Adipocyte-derived exosomes from obstructive sleep apnoea rats aggravate MASLD by TCONS_00039830/miR-455-3p/Smad2 axis.

A correlation exists between obstructive sleep apnoea (OSA) and the severity of metabolic dysfunction-associated steatotic liver disease (MASLD), OSA can induce more severe MASLD. However, the underlying regulatory mechanism between the two is unclear. To this end, this study explored the role and possible molecular mechanisms of adipocyte-derived exosomes under OSA in aggravating MASLD. Through sequencing technology, miR-455-3p was identified as a co-differentially expressed miRNA between the MASLD + OSA and Control groups and between the MASLD + OSA and MASLD groups. Upregulation of TCONS-00039830 and Smad2 and downregulation of miR-455-3p in the MASLD and MASLD + OSA groups were validated in vivo and in vitro. TCONS-00039830, as a differentially expressed LncRNA in exosomes found in the sequencing results, transfection notably downregulated miR-455-3p and upregulated Smad2 in hepatocytes. TCONS_00039830 overexpression increased fat, triglyceride and cholesterol levels, while miR-455-3p overexpression decreased these levels. Furthermore, exosome administration promoted the accumulation of fat, triglyceride and cholesterol, upregulated TCONS_00039830 and Smad2, and downregulated miR-455-3p. Overexpression of miR-455-3p reversed the increased fat accumulation and upregulated TCONS_00039830 and Smad2. In conclusion, OSA-derived exosomes promoted hepatocyte steatosis by regulating TCONS_00039830/miR-455-3p/Smad2 axis, thereby aggravating liver damage in MASLD.

The immune regulation and therapeutic potential of the SMAD gene family in breast cancer.

Breast cancer is a serious threat to human health. The transforming growth factor-ß signaling pathway is an important pathway involved in the occurrence and development of cancer. The SMAD family genes are responsible for the TGF-ß signaling pathway. However, the mechanism by which genes of the SMAD family are involved in breast cancer is still unclear. Therefore, it is necessary to investigate the biological roles of the SMAD family genes in breast cancer. We downloaded the gene expression data, gene mutation data, and clinical pathological data of breast cancer patients from the UCSC Xena database. We used the Wilcox test to estimate the expression of genes of the SMAD family in cancers. And the biological functions of SMAD family genes using the DAVID website. The Pearson correlation method was used to explore the immune cell infiltration and drug response of SMAD family genes. We conducted in biological experiments vitro and vivo. In this study, we integrated the multi-omics data from TCGA breast cancer patients for analysis. The expression of genes of SMAD family was significantly dysregulated in patients with breast cancer. Except for SMAD6, the expression of other SMAD family genes was positively correlated. We also found that genes of the SMAD family were significantly enriched in the TGF-ß signaling pathway, Hippo signaling pathway, cell cycle, and cancer-related pathways. In addition, SMAD3, SMAD6, and SMAD7 were lowly expressed in stage II breast cancer, while SMAD4 and SMAD2 were lowly expressed in stage III cancer. Furthermore, the expression of genes of the SMAD family was significantly correlated with immune cell infiltration scores. Constructing a xenograft tumor mouse model, we found that SMAD3 knockdown significantly inhibited tumorigenesis. Finally, we analyzed the association between these genes and the IC50 value of drugs. Interestingly, patients with high expression of SMAD3 exhibited significant resistance to dasatinib and staurosporine, while high sensitivity to tamoxifen and auranofin. In addition, SMAD3 knockdown promoted the apoptosis of BT-549 cells and decreased cell activity, and BAY-1161909 and XK-469 increased drug efficacy. In conclusion, genes of the SMAD family play a crucial role in the development of breast cancer.

Upregulation of CYR61 by TGF-ß and YAP signaling exerts a counter-suppression of hepatocellular carcinoma.

