Expression Of Smad7 Research Articles

LINC00909 up-regulates pluripotency factors and promotes cancer stemness and metastasis in pancreatic ductal adenocarcinoma by targeting SMAD4

BackgroundPancreatic cancer stem cells are crucial for tumorigenesis and cancer metastasis. Presently, long non-coding RNAs were found to be associated with Pancreatic Ductal Adenocarcinoma stemness characteristics but the underlying mechanism is largely known. Here, we aim to explore the function of LINC00909 in regulating pancreatic cancer stemness and cancer metastasis.MethodsThe expression level and clinical characteristics of LINC00909 were verified in 80-paired normal pancreas and Pancreatic Ductal Adenocarcinoma tissues from Guangdong Provincial People’s Hospital cohort by in situ hybridization. RNA sequencing of PANC-1 cells with empty vector or vector encoding LINC00909 was experimented for subsequent bioinformatics analysis. The effect of LINC00909 in cancer stemness and metastasis was examined by in vitro and in vivo experiments. The interaction between LINC00909 with SMAD4 and the pluripotency factors were studied.ResultsLINC00909 was generally upregulated in pancreatic cancer tissues and was associated with inferior clinicopathologic features and outcome. Over-expression of LINC00909 enhanced the expression of pluripotency factors and cancer stem cells phenotype, while knock-down of LINC00909 decreased the expression of pluripotency factors and cancer stem cells phenotype. Moreover, LINC00909 inversely regulated SMAD4 expression, knock-down of SMAD4 rescued the effect of LINC00909-deletion inhibition on pluripotency factors and cancer stem cells phenotype. These indicated the effect of LINC00909 on pluripotency factors and CSC phenotype was dependent on SMAD4 and MAPK/JNK signaling pathway, another downstream pathway of SMAD4 was also activated by LINC00909. Specifically, LINC00909 was localized in the cytoplasm in pancreatic cancer cells and decreased the stability the SMAD4 mRNA. Finally, we found over-expression of LINC00909 not only accelerated tumor growth in subcutaneous mice models, but also facilitated tumorigenicity and spleen metastasis in orthotopic mice models.ConclusionWe demonstrate LINC00909 inhibits SMAD4 expression at the post-transcriptional level, which up-regulates the expression of pluripotency factors and activates the MAPK/JNK signaling pathway, leading to enrichment of cancer stem cells and cancer metastasis in pancreatic cancer.

Inhibition of miR-25 ameliorates cardiac and skeletal muscle dysfunction in aged mdx/utrn haploinsufficient (+/−) mice

Dystrophic cardiomyopathy is a significant feature of Duchenne muscular dystrophy (DMD). Increased cardiomyocyte cytosolic calcium (Ca2+) and interstitial fibrosis are major pathophysiological hallmarks that ultimately result in cardiac dysfunction. MicroRNA-25 (miR-25) has been identified as a suppressor of both sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) and mothers against decapentaplegic homolog-7 (Smad7) proteins. In this study, we created a gene transfer using an miR-25 tough decoy (TuD) RNA inhibitor delivered via recombinant adeno-associated virus serotype 9 (AAV9) to evaluate the effect of miR-25 inhibition on cardiac and skeletal muscle function in aged dystrophin/utrophin haploinsufficient mice mdx/utrn (+/-), a validated transgenic murine model of DMD. We found that the intravenous delivery of AAV9 miR-25 TuD resulted in strong and stable inhibition of cardiac miR-25 levels, together with the restoration of SERCA2a and Smad7 expression. This was associated with the amelioration of cardiomyocyte interstitial fibrosis as well as recovered cardiac function. Furthermore, the direct quadricep intramuscular injection of AAV9 miR-25 TuD significantly restored skeletal muscle Smad7 expression, reduced tissue fibrosis, and enhanced skeletal muscle performance in mdx/utrn (+/-) mice. These results imply that miR-25 TuD gene transfer may be a novel therapeutic approach to restore cardiomyocyte Ca2+ homeostasis and abrogate tissue fibrosis in DMD.

