injury in Diabetic Nephropathy Rats through TGF-β1/Smads Signal Pathway

Objective: To investigate the therapeutic effect and mechanism of Triptolide on renal injury in diabetic nephropathy rats. Methods: A total of 15 male SD rats aged 8 weeks were randomly divided into five groups (3 rats in each group): control group, model group, Triptolide low-dose (Triptolide-L) group, Triptolide medium-dose (Triptolide-M) group, Triptolide high-dose (Triptolide-H) group. The rats models of diabetic nephropathy (DN) were established by a single intraperitoneal injection of STZ after being fed with high-fat and high-sugar diet for 4 weeks, and the fasting blood glucose (FBG) concentration of rats was detected. After 4 weeks, HE-staining was used to evaluate the renal pathological damage in rats; biochemical analysis was used to determine the blood urea nitrogen (BUN), serum creatinine (SCr), total cholesterol (TC), triglyceride (TG); ELISA was used to measure the serum inflammatory factor levels; Western blot (WB) was used to detect the expression of TGF-β1/Smads pathway proteins. Results: In the four FBG tests (once a week), the FBG concentration in the model group was significantly higher than that in the control group, while Triptolide-treated rats were significantly lower than that in the model group. Rats in Model group showed obvious renal injury, and Triptolide significantly improved the renal injury in DN rats. Compared with the control group, the expression of BUN, SCr, TC, TG, inflammatory factors TNF-α, IL-6 and IL-1β in the model group increased significantly. WB results showed that the expressions of TGF-β1, Smad3, α-SMA and vimentin in the kidney significantly increased, while the Smad7 expression significantly decreased. Triptolide significantly reduced the levels of BUN, SCr, TC, TG and TNF-α, IL-6, IL-1β in diabetic rats, decreased the expression of TGF-β1, Smad3, α-SMA, vimentin, and increased the Smad7 expression. In different doses of Triptolide treatment group, its effect showed a significant concentration dependence. Conclusion: Triptolide alleviates renal injury in diabetic rats by inhibiting the TGF-β1/Smads signaling pathway.

The epigenetic regulator RINF (CXXC5) maintains SMAD7 expression in human immature erythroid cells and sustains red blood cells expansion.

The gene CXXC5, encoding a Retinoid-Inducible Nuclear Factor (RINF), is located within a region at 5q31.2 commonly deleted in myelodysplastic syndrome (MDS) and adult acute myeloid leukemia (AML). RINF may act as an epigenetic regulator and has been proposed as a tumor suppressor in hematopoietic malignancies. However, functional studies in normal hematopoiesis are lacking, and its mechanism of action is unknow. Here, we evaluated the consequences of RINF silencing on cytokineinduced erythroid differentiation of human primary CD34+ progenitors. We found that RINF is expressed in immature erythroid cells and that RINF-knockdown accelerated erythropoietin-driven maturation, leading to a significant reduction (~45%) in the number of red blood cells (RBCs), without affecting cell viability. The phenotype induced by RINF-silencing was TGFs-dependent and mediated by SMAD7, a TGFa-signaling inhibitor. RINF upregulates SMAD7 expression by direct binding to its promoter and we found a close correlation between RINF and SMAD7 mRNA levels both in CD34+ cells isolated from bone marrow of healthy donors and MDS patients with del(5q). Importantly, RINF knockdown attenuated SMAD7 expression in primary cells and ectopic SMAD7 expression was sufficient to prevent the RINF knockdowndependent erythroid phenotype. Finally, RINF silencing affects 5’-hydroxymethylation of human erythroblasts, in agreement with its recently described role as a Tet2- anchoring platform in mouse. Altogether, our data bring insight into how the epigenetic factor RINF, as a transcriptional regulator of SMAD7, may fine-tune cell sensitivity to TGFsssuperfamily cytokines and thus play an important role in both normal and pathological erythropoiesis.

