SMAD Family Member Research Articles

Evaluation of the interaction between tumor growth factor-β and interferon type I pathways in patients with COVID-19: focusing on ages 1 to 90 years

BackgroundEvidence revealed that age could affect immune responses in patients with the acute respiratory syndrome of coronavirus 2 (SARS-CoV-2) infection. This study investigated the impact of age on immune responses, especially on the interaction between the tumor growth factor-β (TGF-β) and interferon type-I (IFN-I) axes in the pathogenesis of novel coronavirus disease 2019 (COVID-19).MethodsThis age-matched case–control investigation enrolled 41 COVID-19 patients and 40 healthy controls categorized into four groups, including group 1 (up to 20 years), group 2 (20–40 years), group 3 (40–60 years), and group 4 (over 60 years). Blood samples were collected at the time of admission. The expression of TGF-βRI, TGF-βRII, IFNARI, IFNARII, interferon regulatory factor 9 (IRF9), and SMAD family member 3 (SMAD3) was measured using the real-time PCR technique. In addition, serum levels of TGF-β, IFN-α, and SERPINE1 were measured by the enzyme-linked immunosorbent assay (ELISA) technique. All biomarkers were measured and analyzed in the four age studies groups.ResultsThe expression of TGF-βRI, TGF-βRII, IFNARI, IFNARII, IRF9, and SMAD3 was markedly upregulated in all age groups of patients compared with the matched control groups. Serum levels of IFN-α and SERPINE1 were significantly higher in patient groups than in control groups. While TGF-β serum levels were only significantly elevated in the 20 to 40 and over 60 years patient group than in matched control groups.ConclusionsThese data showed that the age of patients, at least at the time of admission, may not significantly affect TGF-β- and IFN-I-associated immune responses. However, it is possible that the severity of the disease affects these pathway-mediated responses, and more studies with a larger sample size are needed to verify it.

NcRNA-mediated upregulation of FAM83A is associated with poor prognosis and immune infiltration in pancreatic cancer.

Malignant pancreatic cancer has poor long-term survival. Increasing evidence shows that FAM83A (family with sequence similarity 83 member A) plays a vital role in tumorigenesis and malignant progression in some human cancer types. The present study explored the potential mechanism of FAM83A in improving the prognosis of pancreatic cancer patients. Transcriptomic and clinical data from patients were obtained from The Cancer Genome Atlas while FAM83A expression was measured in tumorous pancreatic tissue compared with normal controls by quantitative real-time PCR and immunohistochemistry. FAM83A is a vital prognostic indicator and potential oncogene in pancreatic cancer via pan-cancer analysis. In silico analysis revealed that AL049555.1/hsa-miR-129-5p axis was the pivotal upstream ncRNA- mediated pathway of FAM83A in pancreatic cancer. Furthermore, FAM83A expression was related to immune cell infiltration through vital immune-related genes including programmed cell death 1 (PDCD1), and tumorigenesis through common mutation genes including KRAS protooncogene GTPase (KRAS), and SMAD family member 4 (SMAD4). In summary, ncRNA-mediated upregulation of FAM83A is associated with poor long-term survival and immune cell infiltration in pancreatic cancer. FAM83A may be used as a novel survival-related and immune-related biomarker. This information suggests that FAM83A may be a novel therapeutic target for combined or individual treatment for patients with pancreatic cancer.

Role of miR-300-3p in Leydig cell function and differentiation: A therapeutic target for obesity-related testosterone deficiency

MicroRNAs (miRNAs) regulate various cellular functions, but their specific roles in the regulation of Leydig cells (LCs) have yet to be fully understood. Here, we found that the expression of miR-300-3p varied significantly during the differentiation from progenitor LCs (PLCs) to adult LCs (ALCs). High expression of miR-300-3p in PLCs inhibited testosterone production and promoted PLC proliferation by targeting the steroidogenic factor-1 (Sf-1) and transcription factor forkhead box O1 (FoxO1) genes, respectively. As PLCs differentiated into ALCs, the miR-300-3p expression level significantly decreased, which promoted testosterone biosynthesis and suppressed proliferation of ALCs by upregulating SF-1 and FoxO1 expression. The LH/METTL3/SMURF2/SMAD2 cascade pathway controlled miR-300-3p expression, in which luteinizing hormone (LH) upregulated SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2) expression through methyltransferase like 3 (METTL3)-mediated Smurf2 N6-methyladenosine modification. The Smurf2 then suppressed miR-300 transcription by inhibiting SMAD family member 2 (SMAD2) binding to the promoter of miR-300. Notably, miR-300-3p was associated with an obesity-related testosterone deficiency in men and the inhibition of miR-300-3p effectively rescued testosterone deficiency in obese mice. These findings suggested that miR-300-3p plays a pivotal role in LC differentiation and function, and could be a promising diagnostic or therapeutic target for obesity-related testosterone deficiency.

