Targeting Smad3 Research Articles

Latency-Associated Transcript-Derived MicroRNAs in Herpes Simplex Virus Type 1 Target SMAD3 and SMAD4 in TGF-β/Smad Signaling Pathway.

During its latent infection, hepatic stellate cell (HSV-1) produces only a micro RNA (miRNA) precursor called latency-associated transcript (LAT), which encodes six distinct miRNAs. Recent studies have suggested that some of these miRNAs could target cellular mRNAs. One of the key cell signaling pathways that can be affected by HSV-1 is the TGF-β/Smad pathway. Herein, we investigated the potential role of the LAT as well as three LAT-derived miRNAs in targeting SMAD3 and SMAD4, as two main mediators in TGF-β/Smad. The selection of LAT-derived miRNAs was based on the search results obtained from an online miRNA prediction tool. HEK293T cells were transfected with each miRNA-expressing lentivector and with the construct-expressing LAT. To survey the effect of LAT on the expression of pro-fibrotic markers, we transfected LX-2 cells with LAT construct. The impact of viral miRNA overexpression on SMADs and fibrotic markers was measured by quantitative PCR and luciferase assays. Among the LAT-derived miRNAs, miR-H2, miR-H3, and miR-H4 were selected for the study. Our results demonstrated that while miR-H2 binds to both SMAD mRNAs, miR-H3 and miR-H4 inhibit only the expression of the SMAD4 and SMAD3, respectively. Transfection of the LX-2 with LAT also decreased pro-fibrotic genes expression. Our findings display that LAT negatively regulates TGF-β/Smad through targeting SMAD3 and SMAD4 by its miRNAs. These viral miRNAs can also contribute to the development of therapeutic interventions in diseases for which prevention or treatment can be achieved through targeting TGF-β pathway.

Latency-Associated Transcript-Derived MicroRNAs in Herpes Simplex Virus Type 1 Target SMAD3 and SMAD4 in TGF-β/Smad Signaling Pathway

Background:During its latent infection, HSV-1 produces only a miRNA precursor called LAT, which encodes six distinct miRNAs. Recent studies have suggested that some of these miRNAs could target cellular mRNAs. One of the key cell signaling pathways that can be affected by HSV-1 is the TGF-β/Smad pathway. Herein, we investigated the potential role of the LAT as well as three LAT-derived miRNAs in targeting SMAD3 and SMAD4, as two main mediators in TGF-β/Smad. Methods:The selection of LAT-derived miRNAs was based on the search results obtained from an online miRNA prediction tool. HEK293T cells were transfected with each miRNA-expressing lentivector and with the construct-expressing LAT. To survey the effect of LAT on the expression of pro-fibrotic markers, we transfected LX-2 cells with LAT construct. The impact of viral miRNA overexpression on SMADs and fibrotic markers was measured by qPCR and luciferase assays. Results:Among the LAT-derived miRNAs, miR-H2, miR-H3, and miR-H4 were selected for the study. Our results demonstrated that while miR-H2 binds to both SMAD mRNAs, miR-H3 and miR-H4 inhibit only the expression of the SMAD4 and SMAD3, respectively. Transfection of the LX-2 with LAT also decreased pro-fibrotic genes expression. Conclusion:Our findings display that LAT negatively regulates TGF-β/Smad through targeting SMAD3 and SMAD4 by its miRNAs. These viral miRNAs can also contribute to the development of therapeutic interventions in diseases for which prevention or treatment can be achieved through targeting TGF-β pathway.

MiR-21-5p in Macrophage-Derived Exosomes Targets Smad7 to Promote Epithelial Mesenchymal Transition of Airway Epithelial Cells.