Transforming growth factor-ß (TGF-ß) and Hippo signaling are two critical pathways engaged in cancer progression by regulating both oncogenes and tumor suppressors, yet how the two pathways coordinately exert their functions in the development of hepatocellular carcinoma (HCC) remains elusive. In this study, we firstly conducted an integrated analysis of public liver cancer databases and our experimental TGF-ß target genes, identifying CYR61 as a pivotal candidate gene relating to HCC development. The expression of CYR61 is downregulated in clinical HCC tissues and cell lines than that in the normal counterparts. Evidence revealed that CYR61 is a direct target gene of TGF-ß in liver cancer cells. In addition, TGF-ß-stimulated Smad2/3 and the Hippo pathway downstream effectors YAP and TEAD4 can form a protein complex on the promoter of CYR61, thereby activating the promoter activity and stimulating CYR61 gene transcription in a collaborative manner. Functionally, depletion of CYR61 enhanced TGF-ß- or YAP-mediated growth and migration of liver cancer cells. Consistently, ectopic expression of CYR61 was capable of impeding TGF-ß- or YAP-induced malignant transformation of HCC cells in vitro and attenuating HCC xenograft growth in nude mice. Finally, transcriptomic analysis indicates that CYR61 can elicit an antitumor program in liver cancer cells. Together, these results add new evidence for the crosstalk between TGF-ß and Hippo signaling and unveil an important tumor suppressor function of CYR61 in liver cancer.

Gulp1 regulates chondrocyte growth arrest and differentiation via the TGF-ß/SMAD2/3 pathway.

Chondrocyte differentiation is crucial for cartilage formation. However, the complex processes and mechanisms coordinating chondrocyte proliferation and differentiation remain incompletely understood. Here, we report a novel function of the adaptor protein Gulp1 in chondrocyte differentiation. Gulp1 expression is upregulated during chondrogenic differentiation. Gulp1 knockdown in chondrogenic ATDC5 cells reduces the expression of chondrogenic and hypertrophic marker genes during differentiation. Furthermore, Gulp1 knockdown impairs cell growth arrest during chondrocyte differentiation and reduces the expression of the cyclin-dependent kinase inhibitor p21. The activation of the TGF-ß/SMAD2/3 pathway, which is associated with p21 expression in chondrocytes, is impaired in Gulp1 knockdown cells. Collectively, these results demonstrate that Gulp1 contributes to cell growth arrest and chondrocyte differentiation by modulating the TGF-ß/SMAD2/3 pathway.

Hypoxia-induced TREM1 promotes mesenchymal-like states of glioma stem cells via alternatively activating tumor-associated macrophages.

The mesenchymal subtype of glioblastoma (GBM) cells characterized by aggressive invasion and therapeutic resistance is thought to be dependent on cell-intrinsic alteration and extrinsic cellular crosstalk. Tumor-associated macrophages (TAMs) are pivotal in tumor progression, chemo-resistance, angiogenesis, and stemness maintenance. However, the impact of TAMs on the shifts in glioma stem cells (GSCs) states remains largely uncovered. Herein, we showed that the triggering receptor expressed on myeloid cells-1 (TREM1) preferentially expressed by M2-like TAMs and induced GSCs into mesenchymal-like states by modulating the secretion of TGFß2, which activated the TGFßR/SMAD2/3 signaling in GSCs. Furthermore, we demonstrated that TREM1 was transcriptionally regulated by HIF1a under the hypoxic environment and thus promoted an immunosuppressive type of TAMs via activating the TLR2/AKT/mTOR/c-MYC axis. Collectively, this study reveals that cellular communication between TAMs and GSCs through the TREM1-mediated TGFß2/TGFßR axis is involved in the mesenchymal-like transitions of GSCs. Our study provides valuable insights into the regulatory mechanisms between the tumor immune microenvironment and the malignant characteristics of GBM, which can lead to potential novel strategies targeting TAMs for tumor control.

TMED3 stabilizes SMAD2 by counteracting NEDD4-mediated ubiquitination to promote ovarian cancer.