Integrated Proteomics Unveils Nuclear PDE3A2 as a Regulator of Cardiac Myocyte Hypertrophy.

Signaling by cAMP is organized in multiple distinct subcellular nanodomains regulated by cAMP-hydrolyzing PDEs (phosphodiesterases). Cardiac β-adrenergic signaling has served as the prototypical system to elucidate cAMP compartmentalization. Although studies in cardiac myocytes have provided an understanding of the location and properties of a handful of cAMP subcellular compartments, an overall view of the cellular landscape of cAMP nanodomains is missing. Here, we combined an integrated phosphoproteomics approach that takes advantage of the unique role that individual PDEs play in the control of local cAMP, with network analysis to identify previously unrecognized cAMP nanodomains associated with β-adrenergic stimulation. We then validated the composition and function of one of these nanodomains using biochemical, pharmacological, and genetic approaches and cardiac myocytes from both rodents and humans. We demonstrate the validity of the integrated phosphoproteomic strategy to pinpoint the location and provide critical cues to determine the function of previously unknown cAMP nanodomains. We characterize in detail one such compartment and demonstrate that the PDE3A2 isoform operates in a nuclear nanodomain that involves SMAD4 (SMAD family member 4) and HDAC-1 (histone deacetylase 1). Inhibition of PDE3 results in increased HDAC-1 phosphorylation, leading to inhibition of its deacetylase activity, derepression of gene transcription, and cardiac myocyte hypertrophic growth. We developed a strategy for detailed mapping of subcellular PDE-specific cAMP nanodomains. Our findings reveal a mechanism that explains the negative long-term clinical outcome observed in patients with heart failure treated with PDE3 inhibitors.

Relationship of cytokine gene polymorphism and the development of liver fibrosis in patients with chronic hepatitis B&C

One of the features of the spread of infectious diseases is an increase in the number of mixed infections caused by various types of microorganisms: bacteria, viruses, protozoa, fungi, mycoplasmas, chlamydia, etc. In mixed infections, the simultaneous presence of several pathogens leads to not only to the complex interaction of pathological processes, but also complicates the diagnosis and choice of treatment. Mixed hepatitis is characterized by high biochemical activity and histological changes, the presence of signs of portal hypertension. Infection with two viruses increases the risk of developing hepatocellular carcinoma. It should be noted that in patients with HBV infection, the development of hepatocellular carcinoma is possible at the stage of chronic hepatitis before the onset of liver cirrhosis. The aim of the study: to investigate a comprehensive assessment of the allelic polymorphism of the IL-4 (rs2243250), IL-10 (rs1800896), TNFα (rs1800620), SMAD family member 7 (rs4939827) and eIF3h (rs16892766) genes with the degree of fibrosis in patients with chronic hepatitis B&C. Materials and methods. In the course of the study, 62 patients with chronic hepatitis B&C were examined before the start of treatment and after the end of the course of antiviral therapy. The degree of liver fibrosis was established by FibroScan. Molecular genetic studies included the determination of polymorphic variants of the IL-4 (rs2243250), IL10 (rs1800896), TNFα (rs1800620), SMAD family member 7 (rs4939827), eIF3h (rs16892766) genes. Polymorphism was studied by amplification of the corresponding regions of the genome by PCR. Results and discussions. Genotypes CC IL-4 (rs2243250), GG IL-10 (rs1800896), GG TNFα (rs1800620), AA SMAD family member 7 (rs4939827), TT eIF3h (rs16892766) have a protective effect on the course of chronic hepatitis B&C, as they are established in patients with chronic hepatitis B&C with a lower degree of fibrosis. Heterozygous CT IL-4 (rs2243250), GA IL-10 (rs1800896), GA TNFα (rs1800620), CC SMAD family member 7 (rs4939827), CC eIF3h (rs16892766) have a profibrotic effect on the course of chronic hepatitis B&C, therefore that they are established in patients with chronic hepatitis B&C with a greater degree of fibrosis.In patients with a low stage of fibrosis, the genotypes CC IL-4 (rs2243250), GG IL-10 (rs1800896), GG TNFα (rs1800620), AA SMAD family member 7 (rs4939827), TT eIF3h (rs16892766), are most often determined. In patients with a degree of fibrosis, other genotypes were detected, namely CT IL-4 (rs2243250), GA IL-10 (rs1800896), GA TNFα (rs1800620), CC SMAD family member 7 (rs4939827), CC eIF3h (rs16892766). Сonclusions. The revealed differences in the frequency of occurrence of various gene polymorphisms in healthy subjects and patients with chronic hepatitis indicate a certain predisposition to the development of a chronic inflammatory process in the liver.