BackgroundAsthma is usually associated with airway inflammation and airway remodeling. Epithelial mesenchymal transition (EMT) often occurs in airway remodeling. The purpose of this study is to identify the effect of miR-21-5p and Smad7 signaling pathway in macrophage-derived exosomes on EMT of airway epithelial cells.MethodsHE staining and Masson staining were used to verify the successful establishment of the asthma model. The levels of epithelial cell adhesion factor and stromal cell markers were detected by Western blot. The levels of miR-21-5p were detected by qRT-PCR. The expression of miR-21-5p in lung tissue was further verified by fluorescence in situ hybridization (FISH). Exosome morphology was observed by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Luciferase reporter assay was applied to analyze the interaction of miR-21-5p with Smad7.ResultsThe expression of miR-21-5p was upregulated in macrophages of rats in vivo with OVA-induced asthma. In vitro cultured alveolar macrophages stimulated by LPS could secrete exosomes with high levels of miR-21-5p. The exosome-derived miR-21-5p promotes EMT in rat tracheal epithelial cells through TGFβ1/Smad signaling pathway by downregulating Smad7. This process can be blocked by miR-21-5p inhibitor.ConclusionRat alveolar macrophages produced high levels of miR-21-5p-containing exosomes, which transported miR-21-5p to tracheal epithelial cells, thus promoting EMT through TGF-β1/Smad signaling pathway by targeting Smad7.

MiR-144 Suppresses Cell Proliferation, Migration, and Invasion in Hepatocellular Carcinoma by Targeting SMAD4 [Retraction

[This retracts the article DOI: 10.2147/OTT.S88233.].

Downregulation of miR-17 suppresses TGF-β1-mediated renal fibrosis through targeting Smad7.

MiR-17 is found upregulated in diabetic mice; however, its effect(s) on renal fibrosis of diabetic nephropathy remain(s) unknown. This study aimed to explore the mechanism underlying the downregulation of miR-17 in renal fibrosis of diabetic nephropathy (DN). Patients with diabetes mellitus (DM) and DN and normal healthy individual controls, mice (db/db, db/m), and human mesangial cells (HMCs) and human proximal tubule epithelial cells (HK-2) were used as research subjects in the study. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to measure the expression of miR-17 in the serum samples, renal tissues and cells. Acid-Schiff (PAS) and Masson staining experiments were performed to detect glomerular mesangial matrix and collagen deposition. Levels of fibrosis-related proteins (E-Cadherin (E-cad), vimentin, fibronectin and collagen I) were measured by Western blot (WB). The target gene of miR-17 was predicted by TargetScan 7.2 and confirmed by dual-luciferase reporter analysis. The study found that miR-17 expression was elevated in the serums of DN patients as well as in the serums and kidney tissues of db/db mice. db/db mice showed a severe renal fibrosis condition. The levels of E-cad in db/db mice, HMC and HK-2 cells were increased by downregulating miR-17 expression, while expressions of vimentin, fibronectin and collagen I were reduced. Smad7 was predicted to be the target gene of miR-17, and its expression was promoted by downregulation of miR-17. Moreover, the reduced Smad7 expression could inhibit the expressions of fibrosis-related proteins, which, however, can be ameliorated by the downregulation of miR-17. In addition, downregulation of miR-17 could suppress renal fibrosis mediated by TGF-β1 through targeting Smad7, which might be a clinical therapeutic target for patients with DN.

Deletion of Smad3 protects against diabetic myocardiopathy in db/db mice

Diabetic cardiomyopathy (DCM) is a common diabetic complication characterized by diastolic relaxation abnormalities, myocardial fibrosis and chronic heart failure. Although TGF‐β/Smad3 signalling has been shown to play a critical role in chronic heart disease, the role and mechanisms of Smad3 in DCM remain unclear. We reported here the potential role of Smad3 in the development of DCM by genetically deleting the Smad3 gene from db/db mice. At the age of 32 weeks, Smad3WT‐db/db mice developed moderate to severe DCM as demonstrated by a marked increase in the left ventricular (LV) mass, a significant fall in the LV ejection fraction (EF) and LV fractional shortening (FS), and progressive myocardial fibrosis and inflammation. In contrast, db/db mice lacking Smad3 (Smad3KO‐db/db) were protected against the development of DCM with normal cardiac function and undetectable myocardial inflammation and fibrosis. Interestingly, db/db mice with deleting one copy of Smad3 (Smad3 ± db/db) did not show any cardioprotective effects. Mechanistically, we found that deletion of Smad3 from db/db mice largely protected cardiac Smad7 from Smurf2‐mediated ubiquitin proteasome degradation, thereby inducing IBα to suppress NF‐kB‐driven cardiac inflammation. In addition, deletion of Smad3 also altered Smad3‐dependent miRNAs by up‐regulating cardiac miR‐29b while suppressing miR‐21 to exhibit the cardioprotective effect on Smad3KO‐db/db mice. In conclusion, results from this study reveal that Smad3 is a key mediator in the pathogenesis of DCM. Targeting Smad3 may be a novel therapy for DCM.