Ovarian cancer is a major cause of death among cancer patients. Recent research has shown that the transmembrane emp24 domain (TMED) protein family plays a role in the progression of various types of cancer. In this study, we investigated the expression of TMED3 in ovarian cancer tumors compared to nontumor tissues using immunohistochemical staining. We found that TMED3 was overexpressed in ovarian cancer tumors, and its high expression was associated with poor disease-free and overall survival. To understand the functional implications of TMED3 overexpression in ovarian cancer, we conducted experiments to knockdown TMED3 using short hairpin RNA (shRNA). We observed that TMED3 knockdown resulted in reduced cell viability and migration, as well as increased cell apoptosis. Additionally, in subcutaneous xenograft models in BALB-c nude mice, TMED3 knockdown inhibited tumor growth. Further investigation revealed that SMAD family member 2 (SMAD2) was a downstream target of TMED3, driving ovarian cancer progression. TMED3 stabilized SMAD2 by inhibiting the E3 ligase NEDD4-mediated ubiquitination of SMAD2. To confirm the importance of SMAD2 in TMED3-mediated ovarian cancer, we performed functional rescue experiments and found that SMAD2 played a critical role in this process. Moreover, we discovered that the PI3K-AKT pathway was involved in the promoting effects of TMED3 overexpression on ovarian cancer cells. Overall, our study identifies TMED3 as a prognostic indicator and tumor promoter in ovarian cancer. Its function is likely mediated through the regulation of the SMAD2 and PI3K-AKT signaling pathway. These findings contribute to our understanding of the molecular mechanisms underlying ovarian cancer progression and provide potential targets for therapeutic intervention.

FBXO28 promotes cell proliferation, migration and invasion via upregulation of the TGF-beta1/SMAD2/3 signaling pathway in ovarian cancer.

BACKGROUND: Ovarian cancer is one of the most common gynecological malignancies due to the lack of early symptoms, early diagnosis and limited screening. Therefore, it is necessary to understand the molecular mechanism underlying the occurrence and progression of ovarian cancer and to identify a basic biomarker for the early diagnosis and clinical treatment of ovarian cancer. METHODS: The association between FBXO28 and ovarian cancer prognosis was analyzed using Kaplan‒Meier survival analysis. The difference in FBXO28 mRNA expression between normal ovarian tissues and ovarian tumor tissues was obtained from The Cancer Genome Atlas (TCGA), and Genotype-Tissue Expression (GTEx) cohorts. The expression levels of the FBXO28 protein in ovarian cancer tissues and normal ovarian tissues were measured via immunohistochemical staining. Western blotting was used to determine the level of FBXO28 expression in ovarian cancer cells. The CCK-8, the colony formation, Transwell migration and invasion assays were performed to evaluate cell proliferation and motility. RESULTS: We found that a higher expression level of FBXO28 was associated with poor prognosis in ovarian cancer patients. Analysis of the TCGA and GTEx cohorts showed that the FBXO28 mRNA level was lower in normal ovarian tissue samples than in ovarian cancer tissue samples. Compared with that in normal ovarian tissues or cell lines, the expression of FBXO28 was greater in ovarian tumor tissues or tumor cells. The upregulation of FBXO28 promoted the viability, proliferation, migration and invasion of ovarian cancer cells. Finally, we demonstrated that FBXO28 activated the TGF-beta1/Smad2/3 signaling pathway in ovarian cancer. CONCLUSIONS: In conclusion, FBXO28 enhanced oncogenic function via upregulation of the TGF-beta1/Smad2/3 signaling pathway in ovarian cancer.

MET exon 14 skipping mutation drives cancer progression and recurrence via activation of SMAD2 signalling.