The Differentially Expressed Genes Responsible for the Development of T Helper 9 Cells From T Helper 2 Cells in Various Disease States: Immuno-Interactomics Study

Background T helper (Th) 9 cells are a novel subset of Th cells that develop independently from Th2 cells and are characterized by the secretion of interleukin (IL)-9. Studies have suggested the involvement of Th9 cells in variable diseases such as allergic and pulmonary diseases (eg, asthma, chronic obstructive airway disease, chronic rhinosinusitis, nasal polyps, and pulmonary hypoplasia), metabolic diseases (eg, acute leukemia, myelocytic leukemia, breast cancer, lung cancer, melanoma, pancreatic cancer), neuropsychiatric disorders (eg, Alzheimer disease), autoimmune diseases (eg, Graves disease, Crohn disease, colitis, psoriasis, systemic lupus erythematosus, systemic scleroderma, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, atopic dermatitis, eczema), and infectious diseases (eg, tuberculosis, hepatitis). However, there is a dearth of information on its involvement in other metabolic, neuropsychiatric, and infectious diseases. Objective This study aims to identify significant differentially altered genes in the conversion of Th2 to Th9 cells, and their regulating microRNAs (miRs) from publicly available Gene Expression Omnibus data sets of the mouse model using in silico analysis to unravel various pathogenic pathways involved in disease processes. Methods Using differentially expressed genes (DEGs) identified from 2 publicly available data sets (GSE99166 and GSE123501) we performed functional enrichment and network analyses to identify pathways, protein-protein interactions, miR-messenger RNA associations, and disease-gene associations related to significant differentially altered genes implicated in the conversion of Th2 to Th9 cells. Results We extracted 260 common downregulated, 236 common upregulated, and 634 common DEGs from the expression profiles of data sets GSE99166 and GSE123501. Codifferentially expressed ILs, cytokines, receptors, and transcription factors (TFs) were enriched in 7 crucial Kyoto Encyclopedia of Genes and Genomes pathways and Gene Ontology. We constructed the protein-protein interaction network and predicted the top regulatory miRs involved in the Th2 to Th9 differentiation pathways. We also identified various metabolic, allergic and pulmonary, neuropsychiatric, autoimmune, and infectious diseases as well as carcinomas where the differentiation of Th2 to Th9 may play a crucial role. Conclusions This study identified hitherto unexplored possible associations between Th9 and disease states. Some important ILs, including CCL1 (chemokine [C-C motif] ligand 1), CCL20 (chemokine [C-C motif] ligand 20), IL-13, IL-4, IL-12A, and IL-9; receptors, including IL-12RB1, IL-4RA (interleukin 9 receptor alpha), CD53 (cluster of differentiation 53), CD6 (cluster of differentiation 6), CD5 (cluster of differentiation 5), CD83 (cluster of differentiation 83), CD197 (cluster of differentiation 197), IL-1RL1 (interleukin 1 receptor-like 1), CD101 (cluster of differentiation 101), CD96 (cluster of differentiation 96), CD72 (cluster of differentiation 72), CD7 (cluster of differentiation 7), CD152 (cytotoxic T lymphocyte–associated protein 4), CD38 (cluster of differentiation 38), CX3CR1 (chemokine [C-X3-C motif] receptor 1), CTLA2A (cytotoxic T lymphocyte–associated protein 2 alpha), CTLA28, and CD196 (cluster of differentiation 196); and TFs, including FOXP3 (forkhead box P3), IRF8 (interferon regulatory factor 8), FOXP2 (forkhead box P2), RORA (RAR-related orphan receptor alpha), AHR (aryl-hydrocarbon receptor), MAF (avian musculoaponeurotic fibrosarcoma oncogene homolog), SMAD6 (SMAD family member 6), JUN (Jun proto-oncogene), JAK2 (Janus kinase 2), EP300 (E1A binding protein p300), ATF6 (activating transcription factor 6), BTAF1 (B-TFIID TATA-box binding protein associated factor 1), BAFT (basic leucine zipper transcription factor), NOTCH1 (neurogenic locus notch homolog protein 1), GATA3 (GATA binding protein 3), SATB1 (special AT-rich sequence binding protein 1), BMP7 (bone morphogenetic protein 7), and PPARG (peroxisome proliferator–activated receptor gamma, were able to identify significant differentially altered genes in the conversion of Th2 to Th9 cells. We identified some common miRs that could target the DEGs. The scarcity of studies on the role of Th9 in metabolic diseases highlights the lacunae in this field. Our study provides the rationale for exploring the role of Th9 in various metabolic disorders such as diabetes mellitus, diabetic nephropathy, hypertensive disease, ischemic stroke, steatohepatitis, liver fibrosis, obesity, adenocarcinoma, glioblastoma and glioma, malignant neoplasm of stomach, melanoma, neuroblastoma, osteosarcoma, pancreatic carcinoma, prostate carcinoma, and stomach carcinoma.