Plasma Small Extracellular Vesicle‐Carried miRNA‐501‐5p Promotes Vascular Smooth Muscle Cell Phenotypic Modulation‐Mediated In‐Stent Restenosis

Vascular smooth muscle cell (VSMC) phenotypic modulation plays an important role in the occurrence and development of in-stent restenosis (ISR), the underlying mechanism of which remains a key issue needing to be urgently addressed. This study is designed to investigate the role of plasma small extracellular vesicles (sEV) in VSMC phenotypic modulation. sEV were isolated from the plasma of patients with ISR (ISR-sEV) or not (Ctl-sEV) 1 year after coronary stent implantation using differential ultracentrifugation. Plasma sEV in ISR patients are elevated markedly and decrease the expression of VSMC contractile markers α-SMA and calponin and increase VSMC proliferation. miRNA sequencing and qRT-PCR validation identified that miRNA-501-5p was the highest expressed miRNA in the plasma ISR-sEV compared with Ctl-sEV. Then, we found that sEV-carried miRNA-501-5p level was significantly higher in ISR patients, and the level of plasma sEV-carried miRNA-501-5p linearly correlated with the degree of restenosis (R2 = 0.62). Moreover, miRNA-501-5p inhibition significantly increased the expression of VSMC contractile markers α-SMA and calponin and suppressed VSMC proliferation and migration; in vivo inhibition of miRNA-501-5p could also blunt carotid artery balloon injury induced VSMC phenotypic modulation in rats. Mechanically, miRNA-501-5p promoted plasma sEV-induced VSMC proliferation by targeting Smad3. Notably, endothelial cells might be the major origins of miRNA-501-5p. Collectively, these findings showed that plasma sEV-carried miRNA-501-5p promotes VSMC phenotypic modulation-mediated ISR through targeting Smad3.

MiR-21-3p alleviates neuronal apoptosis during cerebral ischemiareperfusion injury by targeting SMAD2

Cerebral ischemia is due to the formation of blood clots or embolisms in the brain arteries, which leads to local brain tissue necrosis and neural cell apoptosis. Recent studies have shown that microRNA (miRNA) plays an important regulatory role in the pathological process of ischemic injury. The aim of this study is to investigate the role and the mechanism of miR-21-3p and drosophila mothers against decapentaplegic 2 (SMAD2) in cerebral ischemic reperfusion injured (CIRI) neural cells. The CIRI model was established by oxygen-glucose deprivation and recovery process for N2a cells. The cell viability and the apoptotic was evaluated by MTT assay and the Flow Cytometer, respectively. The expression of miR-21-3p and SMAD2 mRNA was detected by real-time fluorescence quantitative PCR (qRT-PCR), and the expression of SMAD2 and apoptotic-related proteins were detected by Western Blotting. Our results showed that miR-21-3p is down-regulated, and SMAD2 is up-regulated in CIRI. Overexpression of miR- 21-3p inhibits the apoptosis of neural cells in CIRI. miR-21-3p targets SMAD2 and inhibits SMAD2 expression. Overexpression of SMAD2 eliminates the protective effect of over-expression of miR-21-3p on neural cells in CIRI. Token together, this study provides a theoretical basis for the mechanism of ischemic reperfusion injury in neural cells and a new molecular target for ischemic stroke therapy.

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.