Single-cell transcriptomic sequencing data reveal aberrant DNA methylation in SMAD3 promoter region in tumor-associated fibroblasts affecting molecular mechanism of radiosensitivity in non-small cell lung cancer

ObjectiveNon-small cell lung cancer (NSCLC) often exhibits resistance to radiotherapy, posing significant treatment challenges. This study investigates the role of SMAD3 in NSCLC, focusing on its potential in influencing radiosensitivity via the ITGA6/PI3K/Akt pathway.MethodsThe study utilized gene expression data from the GEO database to identify differentially expressed genes related to radiotherapy resistance in NSCLC. Using the GSE37745 dataset, prognostic genes were identified through Cox regression and survival analysis. Functional roles of target genes were explored using Gene Set Enrichment Analysis (GSEA) and co-expression analyses. Gene promoter methylation levels were assessed using databases like UALCAN, DNMIVD, and UCSC Xena, while the TISCH database provided insights into the correlation between target genes and CAFs. Experiments included RT-qPCR, Western blot, and immunohistochemistry on NSCLC patient samples, in vitro studies on isolated CAFs cells, and in vivo nude mouse tumor models.ResultsFifteen key genes associated with radiotherapy resistance in NSCLC cells were identified. SMAD3 was recognized as an independent prognostic factor for NSCLC, linked to poor patient outcomes. High expression of SMAD3 was correlated with low DNA methylation in its promoter region and was enriched in CAFs. In vitro and in vivo experiments confirmed that SMAD3 promotes radiotherapy resistance by activating the ITGA6/PI3K/Akt signaling pathway.ConclusionHigh expression of SMAD3 in NSCLC tissues, cells, and CAFs is closely associated with poor prognosis and increased radiotherapy resistance. SMAD3 is likely to enhance radiotherapy resistance in NSCLC cells by activating the ITGA6/PI3K/Akt signaling pathway.

Clonal analysis of metachronous double biliary tract cancers.

The molecular mechanisms underpinning the development of metachronous tumors in the remnant bile duct following surgical resection of primary biliary tract carcinomas (BTCs) are unknown. This study aimed to elucidate these mechanisms by evaluating the clinicopathologic features of BTCs, the alterations to 31 BTC-related genes on targeted sequencing, and the aberrant expression of p53, p16, SMAD4, ARID1A and β-catenin on immunohistochemistry. Twelve consecutive patients who underwent resection of metachronous BTCs following primary BTC resection with negative bile duct margins were enrolled. Among the 12 metachronous tumors, six exhibited anterograde growth in the lower portion and six exhibited retrograde growth in the upper portion of the biliary tree. Surgical resection of metachronous BTCs resulted in recurrence-free survival in seven, local recurrence in five, and death in two patients. Nine achieved 5-year overall survival after primary surgery. Molecular analyses revealed that recurrently altered genes were: TP53, SMAD4, CDKN2A, ELF3, ARID1A, GNAS, NF1, STK11, RNF43, KMT2D and ERBB3. Each of these was altered in at least three cases. A comparison of the molecular features between 12 paired primary and metachronous BTCs indicated that 10 (83%) metachronous tumors developed in clonal association with corresponding primary tumors either successionally or phylogenically. The remaining two (17%) developed distinctly. The successional tumors consisted of direct or evolved primary tumor clones that spread along the bile duct. The phylogenic tumors consisted of genetically unstable clones and conferred a poor prognosis. Metachronous tumors distinct from their primaries harbored fewer mutations than successional and phylogenic tumors. In conclusion, over 80% of metachronous BTCs that develop following primary BTC resection are probably molecularly associated with their primaries in either a successional or a phylogenetic manner. Comparison between the molecular features of a metachronous tumor and those of a preceding tumor may provide effective therapeutic clues for the treatment of metachronous BTC. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Retraction: miR-212/132 downregulates SMAD2 expression to suppress the G1/S phase transition of the cell cycle and the epithelial to mesenchymal transition in cervical cancer cells.

Retraction: 'miR-212/132 downregulates SMAD2 expression to suppress the G1/S phase transition of the cell cycle and the epithelial to mesenchymal transition in cervical cancer cells' by Jian-Li Zhao , Le Zhang, Xu Guo , Jing-Hua Wang , Wen Zhou , Min Liu , Xin Li and Hua Tang , IUBMB Life 2015, 67, 380-394 : The above article, published online on 15 May 2015 on Wiley Online Library (https://doi.org/10.1002/iub.1381) has been retracted by agreement between the journal's Editor in Chief, Dr. Efstathios S. Gonos, and Wiley Periodicals LLC. The retraction has been agreed following an investigation based on allegations raised by a third party. Several flaws and inconsistencies were found, including image manipulation in Figures 3C, 4C, 5B, 6B, 7C, 7D, 8B, 8C and 8D, and the editors consider the conclusions of this article to be invalid. The authors were contacted regarding the findings and the proposed retraction, but we did not receive a response.