Rebalancing SMAD7/SMAD3 Signaling Reduces Adhesion Formation during Flexor Tendon Healing.

Transforming growth factor-β is a key factor in regulating adhesion formation during tendon healing. We investigated the effectiveness of SMAD family members, SMAD7 and SMAD3, in the TGF-β/Smad signaling during flexor tendon repair. Mouse flexor toe deep tendon rupture anastomosis models were made. On days 3, 7, 14, 21, and 28, the expressions of smad7 and smad3 in flexor tendon tissues were detected by RT-qPCR and western blot. Furthermore, postoperative intraperitoneal injections of SMAD7 agonists or SMAD3 antagonists were given. The degree of tendon healing was evaluated by adhesion testing and biomechanical experiments. Hematoxylin and eosin (HE) staining was used to observe the pathological changes. Immunohistochemistry was used to evaluate the expressions of collagen III, SMAD3, and SMAD7. The mRNA levels of matrix metalloproteinases, Mmp2 and Mmp9, and scleraxis (SCX) in flexor tendon tissue were detected by RT-qPCR. Smad3 expression increased and Smad7 expression decreased in flexor tendon tissue after injury. In addition, the SMAD7 agonist blocked SMAD3 phosphorylation. SMAD7 agonist and SMAD3 antagonist both improved adhesion formation during flexor tendon healing, and decreased the expressions of collagen III, Mmp9, and SCX, while increasing Mmp2 expression. This study provides a possible theoretical basis for the SMAD7-SMAD3 signal cascade during flexor tendon adhesion healing.

Nanoparticles Targeting Delivery Antagomir-483-5p to Bone Marrow Mesenchymal Stem Cells Treat Osteoporosis by Increasing Bone Formation.

Promoting bone marrow mesenchymal stem cell (BMSC) osteoblastic differentiation is a promising therapeutic strategy for osteoporosis (OP). The present study demonstrates that miR- 483-5p inhibits the osteogenic differentiation of BMSCs. Therefore, selectively delivering the nanoparticles carrying antagomir-483-5p (miR-483-5p inhibitor) to BMSCs is expected to become an effective treatment drug for OP. Real-time PCR assays were used to analyze miR-483-5p, ALP and Bglap levels in BMSCs of ovariectomized and aged osteoporotic mice. Immunoglobulin G and poloxamer-188 encapsulated the functional small molecules, and a BMSC-targeting aptamer was employed to confirm the direction of the nanoparticles to selectively and efficiently deliver antagomir-483-5p to BMSCs in vivo. Luciferase assays were used to determine the target genes of miR-483-5p. Western blot assays and immunohistochemistry staining were used to detect the targets in vitro and in vivo. miR-483-5p levels were increased in BMSCs of ovariectomized and aged osteoporotic mice. Inhibiting miR-483-5p levels in BMSCs by antagomir-483-5p in vitro promoted the expression of bone formation markers, such as ALP and Bglap. The FAM-BMSC-aptamer-nanoparticles carrying antagomir- 483-5p were taken up by BMSCs, resulting in stimulation of BMSC osteoblastic differentiation in vitro and osteoporosis prevention in vivo. Furthermore, our research demonstrated that mitogen-activated protein kinase 1 (MAPK1) and SMAD family member 5 (Smad5) were direct targets of miR-483-5p in regulating BMSC osteoblastic differentiation and osteoporosis pathological processes. The important therapeutic role of FAM-BMSC-aptamer-nanoparticles carrying antagomir- 483-5p in osteoporosis was established in our study. These nanoparticles are a novel candidate for the clinical prevention and treatment of osteoporosis. The optimized, targeted drug delivery platform for small molecules will provide new ideas for treating clinical diseases.

Kalkitoxin attenuates calcification of vascular smooth muscle cells via RUNX-2 signaling pathways.