LncRNA-MALAT1/miRNA-204-5p/Smad4 Axis Regulates Epithelial–Mesenchymal Transition, Proliferation and Migration of Lens Epithelial Cells

ABSTRACT Purpose: Posterior capsular opacification (PCO), a common complication after cataract surgery, primarily originated from the epithelial–mesenchymal transition (EMT), proliferation, and migration of human lens epithelial cells (LECs). This study aimed to explore whether the long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1)/miR-204-5p/Smad4 axis are involved in EMT of LECs. Materials and methods LECs were cultured and induced with TGF-β2 (10 ng/mL). SiRNA against MALAT1 (Si‐MALAT1) was transfected into LECs to knockdown the expression of MALAT1. To overexpress or knockdown miR‐204-5p, miR-204-5p mimics (miR-204-5p mimics) and anti-miR-204-5p (miR-204-5p inhibitor) were transfected into LECs. We used RNA FISH to identify the location of MALAT1. RNA levels of MALAT1 and miR-204-5p were analyzed by RT-qPCR. Additionally, target protein levels of Smad4, epithelial differentiation and mesenchymal markers were analyzed with Western blot. We employed EdU Labeling to measured cell proliferation and performed Transwell Assay to analyze the cell migration. Dual-luciferase reporter assays in LECs were conducted to verify whether miRNA-204-5p was negatively regulated by MALAT1 and Smad4 was a direct target of miR-204-5p. Results The expression of MALAT1 was upregulated in PCO specimens. MALAT1 was overexpressed in TGF-β2 induced LECs, and the knockdown of MALAT1 could attenuate TGF-β2 induced EMT. Besides, the upregulation of MALAT1 was correlated with the downregulation of miR-204-5p and upregulation of Smad4. Importantly, MALAT1 was revealed to be located in the cytoplasm of LECs. Furthermore, luciferase reporter assays confirmed that MALAT1 could negatively regulate the expression of miR-204-5p and then regulate its direct target Smad4. Finally, the knockdown of MALAT1 could inhibit the EMT, proliferation, and migration of LECs; however, those can be reversed by anti-miR-204-5p. Conclusions Our findings reveal that MALAT1 may regulate EMT, proliferation, and migration of LECs as a ceRNA by “sponging” miR-204-5p and targeting Smad4, and serve as a promising therapeutic target in preventing PCO.

Bleomycin: A novel osteogenesis inhibitor of dental follicle cells via a TGF-β1/SMAD7/RUNX2 pathway.

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. 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. 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. 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.

Identifying miRNA modules associated with progression of keloids through weighted gene co-expression network analysis and experimental validation in vitro

Keloid is a type of skin fibroproliferative disease, characterized by excessive deposition of collagen in the extracellular matrix, myofibroblast activation and invasive growth to the surrounding normal skin tissue. However, the specific pathogenesis of keloids is not yet fully understood and existing treatment strategies are unsatisfied. It is therefore urgent to explore new biomarkers associated with its progression for keloids. In this study, the microarray dataset GSE113620 was downloaded from the Gene Expression Omnibus (GEO) database to screen out the differential expression of miRNAs (DEMs). The DEMs with large variance were applied to construct a weighted gene co-expression network to identify miRNA modules that are closely relevant to keloid progression. It is worth noting that miR-424-3p in the blue module (r = 0.98, p = 1e-18) is considered to be the ultimate target most relevant to keloid progression through co-expressed network analysis. Subsequently, the results of molecular biology experiments determine that miR-424-3p targeting Smad7 significantly enhanced the ability of cell proliferation, migration and collagen secretion after transfection with miR-424-3p mimic, while the apoptosis rate was significantly reduced. On the contrary, the miR-424-3p inhibitor performs the exact opposite function.

Circular RNA hsa_circ_0004872 inhibits gastric cancer progression via the miR-224/Smad4/ADAR1 successive regulatory circuit