Smad4 regulates TGF-β1-mediated hedgehog activation to promote epithelial-to-mesenchymal transition in pancreatic cancer cells by suppressing Gli1 activity

Pancreatic cancer (PC) is an aggressive and metastatic gastrointestinal tumor with a poor prognosis. Persistent activation of the TGF-β/Smad signaling induces PC cell (PCC) invasion and infiltration via epithelial-to-mesenchymal transition (EMT). Hedgehog signaling is a crucial pathway for the development of PC via the transcription factors Gli1/2/3. This study aimed to investigate the underlying molecular mechanisms of action of hedgehog activation in TGF-β1-triggered EMT in PCCs (PANC-1 and BxPc-3). In addition, overexpression and shRNA techniques were used to evaluate the role of Smad4 in TGF-β1-treated PCCs. Our data showed that TGF-β1 promoted PCC invasion and infiltration via Smad2/3-dependent EMT. Hedgehog-Gli signaling axis in PCCs was activated upon TGF-β1 stimulation. Inhibition of hedgehog with cyclopamine effectively antagonized TGF-β1-induced EMT, thereby suggesting that the hedgehog signaling may act as a downstream cascade signaling of TGF-β1. As a key protein that assists the nuclear translocation of Smad2/3, Smad4 was highly expressed in PANC-1 cells, but not in BxPc-3 cells. Conversely, Gli1 expression was low in PANC-1 cells, but high in BxPc-3 cells. Furthermore, knockdown of Smad4 in PANC-1 cells by shRNA inhibited TGF-β1-mediated EMT and collagen deposition. Overexpression of Smad4 did not affect TGF-β1-mediated EMT due to the lack of significant increase in nuclear expression of Smad4. Importantly, Gli1 activity was upregulated by Smad4 knockdown in PANC-1 cells and downregulated by Smad4 overexpression in BxPc-3 cells, indicating that Gli1 may be a negative target protein downstream of Smad4. Thus, Smad4 regulates TGF-β1-mediated hedgehog activation to promote EMT in PCCs by suppressing Gli1 activity.

Aberrant TIMP-1 production in tumor-associated fibroblasts drives the selective benefits of nintedanib in lung adenocarcinoma.

The fibrotic tumor microenvironment is a pivotal therapeutic target. Nintedanib, a clinically approved multikinase antifibrotic inhibitor, is effective against lung adenocarcinoma (ADC) but not squamous cell carcinoma (SCC). Previous studies have implicated the secretome of tumor-associated fibroblasts (TAFs) in the selective effects of nintedanib in ADC, but the driving factor(s) remained unidentified. Here we examined the role of tissue inhibitor of metalloproteinase-1 (TIMP-1), a tumor-promoting cytokine overproduced in ADC-TAFs. To this aim, we combined genetic approaches with invitro and invivo preclinical models based on patient-derived TAFs. Nintedanib reduced TIMP-1 production more efficiently in ADC-TAFs than SCC-TAFs through a SMAD3-dependent mechanism. Cell culture experiments indicated that silencing TIMP1 in ADC-TAFs abolished the therapeutic effects of nintedanib on cancer cell growth and invasion, which were otherwise enhanced by the TAF secretome. Consistently, co-injecting ADC cells with TIMP1-knockdown ADC-TAFs into immunocompromised mice elicited a less effective reduction of tumor growth and invasion under nintedanib treatment compared to tumors bearing unmodified fibroblasts. Our results unveil a key mechanism underlying the selective mode of action of nintedanib in ADC based on the excessive production of TIMP-1 in ADC-TAFs. We further pinpoint reduced SMAD3 expression and consequent limited TIMP-1 production in SCC-TAFs as key for the resistance of SCC to nintedanib. These observations strongly support the emerging role of TIMP-1 as a critical regulator of therapy response in solid tumors.