Kalkitoxin (KT) is an active lipopeptide isolated from the cyanobacterium Lyngbya majuscula found in the bed of the coral reef. Although KT suppresses cell division and inflammation, KT's mechanism of action in vascular smooth muscle cells (VSMCs) is unidentified. Therefore, our main aim was to investigate the impact of KT on vascular calcification for the treatment of cardiovascular disease. Using diverse calcification media, we studied the effect of KT on VSMC calcification and the underlying mechanism of this effect. VSMC was isolated from the 6 weeks ICR mice. Then VSMCs were treated with different concentrations of KT to check the cell viability. Alizarin red and von Kossa staining were carried out to examine the calcium deposition on VSMC. Thoracic aorta of 6 weeks mice were taken and treated with different concentrations of KT, and H and E staining was performed. Real-time polymerase chain reaction and western blot were performed to examine KT's effect on VSMC mineralization. Calcium deposition on VSMC was examined with a calcium deposition quantification kit. Calcium deposition, Alizarin red, and von Kossa staining revealed that KT reduced inorganic phosphate-induced calcification phenotypes. KT also reduced Ca++-induced calcification by inhibiting genes that regulate osteoblast differentiation, such as runt-related transcription factor 2 (RUNX-2), SMAD family member 4, osterix, collagen 1α, and osteopontin. Also, KT repressed Ca2+-induced bone morphogenetic protein 2, RUNX-2, collagen 1α, osteoprotegerin, and smooth muscle actin protein expression. Likewise, Alizarin red and von Kossa staining showed that KT markedly decreased the calcification of ex vivo ring formation in the mouse thoracic aorta. This experiment demonstrated that KT decreases vascular calcification and may be developed as a new therapeutic treatment for vascular calcification and arteriosclerosis.

Hydrogen sulfide alleviates hypothyroidism-induced myocardial fibrosis in rats through stimulating autophagy and inhibiting TGF-β1/Smad2 pathway.

Hypothyroidism alone can lead to myocardial fibrosis and result in heart failure, but traditional hormone replacement therapy does not improve the fibrotic situation. Hydrogen sulfide (H2S), a new gas signaling molecule, possesses anti-inflammatory, antioxidant, and anti-fibrotic capabilities. Whether H2S could improve hypothyroidism-induced myocardial fibrosis are not yet studied. In our study, H2S could decrease collagen deposition in the myocardial tissue of rats caused by hypothyroidism. Furthermore, in hypothyroidism-induced rats, we found that H2S could enhance cystathionine-gamma-lyase (CSE), not cystathionine β-synthase (CBS), protein expressions. Finally, we noticed that H2S could elevate autophagy levels and inhibit the transforming growth factor-β1 (TGF-β1) signal transduction pathway. In conclusion, our experiments not only suggest that H2S could alleviate hypothyroidism-induced myocardial fibrosis by activating autophagy and suppressing TGF-β1/SMAD family member 2 (Smad 2) signal transduction pathway, but also show that it can be used as a complementary treatment to conventional hormone therapy.

ALKBH1-mediated m1 A demethylation of METTL3 mRNA promotes the metastasis of colorectal cancer by downregulating SMAD7 expression.

Colorectal cancer (CRC) is one of the most common malignancies, and the main cause of death from CRC is tumor metastasis. m1 A RNA modification plays critical role in many biological processes. However, the role of m1 A modification in CRC remains unclear. Here, we find that the m1 A demethylase alkB homolog 1, histone H2A dioxygenase (ALKBH1) is overexpressed in CRC and is associated with metastasis and poor prognosis. Upregulation of ALKBH1 expression promotes CRC metastasis in vitro and in vivo. Mechanistically, knockdown of ALKBH1 results in a decrease in methyltransferase 3, N6-adenosine-methyltransferase complex catalytic subunit (METTL3) expression, probably due to m1 A modification of METTL3 mRNA, followed by m6 A demethylation of SMAD family member 7 (SMAD7) mRNA. In addition, downregulation of SMAD7 establishes an aggressive phenotype. More importantly, the cell migration and invasion defects caused by ALKBH1 depletion or METTL3 depletion are significantly reversed by SMAD7 silencing. Considering these results collectively, we propose that ALKBH1 promotes CRC metastasis by destabilizing SMAD7 through METTL3.

In the Quest for Potent and Selective Malic Enzyme 3 Inhibitors for the Treatment of Pancreatic Ductal Adenocarcinoma.