BackgroundEmerging evidence has shown that circular RNAs (circRNAs) play a crucial regulatory role in the occurrence and development of cancer. Exploring the roles and mechanisms of circRNAs in tumorigenesis and progression may help to identify new diagnostic markers and therapeutic targets. In the present study, we investigated the role and regulatory mechanism of hsa_circ_0004872 in gastric cancer (GC).MethodsqRT-PCR was used to determine the expression of hsa_circ_0004872 in GC tissues and cells. EdU, CCK-8, transwell and scratch wound healing assays were used to assess the role of hsa_circ_0004872 in GC cell proliferation, invasion and migration, respectively. Subcutaneous and tail vein tumor injections in nude mice were used to assess the role of hsa_circ_0004872 in vivo. RIP assay, biotin-coupled probe pull-down assay, FISH and luciferase reporter assay were performed to confirm the relationship between hsa_circ_0004872 and the identified miRNA. ChIP assay, luciferase reporter assay and western blot were used to determine the direct binding of Smad4 to the promoter of the ADAR1 gene.ResultsIn this study, we found that hsa_circ_0004872 was dramatically downregulated in GC tissues compared with adjacent noncancerous tissues. The expression level of hsa_circ_0004872 was associated with tumor size and local lymph node metastasis. Enforced expression of hsa_circ_0004872 inhibited the proliferation, invasion and migration of GC cells, whereas knockdown of hsa_circ_0004872 had the opposite effects. Nude mice experiments showed that ectopic expression of hsa_circ_0004872 dramatically inhibited tumor growth and metastasis in vivo. Moreover, we demonstrated that hsa_circ_0004872 acted as a “molecular sponge” for miR-224 to upregulate the expression of the miR-224 downstream targets p21 and Smad4. Importantly, we found that the RNA-editing enzyme ADAR1 inhibited hsa_circ_0004872 expression and further led to the upregulation of miR-224. Smad4, the downstream target of miR-224, could further affect hsa_circ_0004872 levels by directly binding to the promoter region of ADAR1 to inhibit ADAR1 expression.ConclusionsOur findings showed that hsa_circ_0004872 acted as a tumor suppressor in GC by forming a negative regulatory loop consisting of hsa_circ_0004872/miR-224/Smad4/ADAR1. Thus, hsa_circ_0004872 may serve as a potential biomarker and therapeutic target for GC.

Transgenic Mice Overexpressing Erythroferrone, a Novel Erythrokine, Develop Iron Overload and Multi-Organ Iron-Independent Abnormalities

Erythroferrone (ERFE) is a hormone secreted by erythroblasts in response to endogenous or exogenous erythropoietic stimuli. ERFE suppresses the production of the iron-regulatory hormone hepcidin by hepatocytes, thereby allowing for increased iron absorption and iron mobilization to support intensified erythropoiesis. In disorders of ineffective erythropoiesis, such as β-thalassemia or congenital dyserythropoietic anemias, chronic suppression of hepcidin results in excessive iron absorption, toxic iron accumulation and a range of systemic manifestations, some of which appear unrelated to iron overload. Recent evidence suggests that ERFE is a ligand trap for select bone morphogenetic proteins (BMPs). BMPs regulate iron homeostasis, but also modulate diverse physiologic processes including embryonic development, kidney growth, and bone homeostasis. Existing mouse models of β-thalassemia used to study the effects of elevated ERFE in vivo are confounded by chronic anemia and hemolysis, complicating attribution of observed phenotypes directly to the action of ERFE. Additionally, serum ERFE levels in these mouse models are much lower than those measured in human patients with β-thalassemia, resulting in a potential underestimation of the pathophysiologic effects of elevated ERFE. To determine the specific effect of elevated ERFE levels in vivo, we generated multiple lines of novel transgenic mice that selectively overexpress graded levels of Erfe in erythroid cells. As expected, Erfe transgenic mice, relative to wild-type littermates, displayed a dose-dependent phenotype of liver iron loading with inappropriately low hepcidin expression. At 6 weeks of age liver nonheme iron levels in transgenic mice ranged from ~2 times that of wild-type littermates, in the lowest expressing line, to ~4 times that of wild-type littermates in the highest-expressing line (line H mice). However, elevated Erfe expression did not consistently alter expression of the BMP target genes Id1 or Smad7 in either the liver or bone marrow. Line-H transgenic mice had increased hemoglobin, hematocrit, mean corpuscular volume, and serum iron suggesting that elevated ERFE levels promoted erythropoiesis, at least in part, by suppressing hepcidin and mobilizing more iron, generating a phenotype similar to mouse models of hereditary hemochromatosis. We also observed phenotypic features that appeared unrelated to iron excess. Line-H breeding diverged from the expected mendelian inheritance ratio of 50% transgenic pups for male mice at weaning age, yielding 37% transgenic pups (N = 108, p =0.0035). Transgenic mice from line-H also had lower body weights and reduced kidney size, accompanied by higher serum urea levels. Additionally, approximately a third of line-H transgenic mice displayed an unusual behavior characterized by repetitive circling and an impaired righting reflex, suggesting vestibular dysfunction. These findings of stunted growth, renal impairment and behavioral abnormalities raise the possibility that elevated ERFE levels may exert iron-independent adverse effects in congenital anemias with ineffective erythropoiesis and in β-thalassemia, perhaps by inhibiting BMP-dependent developmental signals. Future studies using the graded expression of the Erfe-transgene combined with mouse models of dyserythropoiesis will facilitate analysis of the contribution of varying levels of ERFE to the pathophysiology of ineffective erythropoiesis and lay the foundation for pharmacologically targeting ERFE for the amelioration of the hematologic and nonhematologic manifestations of anemias with ineffective erythropoiesis. Disclosures Nemeth: Vifor: Consultancy; Protagonist: Consultancy; Ionis Pharmaceuticals: Consultancy; Silarus Therapeutics: Current equity holder in private company; Intrinsic LifeSciences: Current equity holder in private company. Ganz:Disc Medicine: Consultancy; Silarus Therapeutics: Current equity holder in private company; Intrinsic LifeSciences: Current equity holder in private company; Ambys: Consultancy; Sierra Oncology: Consultancy; Rockwell: Consultancy; Gossamer Bio: Consultancy; American Regent: Consultancy; Global Blood Therapeutics: Consultancy; Astellas: Consultancy; Akebia: Consultancy; Vifor: Consultancy; Ionis Pharmaceuticals: Consultancy.