The Therapeutic Effect of Ginsenoside Rb1 against Mechanical Trauma in a Rat Model of Postpartum Stress Urinary Incontinence

Aims. The aim of this study was to confirm the repairing effect of ginsenoside Rb1 (GS-Rb1) on mechanical trauma to periurethral tissues caused by childbirth and to explore its potential preventive mechanisms for mechanical trauma-induced stress urinary incontinence. Methods. 48 healthy adult female Sprague–Dawley (SD) rats were randomly divided into four groups: normal control, SUI groups, L-GS-Rb1 groups, and H-GS-Rb1 groups, with 12 rats in each group. The histopathological examinations of the urethral were performed to detect the morphological changes after repair of periurethral traumatic tissue. The TGF-β1/Smad and NRF2/ARE signaling pathways related to periurethral tissue trauma repair were determined by RT-PCR and western blot. The bladder capacity and LPP were examined in rats. Results. GS-Rb1 significantly decreased the number of fragmented and disorganized elastic and muscle fibers in the urethra and anterior vaginal wall of SUI rats. GS-Rb1 also increased the collagen content and reduced damage to the structural integrity of the periurethral myofibers. Furthermore, GS-Rb1 promoted expressions of TGF-β1, Smad2, Smad3, Smad7, p-Smad3, p-Smad2, and collagens I and III. It also increased the protein levels of Nrf2, GPX1, and MnSOD. The bladder capacity and LPP of rats in the L-GS-Rb1 group were close to those of rats in the normal groups. Conclusions. Ginsenoside Rb1 promotes the repair of periurethral tissue trauma in the postpartum period and has a preventive effect on the occurrence of stress urinary incontinence.

Echinacoside ameliorates hepatic fibrosis and tumor invasion in rats with thioacetamide-induced hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) affects approximately 800,000 individuals globally each year. Despite advancements in HCC treatments, there is still a pressing need to identify new drugs that can combat resistance. One potential option is echinacoside, a natural caffeic acid glycoside with antioxidant, anti-inflammatory, antidepressant, and antidiabetic properties. Therefore, we aimed to investigate the ability of echinacoside to exhibit antitumor activity against HCC in rats through ameliorating hepatic fibrosis and tumor invasion. Rats were given thioacetamide to induce HCC, and some were given 30 mg/kg of echinacoside twice a week for 16 weeks. The liver impairment was assessed by measuring serum α-fetoprotein (AFP) and examining liver sections stained with Masson trichrome or anti-transforming growth factor (TGF)-β1 antibodies. The hepatic expression of mRNA and protein levels of TGF-β1, β-catenin, SMAD4, matrix metalloproteinase-9 (MMP9), phosphoinositide 3-kinases (PI3K), mammalian target of rapamycin (mTOR), connective tissue growth factor 2 (CCN2), E-Cadherin, platelets derived growth factor (PDGF)-B and fascin were also analyzed. Echinacoside improved the survival rate of rats by decreasing serum AFP and the number of hepatic nodules. Examination of micro-images indicated that echinacoside can reduce fibrosis. It also significantly decreased the expression of TGF-β1, β-catenin, SMAD4, MMP9, PI3K, mTOR, CCN2, PDGF-B, and fascin while enhancing the expression of E-Cadherin. In conclusion, echinacoside exhibits a protective effect against HCC by increasing survival rates and decreasing tumor growth. It also acts as an inhibitor of the hepatic tissue fibrosis pathway by reducing the expression of TGF-β1, β-catenin, SMAD4, PI3K, CCN2, PDGF-B and mTOR. Additionally, it prevents tumor invasion by suppressing MMP9 and fascin, and increasing the expression of E-Cadherin.

TGFβ signaling pathway in salivary gland tumors

ObjectivePleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) are the most prevalent salivary gland tumors. Their pathogenesis has been recently associated with complex molecular cascades, including the TGFβ signaling pathway. The aim of this study was to evaluate the expression of genes associated with the TGFβ signaling pathway (TGFB1, ITGB6, SMAD2, SMAD4, FBN1, LTBP1, and c-MYC) to map possible downstream alterations in the TGFβ cascade. DesignThirteen PA, 17 MEC, 13 ACC, and 10 non-neoplastic salivary gland samples were analyzed by real-time RT-PCR. ResultsCases of PA presented increased TGFB1, LTPB1, c-MYC, and FBN1 expressions, whereas SMAD2 expression was decreased when compared to non-neoplastic tissue. MEC patients displayed increased expressions of TGFB1, ITGB6, FBN1, and c-MYC and decreased expressions of SMAD2 and SMAD4. ACC cases exhibited elevated expressions of the investigated genes except TGFB1. The present results suggest that decreased expression of SMAD2 and SMAD4 does not impede the transcriptional regulation of c-MYC, especially in PA and MEC. Increased expressions of ITGB6, TGFB1, LTBP1, and FBN1 appear to be related to the regulation of the TGFβ signaling pathway in these tumors. Additionally, we observed a higher expression of SMAD4 in ACC and a raised expression of ITGB6 and lowered expression of SMAD2 in MEC. ConclusionsOur study demonstrated the differential expression of TGFβ cascade members in salivary gland tumors such as SMAD2/SMAD4 and c-MYC as well as the participation of ITGB6, TGFB1, LTBP1, and FBN1, contributing to the understanding of the mechanisms involved in tumor progression.