The genome of pancreatic ductal adenocarcinoma (PDAC) is associated with frequent deletion of the tumor suppressor gene SMAD family member 4 (SMAD4) with collateral deletion of its chromosomal neighbor malic enzyme 2 (ME2). In SMAD4 -/- /ME2 -/- PDAC cells, ME3 takes over the function of the ME2 enzyme, and hence therapeutic targeting of ME3 is expected to arrest tumor growth. Hitherto no selective small molecule inhibitor of ME3 has been reported in the context of PDAC. Based on the molecular docking studies and structure-activity relationships with the reported ME1 inhibitor, several analogues of 6-piperazin-1-ylpyridin-3-ol amides have been synthesized and screened for their ME inhibition activity. Among them, compound 16b is identified as the most potent and selective ME3 inhibitor with an IC50 of 0.15 μM on ME3, and with 15- and 9-fold selectivity over ME1 and ME2, respectively. In the cell viability assay, compound 16b exhibited an IC50 of 3.5 μM on ME2-null PDAC cells, viz., BxPC-3.

The activation of TGF-β signaling promotes cell migration and invasion of ectopic endometrium by targeting NRP2.

Endometriosis is a gynecological disorder seriously affecting the health and life of women of reproductive age. Neuropilin 2 (NRP2) has been indicated to display a high level in ectopic endometrium. Nevertheless, the specific function of NRP2 in endometriosis is unanswered. RT-qPCR was utilized to detect expression of NRP2 and SMAD family member 2 (SMAD2) in endometrial tissues or endometrial stromal cells (ESCs). Protein levels of transforming growth factor beta (TGF-β) signaling-associated markers and epithelial-mesenchymal transition (EMT)-related markers were examined by western blotting. Transwell assays were utilized for detecting the impact of NRP2 on ectopic ESC phenotypes. ChIP and luciferase reporter assays were performed for identification of the relationship between NRP2 and SMAD2. In this study, NRP2 was overexpressed in ectopic endometria in comparison to eutopic endometria. Depletion of NRP2 restrained ectopic ESC migration, invasiveness and EMT. TGF-β signaling-mediated activation of SMAD2 transcriptionally upregulated NRP2 expression in ectopic ESCs. TGF-β treatment could rescue NRP2 silencing-induced suppressive impact on the behaviors of ectopic ESCs. Overall, the activation of TGF-β signaling contributes to the migration and invasiveness of ectopic ESCs by targeting NRP2.

The Interaction Between SMAD1 and YAP1 Is Correlated with Increased Resistance of Gastric Cancer Cells to Cisplatin.

Drug resistance is a major obstacle leading to treating failure and poor outcome in gastric cancer (GC). This study explores the interaction between SMAD family member 1 (SMAD1) and Yes1-associated transcriptional regulator (YAP1) and their roles in cisplatin (DDP) resistance in GC. Transcriptome analysis predicted that SMAD1 is highly expressed in DDP-resistant cells. Elevated SMAD1 expression was detected in GC tissue and cells, especially in DDP-resistant cells (MKN-45/DDP and AGS/DDP). SMAD1 downregulation in cells decreased 50% inhibition value of DDP, reduced proliferation, migration, and invasion, and promoted cell cycle arrest and apoptosis. A protein-protein interaction network suggested a possible SMAD1 and YAP1 interaction in GC. The SMAD1 and YAP1 interaction was validated by chromatin immunoprecipitation (ChIP), co-immunoprecipitation (Co-IP), and luciferase assays. SMAD1 bound to YAP1 and activated its transcription. SMAD1 formed complexes with YAP1 in nucleus, and YAP1 upregulation enhanced SMAD1 activity as well. Upregulation of YAP1 restored the malignant behaviors of GC cells suppressed by SMAD1 silencing. In vivo, SMAD1 silencing suppressed growth and DDP resistance of xenograft tumors in nude mice, and this suppression was blocked by YAP1 overexpression again. In conclusion, this study demonstrates that SMAD1 can interact with YAP1 to enhance the DDP resistance of GC cells.

The 12-bp indel in the SMAD family member 2 gene is associated with goat growth traits

SMAD family member 2 (SMAD2) is a member of the TGFβ signaling pathway and functions as an essential regulator in the processes of development, cell proliferation, and bone formation. A previous observation reported that a 12-bp deletion of this gene affected the litter size in goats. However, according to our knowledge, no study has reported an association between this polymorphism and goat body measurement traits. The purpose of this study was to investigate the association of the insertion/deletion (indel) within the SMAD2 gene with the growth traits of goats. The indel polymorphism was found to be significantly associated with chest width and bust (p < 0.05), while cannon circumference was significantly the strongest compared to other traits (p < 0.01) and individuals with the DD genotypes were more dominant genotypes than other genotypes. In summary, we found evidence that the 12-bp indel within the SMAD2 gene could improve goat body measurement traits, paving the way for marker-assisted selection in the field of goat genetics and breeding.