Chemical proteomics reveals ligustilide targets SMAD3, inhibiting collagen synthesis in aortic endothelial cells

Atherosclerosis is a persistent inflammatory state, while vascular endothelial fibrosis is one of the primary causes of atherosclerosis development. Although ligustilide (Lig) was shown to exert obvious antiatherogenic effects in previous studies, its precise mechanism has not been deeply discussed. In this paper, we designed a Lig-derived photoaffinity labelling (PAL) probe to identify potential therapeutic targets of Lig via chemical proteomics approach. Mothers against decapentaplegic homologue 3 (SMAD3), a signal transmitter of transforming growth factor-β (TGF-β) which promotes the development of vascular fibrosis, was identified as a potential target of Lig. Lig suppressed the phosphorylation and nuclear translocation of SMAD3 by blocking the interaction between SMAD3 and TGF-β receptor 1, thereby inhibiting the collagen synthesis process. Hence, developing a novel SMAD3 inhibitor may present a promising therapeutic option for preventing vascular fibrosis.

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.

MiR-34a-5p Attenuates EMT through targeting SMAD4 in silica-induced pulmonary fibrosis.

Silicosis is an incurable occupational disease, and its pathological feature is diffuse pulmonary fibrosis. Pulmonary epithelial‐mesenchymal transition (EMT) is one of the important events in the pathogenesis of silicosis. Previous studies found that abnormal expression of various microRNAs (miRNAs) involved in the development of lung fibrosis. However, their roles in silicosis have not been elucidated. To research the biological effects of miR‐34a in EMT process in silica‐induced lung fibrosis, we established the silicosis model in mouse and miR‐34a intervention in a cell model of TGF‐β1 stimulated lung epithelial cells (A549). The results showed that miR‐34a expression was down‐regulated in the fibrotic lung tissue after silica treatment, and it was similarly expressed in A549 cells stimulated by TGF‐β1. Meanwhile, silica‐induced EMT process can increase expression of two mesenchymal markers, α‐SMA and vimentin. Furthermore, overexpression miR‐34a markedly inhibited EMT stimulated by TGF‐β1. Mechanistically, SMAD4 was identified as the target of miR‐34a. SMAD4 levels decreased in mRNA and protein levels in A549 cells upon miR‐34a overexpression. In addition, the knockdown of SMAD4 blocked the EMT process. Taken together, miR‐34a regulated EMT, which might be partially realized by targeting SMAD4. Our data might provide new insight into treatment targets for silica‐induced pulmonary fibrosis.