High Smad7 in the early post-operative recurrence of Crohn’s disease

Background In Crohn’s disease (CD), one of the major inflammatory bowel disease (IBD) in human beings, there is over-expression of Smad7, an intracellular inhibitor of the suppressive cytokine TGF-β1. The aim of this study was to assess whether Smad7 over-expression occurs in the early and/or late phases of CD. Methods Mucosal samples were taken from the neo-terminal ileum of CD patients undergoing ileocolonic resection, with or without (early CD) post-operative endoscopic recurrence, and terminal ileum of CD patients with long-standing disease undergoing intestinal resection (late CD). Smad7 was examined by immunohistochemistry and cytokine expression was analysed by flow-cytometry. Results Before the appearance of endoscopic lesions, the mucosa of the neo-terminal ileum contained high number of Smad7-expressing cells in both the epithelial and lamina propria compartments. Transition from this stage to endoscopic recurrence was marked by persistence of high number of Smad7-positive cells, which reduced significantly in the late stages of the disease, where Smad7 expression remained, however, greater than that seen in normal controls. In samples with early lesions, Smad7 expression positively correlated with the number of interferon-γ-secreting cells. Conclusions Smad7 induction is an early event in the inflammatory sequence occurring in CD, thus suggesting that knockdown of Smad7 can help prevent post-operative recurrence.

High Smad7 in the early post-operative recurrence of Crohn’s disease

Background: In Crohn’s disease (CD), one of the major inflammatory bowel disease (IBD) in human beings, there is over-expression of Smad7, an intracellular inhibitor of the suppressive cytokine TGF-β1. The aim of this study was to assess whether Smad7 over-expression occurs in the early and/or late phases of CD. Methods: Mucosal samples were taken from the neo-terminal ileum of CD patients undergoing ileocolonic resection, with or without (early CD) post-operative endoscopic recurrence, and terminal ileum of CD patients with long-standing disease undergoing intestinal resection (late CD). Smad7 was examined by immunohistochemistry and cytokine expression was analysed by flow-cytometry. Results: Before the appearance of endoscopic lesions, the mucosa of the neo-terminal ileum contained high number of Smad7-expressing cells in both the epithelial and lamina propria compartments. Transition from this stage to endoscopic recurrence was marked by persistence of high number of Smad7-positive cells, which reduced significantly in the late stages of the disease, where Smad7 expression remained, however, greater than that seen in normal controls. In samples with early lesions, Smad7 expression positively correlated with the number of interferon-g-secreting cells. Conclusions: Smad7 induction is an early event in the inflammatory sequence occurring in CD, thus suggesting that knockdown of Smad7 can help prevent post-operative recurrence.

High Smad7 in the early post-operative recurrence of Crohn’s disease

Background: In Crohn’s disease (CD), one of the major inflammatory bowel disease (IBD) in human beings, there is over-expression of Smad7, an intracellular inhibitor of the suppressive cytokine TGF-β1. The aim of this study was to assess whether Smad7 over-expression occurs in the early and/or late phases of CD. Methods: Mucosal samples were taken from the neo-terminal ileum of CD patients undergoing ileocolonic resection, with or without (early CD) post-operative endoscopic recurrence, and terminal ileum of CD patients with long-standing disease undergoing intestinal resection (late CD). Smad7 was examined by immunohistochemistry and cytokine expression was analysed by flow-cytometry. Results: Before the appearance of endoscopic lesions, the mucosa of the neo-terminal ileum contained high number of Smad7-expressing cells in both the epithelial and lamina propria compartments. Transition from this stage to endoscopic recurrence was marked by persistence of high number of Smad7-positive cells, which reduced significantly in the late stages of the disease, where Smad7 expression remained, however, greater than that seen in normal controls. In samples with early lesions, Smad7 expression positively correlated with the number of interferon-g-secreting cells. Conclusions: Smad7 induction is an early event in the inflammatory sequence occurring in CD, thus suggesting that knockdown of Smad7 can help prevent post-operative recurrence.