Lactate promotes vascular smooth muscle cell switch to a synthetic phenotype by inhibiting miR-23b expression.

Recent research indicates that lactate promotes the switching of vascular smooth muscle cells (VSMCs) to a synthetic phenotype, which has been implicated in various vascular diseases. This study aimed to investigate the effects of lactate on the VSMC phenotype switch and the underlying mechanism. The CCK-8 method was used to assess cell viability. The microRNAs and mRNAs levels were evaluated using quantitative PCR. Targets of microRNA were predicted using online tools and confirmed using a luciferase reporter assay. We found that lactate promoted the switch of VSMCs to a synthetic phenotype, as evidenced by an increase in VSMC proliferation, mitochondrial activity, migration, and synthesis but a decrease in VSMC apoptosis. Lactate inhibited miR-23b expression in VSMCs, and miR-23b inhibited VSMC's switch to the synthetic phenotype. Lactate modulated the VSMC phenotype through downregulation of miR-23b expression, suggesting that overexpression of miR-23b using a miR-23b mimic attenuated the effects of lactate on VSMC phenotype modulation. Moreover, we discovered that SMAD family member 3 (SMAD3) was the target of miR-23b in regulating VSMC phenotype. Further findings suggested that lactate promotes VSMC switch to synthetic phenotype by targeting SMAD3 and downregulating miR-23b. These findings suggest that correcting the dysregulation of miR-23b/SMAD3 or lactate metabolism is a potential treatment for vascular diseases.

Circ_0008529 Contributes to Renal Tubular Cell Dysfunction in High Glucose Stress via miR-185-5p/SMAD2 Pathway in Diabetic Nephropathy.

Circular RNA (circRNA) is key regulator of diabetic nephropathy (DN) progression. However, the role of circ_0008529 in DN progression remains to be better deciphered. Cell viability, cell cycle, apoptosis and inflammation were measured by MTS assay, flow cytometry and corresponding assay kits. RT-qPCR was used to assess the expression of circ_0008529, miR-185-5p and SMAD family member 2 (SMAD2). Also, western blotting was performed to measure protein expression. Target relationship was validated by RNA pull-down assay, dual-luciferase reporter assay and RNA immunoprecipitation assay. Urinary exosome was isolated using ultracentrifugation method and identified by transmission electron microscopy. Receiver operating characteristic curve was used to analyze the diagnostic value of circ_0008529 in DN patients. Circ_0008529 and SMAD2 were upregulated, while miR-185-5p was downregulated in high glucose (HG)-induced renal tubular HK-2 cells. Under HG treatment, cell viability and cell cycle process were suppressed, while apoptosis, inflammation and extracellular matrix accumulation were enhanced. However, interfering circ_0008529 could attenuate HG-induced effects, and this protection was abated by miR-185 inhibition or SMAD2 re-expression. Mechanically, circ_0008529 and SMAD2 were competing endogenous RNAs for miR-185-5p via target binding, and circ_0008529 regulated SMAD2 expression via miR-185-5p. Notably, circ_0008529 expression was upregulated in urinary exosomes of DN patients, and showed diagnostic value (Sensitivity: 70.21%; Specificity: 86.67%). Circ_0008529 might be a potential target for DN, which regulated DN progression via miR-185-5p/SMAD2 pathway.

SMAD3 interacts with vitamin D receptor and affects vitamin D-mediated oxidative stress to ameliorate cerebral ischaemia-reperfusion injury.

Cerebral ischaemia/reperfusion (I/R) injury is caused by blood flow restoration after an ischaemic insult, and effective treatments targeting I/R injury are still insufficient. Oxidative stress plays a critical role in the pathogenesis of cerebral I/R injury. This study investigated whether vitamin D receptor (VDR) could inhibit oxidative stress caused by cerebral I/R injury and explored the detailed mechanism. VDR was highly expressed in brain tissues of mice with cerebral I/R injury. Pretreatment with the active vitamin D calcitriol and synthetic vitamin D analogue paricalcitol (PC) reduced autophagy and apoptosis, improved neurological deficits and decreased infarct size in mice after cerebral I/R. Calcitriol or PC upregulated VDR expression to prevent cerebral I/R injury by affecting oxidative stress. Silencing of VDR reversed the protective effects of calcitriol or PC on brain tissues in mice with cerebral I/R. The bioinformatics analysis revealed that VDR interacted with SMAD family member 3 (SMAD3). It was validated through the chromatin immunoprecipitation assay that SMAD3 can bind to the VDR promoter and VDR can bind to the SMAD3 promoter. Collectively, these findings provide evidence that reciprocal activation between SMAD3 and VDR transcription factors defines vitamin D-mediated oxidative stress to prevent cerebral I/R injury.