BACKGROUND: c-Met encoded by the proto-oncogene MET, also known as hepatocyte growth factor (HGF) receptor, plays a crucial role in cellular processes. MET exon 14 skipping alteration (METΔ14EX) is a newly discovered MET mutation. SMAD2 is an important downstream transcription factor in TGF-ß pathway. Unfortunately, the mechanisms by which METΔ14EX leads to oncogenic transformation are scarcely understood. The relationship between METΔ14EX and SMAD2 has not been studied yet. METHODS: We generate METΔ14EX models by CRISPR-Cas9. In vitro transwell, wound-healing, soft-agar assay, in vivo metastasis and subcutaneous recurrence assay were used to study the role of METΔ14EX in tumour progression. RNA-seq, Western blotting, co-immunoprecipitation (CO-IP) and immunofluorescent were performed to explore the interaction between c-Met and SMAD2. RESULTS: Our results demonstrated that METΔ14EX, independent of HGF, can prolong the constitutive activation of c-Met downstream signalling pathways by impeding c-Met degradation and facilitating tumour metastasis and recurrence. Meanwhile, METΔ14EX strengthens the interaction between c-Met and SMAD2, promoting SMAD2 phosphorylation. Therapeutically, MET inhibitor crizotinib impedes METΔ14EX-mediated tumour metastasis by decreasing SMAD2 phosphorylation. CONCLUSIONS: These data elucidated the previously unrecognised role of METΔ14EX in cancer progression via activation of SMAD2 independent of TGF-ß, which helps to develop more effective therapies for such patients. METΔ14EX alteration significantly triggers tumour progression via activation of SMAD2 signalling that are involved in activating tumour invasion, metastasis and recurrence. On the left, in the MET wild-type (METWT), the juxtamembrane (JM) domain is involved in the regulation of tyrosine kinase activity, receptor degradation, and caspase cleavage. On the right, the METΔ14EX mutation leads to the loss of the juxtamembrane domain, resulting in an abnormal MET protein lacking a CBL-binding site. This causes the accumulation of truncated MET receptors followed by constitutive activation of the MET signalling pathway. Thus, the METΔ14EX-mutated protein has strong binding and phosphorylation to SMAD2, which results in the phosphorylation of a large number of SMAD2/3 proteins that combine with SMAD4 to form a complex in the nucleus, activating downstream signalling pathways, such as EMT and ECM remodelling, resulting in tumour progression and recurrence. TF transcription factor.

SET8 is a novel negative regulator of TGF-ß signaling in a methylation-independent manner.

Transforming growth factor ß (TGF-ß) is a multifunctional cytokine that induces a diverse set of cellular processes principally through Smad-dependent transcription. Transcriptional responses induced by Smads are tightly regulated by Smad cofactors and histone modifications; however, the underlying mechanisms have not yet been elucidated in detail. We herein report lysine methyltransferase SET8 as a negative regulator of TGF-ß signaling. SET8 physically associates with Smad2/3 and negatively affects transcriptional activation by TGF-ß in a catalytic activity-independent manner. The depletion of SET8 results in an increase in TGF-ß-induced plasminogen activator inhibitor-1 (PAI-1) and p21 expression and enhances the antiproliferative effects of TGF-ß. Mechanistically, SET8 occupies the PAI-1 and p21 promoters, and a treatment with TGF-ß triggers the replacement of the suppressive binding of SET8 with p300 on these promoters, possibly to promote gene transcription. Collectively, the present results reveal a novel role for SET8 in the negative regulation of TGF-ß signaling.

TGFß1, SMAD2, CTNNß1, and Wnt3a gene mutational status and serum concentrations in individuals with non-small cell lung cancer.

The objective of the current investigation was to investigate the diagnostic utility of the serum concentrations and mutational status of TGFß1, SMAD2, CTNNß1, and Wnt3a. and the expression levels of human­related genes in patients with non-small cell lung cancer (NSCLC). The serum concentrations were determined using the ELISA technique, and PCR for genotype variations of TGFß1, SMAD2, CTNNß1, and Wnt3a were examined using Sanger sequencing in tissue samples obtained from 93 patients with NSCLC and 84 healthy individuals for blood, and 20 Formalin Fixed Paraffin Embedded (FFPE) from normal samples dissected adjacent to the tumour. The findings of the current investigation indicate that individuals diagnosed with NSCLC exhibited significant elevation in the serum levels of CEA and CYFRA21-1, as well as TGFß1, SMAD2, CTNNß1, and Wnt3a. In total, 325 mutations in four trialled genes (243 mutations in TGFß1, 24 mutations in SMAD2,47 mutation Wnt3a and 11 mutations in CTNNß1) were identified in patients with NSCLC. Furthermore, all mutations were recorded in adenocarcinoma, not squamous and normal adjacent tumour cells. CYFRA21-1 and CEA are more significant between NSCLC and HC, gender, and NSCLC types (p<0.001). In detail, TGFß1 exhibited the highest rate of mutations among other genes and three types of genomic mutations. Elevated levels and genetic polymorphisms of TGFß1, SMAD2, CTNNß1, and Wnt3a may play crucial functions in the pathogenesis and angiogenesis of non-small cell lung cancer (NSCLC). These biomarkers might play a role in future immunologic response and pharmacologically targeted NSCLC therapy.