Circular RNA SIPA1L1 promotes osteogenesis via regulating the miR-617/Smad3 axis in dental pulp stem cells

BackgroundBone regeneration is preferred for bone loss caused by tumors, bone defects, fractures, etc. Recently, mesenchymal stem cells are considered as optimistic tools for bone defect therapy. Dental pulp stem cells (DPSCs) are a promising candidate for regenerative medicine and bone regeneration. Our previous study showed that upregulated circSIPA1L1 during osteogenesis of DPSCs is of significance. In this paper, the potential role of circSIPA1L1 in osteogenesis of DPSCs and its underlying mechanisms are explored.MethodsThe circular structure of circSIPA1L1 was identified by Sanger sequencing and PCR. Regulatory effects of circSIPA1L1 and miR-617 on mineral deposition in DPSCs were assessed by alkaline phosphatase (ALP) and alizarin red S (ARS) staining and in vivo bone formation assay were conducted to verify the biological influences of circSIPA1L1 on DPSCs. Western blot was performed to detect the protein expression of Smad3. Localization of circSIPA1L1 and miR-617 was confirmed by FISH. Dual-luciferase reporter assay and rescue experiments were conducted to investigate the role of the circSIPA1L1/miR-617/Smad3 regulatory axis in osteogenesis of DPSCs.ResultsSanger sequencing and back-to-back primer experiments confirmed the closed-loop structure of circSIPA1L1. CircSIPA1L1 could promote the committed differentiation of DPSCs. MiR-617 was predicted to be the target binding circSIPA1L1 through MiRDB, miRTarBase, and TargetScan database analyses, which was further confirmed by dual-luciferase reporter assay. FISH results showed that circSIPA1L1 and miR-617 colocalize in the cytoplasm of DPSCs. MiR-617 exerted an inhibitory effect on the osteogenesis of DPSCs. Knockdown of circSIPA1L1 or upregulation of miR-617 downregulated phosphorylated Smad3. In addition, rescue experiments showed that knockdown of miR-617 reversed the inhibitory effect of circSIPA1L1 on osteogenesis of DPSCs.ConclusionCircRNASIPA1L1 promotes osteogenesis of DPSCs by adsorbing miR-617 and further targeting Smad3.

MiR-143 Regulates Milk Fat Synthesis by Targeting Smad3 in Bovine Mammary Epithelial Cells.

Simple SummaryThe fat content of milk is the main determinant of its nutritional value, and therefore the study of milk fat synthesis has often received a special focus in research. MicroRNAs (miRNAs) are short RNA sequences which have a crucial function in the synthesis of milk fat. miR-143 is one of the miRNAs closely related to lipid metabolism. In this study, the results showed that miR-143 significantly promoted lipid droplet formation and increased the level of intracellular triglyceride (TAG) via increasing the lipid synthesis related genes such as PPARγ, FASN, SCD1, CEBPβ, and SREBP1 by targeting Smad3. Herein, we constructed a miR-143-Smad3 regulatory network map, which revealed the interactions between miR-143 and Smad3 in milk fat synthesis. These findings provide an insight into understanding the theoretical basis of the genes and can thus be applied in the molecular breeding of dairy cows.Milk fat is the main nutritional component of milk and is also an important indicator for evaluating milk quality. Substantial evidence has implicated miRNAs in the synthesis of milk fat. miR-143 is one of the miRNAs closely related to lipid metabolism. Herein, miR-143 upregulation remarkably promoted the production of lipid droplets and increased the level of intracellular triglyceride (TAG). Meanwhile, miR-143 suppression overtly repressed TAG synthesis and lipid droplet accumulation in bovine mammary epithelial cells (BMECs). At the same time, miR-143 significantly upregulated the genes associated with lipid synthesis, including PPARγ, SCD1, CEBPβ, and SREBP1. To examine the regulatory mechanism of miR-143 in milk fat synthesis, Smad3 was predicted as a new potential miR-143 target gene by TargetScan. Further studies found that miR-143 expression was inversely related to the levels of Smad3 mRNA and protein. Furthermore, luciferase reporter assays confirmed Smad3 to be a miR-143 direct target. Moreover, Smad3 gene silencing significantly increased intracellular TAG level in BMECs. These findings revealed that miR-143 promotes the TAG synthesis in BMECs via increasing the lipid synthesis related gens expression by targeting Smad3. The results of this study can be exploited in devising novel approaches for improving the nutritional value of milk in dairy cows.