LncRNA HOTAIR inhibits the progression of fibroblast-like synoviocytes by sponging miRNA-106b-5p in rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic progressive autoimmune disease of unknown etiology. Human fibroblast-like synoviocytes (HFLSs) are the main effector cells for synovial hyperplasia and invasion in RA. Long non-coding RNAs (lncRNAs) play key roles in several autoimmune diseases, including RA. We investigated the effects of lncRNA HOX transcript antisense intergenic RNA (HOTAIR) on the pathological behavior of HFLSs in RA. The microRNAs (miRNAs) with potential binding sites for lncRNA HOTAIR were predicted using Starbase v2.0. TargetScan (http://www.targetscan.org) was used to analyze the potential target genes of miR-106b-5p. The interactions were further verified using a dual-luciferase reporter assay. RNA and protein expression was determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR) and western blotting. The proliferation, cell invasion and migration, and cell apoptosis of HFLSs in RA was detected by the 3-(4,5-dimethylthiazol)-2,5-diphenyl-tetrazolium bromide (MTT) assay, transwell assay, and flow cytometry (FCM). The dual luciferase reporter assay confirmed the interactions between lncRNA HOTAIR and miR-106b-5p and between miR-106b-5p and SMAD family member 7 (SMAD7). The qRT-PCR results indicated that the expression of lncRNA HOTAIR was markedly decreased and that of miR-106b-5p was markedly increased in HFLSs of RA. Cell proliferation, invasion, and migration of HFLSs were inhibited by lncRNA HOTAIR upregulation, and the expression of miR-106b-5p was negatively regulated by lncRNA HOTAIR in HFLSs. Apoptosis of HFLS cells was improved by the overexpression of lncRNA HOTAIR. All the effects of lncRNA HOTAIR upregulation on HFLSs were reversed after the overexpression of miR-106b-5p. Smad7 was identified as a target gene of miR-106b-5p, and the effects of downregulation of miR-106b-5p on HFLSs could be abolished by silencing Smad7. We found that lncRNA HOTAIR was significantly downregulated in the HFLSs of patients with RA. Moreover, lncRNA HOTAIR influenced cell growth, migration, invasion, and apoptosis in HFLSs through the miR-106b-5p/Smad7 axis.

Melatonin Prevents Chondrocyte Matrix Degradation in Rats with Experimentally Induced Osteoarthritis by Inhibiting Nuclear Factor-κB via SIRT1.

Osteoarthritis (OA) is a common degenerative joint disease characterized by an imbalance of cartilage extracellular matrix (ECM) breakdown and anabolism. Melatonin (MT) is one of the hormones secreted by the pineal gland of the brain and has anti-inflammatory, antioxidant, and anti-aging functions. To explore the role of MT in rats, we established an OA model in rats by anterior cruciate ligament transection (ACLT). Safranin O-fast green staining showed that intraperitoneal injection of MT (30 mg/kg) could alleviate the degeneration of articular cartilage in ACLT rats. Immunohistochemical (IHC) analysis found that MT could up-regulate the expression levels of collagen type II and Aggrecan and inhibit the expression levels of matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), and ADAM metallopeptidase with thrombospondin type 1 motif 4 (ADAMTS-4) in ACLT rats. To elucidate the mechanism of MT in protecting the ECM in inflammatory factor-induced rat chondrocytes, we conducted in vitro experiments by co-culturing MT with a culture medium. Western blot (WB) showed that MT could promote the expression levels of transforming growth factor-beta 1 (TGF-β1)/SMAD family member 2 (Smad2) and sirtuin 2-related enzyme 1 (SIRT1) and inhibit the expression of levels of phosphorylated nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibi-tor (p-p65) and phosphorylated IκB kinase-α (p-IκBα). In addition, WB and real-time PCR (qRT-PCR) results showed that MT could inhibit the expression levels of MMP-3, MMP-13, ADAMTS-4, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in chondrocytes induced by interleukin-1β (IL-1β), and up-regulate the expression of chondroprotective protein type II collagen. We found that in vivo, MT treatment protected articular cartilage in the rat ACLT model. In IL-1β-induced rat chondrocytes, MT could reduce chondrocyte matrix degradation by up-regulating nuclear factor-kB (NF-κB) signaling pathway-dependent expression of SIRT1 and protecting chondrocyte by activating the TGF-β1/Smad2 pathway.