The activation of HAMP promotes colorectal cancer cell proliferation through zinc-mediated upregulation of SMAD4 expression.

Colorectal cancer (CRC) poses a significant challenge in digestive system diseases, and emerging evidence underscores the critical role of zinc metabolism in its progression. This study aimed to investigate the clinical implications of genes at the intersection of zinc metabolism and CRC. We downloaded CRC prognosis-related genes and zinc metabolism-related genes from public databases. Then, the overlapping genes were screened out, and bioinformatics analysis was performed to obtain the hub gene associated with CRC prognosis. Subsequently, in vitro assays were carried out to investigate the expression of this hub gene and its exact mechanism between zinc metabolism and CRC. HAMP was identified as the hub CRC prognostic gene from overlapping zinc metabolism-related and CRC prognostic genes. In vitro analysis showed HAMP was over-expressed in CRC, and its knockdown inhibited RKO and HCT-116 cell invasion and migration significantly. ZnSO4 induced HAMP up-regulation to promote cell proliferation, while TPEN decreased HAMP expression to inhibit cell proliferation. Importantly, we further found that ZnSO4 enhanced SMAD4 expression to augment HAMP promoter activity and promote cell proliferation in CRC. HAMP stands out as an independent prognostic factor in CRC, representing a potential therapeutic target. Its intricate regulation by zinc, particularly through the modulation of SMAD4, unveils a novel avenue for understanding CRC biology. This study provides valuable insights into the interplay between zinc metabolism, HAMP, and CRC, offering promising clinical indicators for CRC patients. The findings present a compelling case for further exploration and development of targeted therapeutic strategies in CRC management.

Bioinformatics analysis and experimental validation of key genes associated with lumbar disc degeneration and biomechanics

BackgroundLumbar disc degeneration (LDD) is an important pathological basis for the development of degenerative diseases of the lumbar spine. Most clinical patients have low back pain as their main symptom. The deterioration of the biomechanical environment is an important cause of LDD. Although there is a large amount of basic research on LDD, there are fewer reports that correlate biomechanical mechanisms with basic research. Our research aims to identify 304 key genes involved in LDD due to biomechanical deterioration, using a bioinformatics approach. We focus on SMAD3, CAV1, SMAD7, TGFB1 as hub genes, and screen for 30 potential target drugs, offering novel insights into LDD pathology and treatment options. MethodsThe Gene Cards, GenCLip3, OMIM and Drugbank databases were explored to obtain genes associated with biomechanics and LDD, followed by making veen plots to obtain both co-expressed genes. GO enrichment analysis and KEGG pathway analysis of the co-expressed genes were obtained using the DAVID online platform and visualised via a free online website. Protein interaction networks (PPI) were obtained through the STRING platform and visualised through Cytoscape 3.9.0. These genes were predicted for downstream interaction networks using the STITCH platform. Then, the GSE56081 dataset was used to validate the key genes. RT-PCR was used to detect mRNA expression of core genes in the degenerated nucleus pulposus (NP) samples and western bolt was used for protein expression. Lastly, the obtained hub genes were searched in the drug database (DGIdb) to find relevant drug candidates. ResultsFrom the perspective of biomechanics-induced LDD, we obtained a total of 304 genes, the GO functional enrichment and KEGG pathway enrichment analysis showed that the functions of these genes are mostly related to inflammation and apoptosis. The PPI network was constructed and four Hub genes were obtained through the plug-in of Cytoscape software, namely SMAD3, CAV1, SMAD7 and TGFB1. The analysis of key genes revealed that biomechanical involvement in LDD may be related to the TGF-β signaling pathway. Validation of the GSE56081 dataset revealed that SMAD3 and TGFB1 were highly expressed in degenerating NP samples. RT-PCR results showed that the mRNA expression of SMAD3 and TGFB1 was significantly increased in the severe degeneration group; Western blot results also showed that the protein expression of TGFB1 and P-SMAD3 was significantly increased. In addition, we identified 30 potential drugs. ConclusionThis study presented a new approach to investigate the correlation between biomechanical mechanisms and LDD. The deterioration of the biomechanical environment may cause LDD through the TGF-β signaling pathway. TGFB1 and SMAD3 are important core targets. The important genes, pathways and drugs obtained in this study provided a new basis and direction for the study, diagnosis and treatment of LDD.