Abstract 6336: Non-canonical activation of Gli-2 by TGF-β/Smad3 signaling pathway in modulation of chemoresistance of hepatocellular carcinoma

Abstract BACKGROUND: It has been demonstrated in our previous study that poorly-differentiated hepatoma cells with low α-feto-protein (AFP) expression and a CD133-/EpCAM- surface profile exhibited aberrant hedgehog signaling (Hh) activity, epithelial-mesenchymal transition and chemoresistance. However, expression of canonical Hh molecules (Sonic ligand and SMO) was not increased in poorly-differentiated hepatoma cells in contrast to well-differentiated hepatoma cells, indicating that Hh transcription factors Gli-1/2 may be activated through a non-classic pathway. The present study aims to investigate whether the TGF-β/Smad3 pathway is responsible for Gli-2 activation through a non-canonical pathway. METHODS: Expression of Sonic Hh (Shh) and Smad3 was assessed in both hepatoma cells and HCC specimens. Gli-2 and Smad3 expression was suppressed by lentiviral vector-mediated RNA interference in poorly-differentiated hepatoma Huh-7-DN and HLE cells. Chemosensitivity was evaluated in hepatoma cells with a Gli-2 or Smad3 inhibitor. RESULTS: Significantly increased Gli-1 and Gli-2 protein levels were observed in 78% (11 out of 14) and 50% (7 out of 14) HCC specimens. In 7 HCC patients with a normal serum AFP value (<20 μg/L), Gli-1/2 protein levels were obviously higher than their pericancerous tissue. Moreover, increased Gli-2 and ABCC1 protein levels were accompanied with increased TGF-β1 and p-Smad3 protein levels in 5 out 7 HCC specimens; whereas Shh protein levels were similar to paired pericancerous tissue. Consistent with the findings from HCC specimens, significantly higher Gli-2 and p-Smad3 nuclear protein levels were associated with increased ABCC1 protein levels in poorly-differentiated hepatoma cells (Huh-7-trans, Huh-7-DN, HLE and HLF); whereas Shh and SMO protein levels were not significantly different from well-differentiated hepatoma Huh-7 and Hep3B cells. Suppressing Gli-2 expression by specific shRNA caused marked decreased expression of Gli-2 and ABCC1 in Huh-7-DN, Huh-7-trans and HLE cells. In addition, expression of Smad3, Gli-2 and ABCC1 was significantly down-regulated in HLE cells after infected with a lentiviral vector harboring shSmad3, indicating that Gli-2 is a downstream target of Smad3. Sorafenib IC50 was significantly reduced from 35.76 to 17.45 μM or 14.04 μM in HLE cells when treated with a GLI inhibitor (GANT-61) or a Smad3 inhibitor (SIS3) at 5 μM. A potential GLI-binding site in the ABCC1 promoter was identified. ABCC1-Gli-luciferase reporter activity was increased up to 39-fold in HLE cells when transfected with constructs containing the potential GLI-binding site, compared to the construct that lacked a GLI-binding site. CONCLUSION: TGF-β/Smad3 activated Gli-2 and its direct transcriptional target ABCC1 in poorly-differentiated and chemoresistant hepatoma cells. Suppressing TGF-β/Smad3/Gli-2 signaling activity confers a potential molecular target in partially alleviating Sorafenib resistance for the treatment of refractory hepatoma. Citation Format: Jia Ding, Hui-yan Li, Li Zhang, Xiao-tian Zhou, Jian Wu. Non-canonical activation of Gli-2 by TGF-β/Smad3 signaling pathway in modulation of chemoresistance of hepatocellular carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6336.