Mechanisms of the TGF-β1/Smad3-signaling pathway in gender differences in alcoholic liver fibrosis

The TGF-β1/Smad3-signaling pathway and gender differences were investigated in alcoholic liver fibrosis. Mice were divided into female normal, female model, male normal, and male model groups. Liver injury and fibrosis were assessed using histopathology and serology. Western blotting was performed to analyze the expression of relevant factors. HSC-T6 cells were divided into estradiol + saline, estradiol + ethanol, testosterone + saline, and testosterone + ethanol groups, and similar assessments were conducted in vitro. Compared with the female model group, the male model group exhibited significantly increased GPT, GOT, TNF-α, IL-6, and testosterone levels, fibrosis rate, and TGF-β1, Smad3, and PCNA expression, and significantly decreased estradiol levels and Caspase-3 expression. The apoptosis rate was higher in the estradiol + ethanol group than in the testosterone + ethanol group, although the testosterone + ethanol group exhibited significantly increased TNF-α, IL-6, Collagen-I, α-SMA, TGF-β1, Smad3, and PCNA expression, and significantly decreased Caspase-3 expression. Alcoholic liver fibrosis showed significant gender differences associated with the TGF-β1/Smad3-signaling pathway.

MiR-324-3p Suppresses Hepatic Stellate Cell Activation and Hepatic Fibrosis Via Regulating SMAD4 Signaling Pathway.

In hepatic fibrosis (HF), hepatic stellate cells (HSCs) form the extracellular matrix (ECM), and the pathological accumulation of ECM in the liver leads to inflammation. Our previous research found that miR-324-3p was down-regulated in culture-activated human HSCs. However, the precise effect of miR-324-3p on HF has not been elucidated. In this study, the HF mouse models were induced through directly injecting carbon tetrachloride (CCl4) into mice; the HF cell models were constructed using TGF-β1-treated LX-2 cells. Next, real-time-quantitative polymerase chain reaction (RT-qPCR), western blot (WB) and immunohistochemistry (IHC) were applied to assess the expression levels of miR-324-3p, α-smooth muscle actin (α-SMA), Vimentin or SMAD4; hematoxylin and eosin (H&E), Masson' s trichrome and Sirius red staining to evaluate the liver injury; luciferase reporter assay to verify the targeting relationship between miR-324-3p and SMAD4; enzyme-linked immunosorbent assay (ELISA) to determine the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST); and cell counting kit-8 (CCK-8) and flow cytometry to evaluate the effects of miR-324-3p on cell proliferation and cycle/apoptosis, respectively. The experimental results showed a reduction in miR-324-3p level in CCl4-induced HF mice as well as transforming growth factor (TGF)-β1-activated HSCs. Interestingly, the miR-324-3p level was rescued following the HF recovery process. In HF mice induced by CCl4, miR-324-3p overexpression inhibited liver tissue damage, decreased serum ALT and AST levels, and inhibited fibrosis-related biomarkers (α-SMA, Vimentin) expression, thereby inhibiting HF. Similarly, miR-324-3p overexpression up-regulated α-SMA and Vimentin levels in HF cells, while knockdown of miR-324-3p had the opposite effect. Besides, miR-324-3p played an antifibrotic role through inhibiting the proliferation of hepatocytes. Further experiments confirmed that miR-324-3p targeted and down-regulated SMAD4 expression. SMAD4 was highly expressed in HF cells, and silencing SMAD4 significantly decreased the α-SMA and Vimentin levels in HF cells. Collectively, the miR-324-3p may suppress the activation of HSCs and HF by targeting SMAD4. Therefore, miR-324-3p is identified as a potential and novel therapeutic target for HF.

Evaluation of 6-PPD quinone toxicity on lung of male BALB/c mice by quantitative proteomics

N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine quinone (6-PPDQ), a transformation product of tyre-derived 6-PPD, has been frequently detected in different environments. After 6-PPDQ exposure, we here aimed to examine dynamic lung bioaccumulation, lung injury, and the underlying molecular basis in male BALB/c mice. After single injection at concentration of 4 mg/kg, 6-PPDQ remained in lung up to day 28, and higher level of 6-PPDQ bioaccumulation in lung was observed after repeated injection. Severe inflammation was observed in lung after both single and repeated 6-PPDQ injection as indicated by changes of inflammatory cytokines (TNF-α, IL-6 and IL-10). Sirius red staining and hydroxyproline content analysis indicated that repeated rather than single 6-PPDQ injection induced fibrosis in lung. Repeated 6-PPDQ injection also severely impaired lung function in mice by influencing chord compliance (Cchord) and enhanced pause (Penh). Proteomes analysis was further carried out to identify molecular targets of 6-PPDQ after repeated injection, which was confirmed by transcriptional expression analysis and immunohistochemistry staining. Alterations in Ripk1, Fadd, Il-6st, and Il-16 expressions were identified to be associated with inflammation induction of lung after repeated 6-PPDQ injection. Alteration in Smad2 expression was identified to be associated with fibrosis formation in lung of 6-PPDQ exposed mice. Therefore, long-term and repeated 6-PPDQ exposure potentially resulted in inflammation and fibrosis in lung by affecting certain molecular signals in mammals. Our results suggested several aspects of lung injury caused by 6-PPDQ and provide the underlying molecular basis. These observations implied the possible risks of long-term 6-PPDQ exposure to human health.

The underlying molecular mechanism of ciliated epithelium dysfunction and TGF-β signaling in children with congenital pulmonary airway malformations

The aim of this study was to investigate the variation in gene expression in the complete transcripts of Congenitalpulmonary airwaymalformation (CPAM) of the lung using Next Generation Sequencing (NGS) technology. There were 20 cases involving children with CPAM were used for selection of study sample. NGS was used to establish RNA-Seq libraries for the two groups of samples separately, and both groups were conducted to differential expression analysis and Gene Ontology (GO) functional enrichment analysis. The pathways of the differential genes were analyzed to find the enriched target pathways. A total of 592 genes were expressed with significant differences (CPAM vs. normal tissue, P < 0.05). GO functional analysis of DEGs indicated that abnormal ciliary function played a role in the development of CPAM. Subsequently, analysis of these genes pathways showed the TGF-β signaling pathway was significantly enriched. Finally, the results of immunohistochemical analysis of some DEGs showed that a significant reduction in the expression of SMAD6, a gene related to the TGF-β signaling pathway, led to abnormal activation of the pathway. TGF-β signaling pathway involved in the evolution of the disease obtained by DEGs enrichment pathway analysis. SMAD6, a gene involved in this pathway, might be a potential biomarker for the diagnosis and treatment of CPAM.

Early intervention of carbon dioxide fractional laser in hypertrophic scar through TGFβ-1/ Smad3 signaling pathway.

Hypertrophic scars are usually the result of surgical trauma or burn,and more common in individuals with a darker skin color. They appear as red and raised lesions around the wound that continually expand over a period of weeks or months, causing itching, pain, burning sensation and discomfort. Severe scarring affects interpersonal and social relationships, and decreases the quality of life of the patients.The aim of this study was to evaluate the effect of carbon dioxide fractional laser as an early intervention against hypertrophic scars using a rabbit ear scar model, and explore the role of the TGFβ-1/ Smad3 signaling pathway in scar hyperplasia. Four wounds were made into each ear of rabbits, and divided into the untreated control and three laser-treatment groups. The experimental groups received laser intervention once, twice and thrice respectively. laser treatment significantly inhibited the formation of hypertrophic scars, and maximum benefits were seen in the wounds that received three laser treatments. Immunohistochemical staining showed that the in situ expression of TGFβ-1 and Smad3 in the scars decreased by varying degrees after laser intervention, and was most obvious after three laser interventions. Furthermore, the expression levels were the lowest at the end of 6 months after modeling. Therefore, we can assume that early intervention with carbon dioxide fractional laser can prevent formation of hypertrophic scars by regulating the TGF-β1/Smad3 pathway.

The evaluation of the SMAD1 rs1016792 polymorphism and gene expression on pulmonary hypertension due to congenital heart disease in children: a preliminary study

Smad Family Member (SMAD), a protein family responsible for transducing the signal induced by TGF-β into the nucleus, is thought to play a role in the pathology of many heart diseases. Therefore, we aimed to evaluate the influence of the SMAD1 rs1016792 polymorphism and gene expression on pulmonary arterial hypertension (PAH) due to congenital heart disease (CHD) in children. A total of 90 children, 45 of whom were PAH-CHD children and 45 healthy children, were included in the study. Patients were selected from those who were diagnosed and followed in the Department of Pediatric Cardiology.The SMAD1 rs1016792 genotyping and expression analysis was performed using a real-time polymerase chain reaction (RT-PCR)-based system. It was determined that the left ventricular end-diastolic diameter (LVEDD) value was lower in the patient group than in the control group, while the pulmonary artery pressure (PAP) value was higher in the patient group than in the control group. When the SMAD1 gene expression level was examined, a statistically significant difference was found between the patient and control groups. Patients had decreased SMAD1 expression compared to controls (p˂0.001). We found no significant difference between the patient and control groups in terms of SMAD1 rs1016792 genotype distribution or allele frequency (p > 0.05). There was no difference between genotype distribution and SMAD1 expression levels in the groups. In this study, we showed for the first time that SMAD1 expression is decreased in children with PAH-CHD. These results will be a preliminary step toward understanding the role of SMAD1 in the etiopathogenesis of CHD.

BMP signaling to pharyngeal muscle in the C. elegans response to a bacterial pathogen regulates anti-microbial peptide expression and pharyngeal pumping.

Host response to pathogens recruits multiple tissues in part through conserved cell signaling pathways. In Caenorhabditis elegans, the bone morphogenetic protein (BMP) like DBL-1 signaling pathway has a role in the response to infection in addition to other roles in development and postdevelopmental functions. In the regulation of body size, the DBL-1 pathway acts through cell autonomous signal activation in the epidermis (hypodermis). We have now elucidated the tissues that respond to DBL-1 signaling upon exposure to two bacterial pathogens. The receptors and Smad signal transducers for DBL-1 are expressed in pharyngeal muscle, intestine, and epidermis. We demonstrate that expression of receptor-regulated Smad (R-Smad) gene sma-3 in the pharynx is sufficient to improve the impaired survival phenotype of sma-3 mutants and that expression of sma-3 in the intestine has no effect when exposing worms to bacterial infection of the intestine. We also show that two antimicrobial peptide genes - abf-2 and cnc-2 - are regulated by DBL-1 signaling through R-Smad SMA-3 activity in the pharynx. Finally, we show that pharyngeal pumping activity is reduced in sma-3 mutants and that other pharynx-defective mutants also have reduced survival on a bacterial pathogen. Our results identify the pharynx as a tissue that responds to BMP signaling to coordinate a systemic response to bacterial pathogens.

2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione isolated from Averrhoa carambola L. root inhibits high glucose-induced EMT in HK-2 cells through targeting the regulation of miR-21–5p/Smad7 signaling pathway

Objective2-Dodecyl-6-Methoxycyclohexa-2, 5-Diene-1, 4-Dione (DMDD) isolated from Averrhoa carambola L. root, has been proven therapeutic effects on diabetic kidney disease (DKD). This research aims to assess DMDD’s effects on DKD and to investigate its underlying mechanisms, to establish DMDD as a novel pharmaceutical agent for DKD treatment. MethodsThe human renal tubular epithelial (HK-2) cells were induced by high glucose (HG) to mimic DKD and followed by DMDD treatment. The cytotoxicity of DMDD was assessed using the Cell Counting Kit-8 (CCK-8) assay. The migratory capacity of HK-2 cells was evaluated through transwell and scratch-wound assays. To investigate the effect of Smad7 and miR-21–5p, lentiviral transfection was employed in HK-2 cells. Additionally, the expression of proteins related to epithelial-mesenchymal transition (EMT) and TGFβ1/Smad2/3 pathway was checked by QRT-PCR, Western blot, and immunofluorescence techniques. ResultsThis study has shown that DMDD significantly suppresses cell migration and the expression of Vimentin, α-SMA, TGFβ1, and p-Smad2/3 in HK-2 cells under HG conditions. Concurrently, DMDD enhances the protein expression of E-cadherin and Smad7. Intriguingly, the therapeutic effect of DMDD was abrogated upon Smad7 silencing. Further investigations revealed that DMDD effectively inhibits miR-21–5p expression, which is upregulated by HG. Downregulation of miR-21–5p inhibits the activation of the TGFβ1/Smad2/3 pathway and EMT induced by HG. In contrast, overexpression of miR-21–5p negates DMDD’s therapeutic benefits. ConclusionDMDD mitigates EMT in HG-induced HK-2 cells by modulating the miR-21–5p/Smad7 pathway, thereby inhibiting renal fibrosis in DKD. These findings suggest that DMDD holds promise as a potential therapeutic agent for DKD.