Hypoxia enhances anti-fibrotic properties of extracellular vesicles derived from hiPSCs via the miR302b-3p/TGFß/SMAD2 axis.

BACKGROUND: Cardiac fibrosis is one of the top killers among fibrotic diseases and continues to be a global unaddressed health problem. The lack of effective treatment combined with the considerable socioeconomic burden highlights the urgent need for innovative therapeutic options. Here, we evaluated the anti-fibrotic properties of extracellular vesicles (EVs) derived from human induced pluripotent stem cells (hiPSCs) that were cultured under various oxygen concentrations. METHODS: EVs were isolated from three hiPSC lines cultured under normoxia (21% O2; EV-N) or reduced oxygen concentration (hypoxia): 3% O2 (EV-H3) or 5% O2 (EV-H5). The anti-fibrotic activity of EVs was tested in an in vitro model of cardiac fibrosis, followed by a detailed investigation of the underlying molecular mechanisms. Sequencing of EV miRNAs combined with bioinformatics analysis was conducted and a selected miRNA was validated using a miRNA mimic and inhibitor. Finally, EVs were tested in a mouse model of angiotensin II-induced cardiac fibrosis. RESULTS: We provide evidence that an oxygen concentration of 5% enhances the anti-fibrotic effects of hiPS-EVs. These EVs were more effective in reducing pro-fibrotic markers in activated human cardiac fibroblasts, when compared to EV-N or EV-H3. We show that EV-H5 act through the canonical TGFß/SMAD pathway, primarily via miR-302b-3p, which is the most abundant miRNA in EV-H5. Our results show that EV-H5 not only target transcripts of several profibrotic genes, including SMAD2 and TGFBR2, but also reduce the stiffness of activated fibroblasts. In a mouse model of heart fibrosis, EV-H5 outperformed EV-N in suppressing the inflammatory response in the host and by attenuating collagen deposition and reducing pro-fibrotic markers in cardiac tissue. CONCLUSIONS: In this work, we provide evidence of superior anti-fibrotic properties of EV-H5 over EV-N or EV-H3. Our study uncovers that fine regulation of oxygen concentration in the cellular environment may enhance the anti-fibrotic effects of hiPS-EVs, which has great potential to be applied for heart regeneration.

Impact of SMAD2 and MET Expression on Lymph Node Metastasis of HER2-positive Gastric Cancer Cells.

BACKGROUND/AIM: Intra-tumoral heterogeneity, which is frequently found in various types of cancers, has been suggested to play an important role in cancer progression and metastasis. The findings of our previous study suggested that p-SMAD2 and c-MET signaling might play important roles in the progression to lymph node metastasis of HER2-positive gastric cancer. In this study, we confirmed the effect of SMAD2/MET signaling in the progression of HER2-positive gastric cancer in an animal model. MATERIALS AND METHODS: NCI-N87 cells over-expressing ERBB2, SMAD2, MET were used. To confirm the role of SMAD2 and MET expression on lymph node metastasis of gastric cancer, we orthotopically injected NCI-N87 cells with or without the knockdown of both SMAD2 and MET into the gastric walls of BALBc nude mice. RESULTS: The number of metastatic lymph nodes was significantly smaller in the knockdown group compared to that in the control group. However, there was no significant difference in gastric tumor size between the two groups. CONCLUSION: SMAD2 and MET signaling might play important roles specifically in the progression to lymph node metastasis of HER2-positive gastric cancer. c-MET and SMAD2 may be useful targets for preventing lymph node metastasis in patients with HER2-positive gastric cancer.

SIRT2 alleviated renal fibrosis by deacetylating SMAD2 and SMAD3 in renal tubular epithelial cells.

Transforming growth factor-ß (TGF-ß) is the primary factor that drives fibrosis in most, if not all, forms of chronic kidney disease. In kidneys that are obstructed, specific deletion of Sirt2 in renal tubule epithelial cells (TEC) has been shown to aggravate renal fibrosis, while renal tubule specific overexpression of Sirt2 has been shown to ameliorate renal fibrosis. Similarly, specific deletion of Sirt2 in hepatocyte aggravated CCl4-induced hepatic fibrosis. In addition, we have demonstrated that SIRT2 overexpression and knockdown restrain and enhance TGF-ß-induced fibrotic gene expression, respectively, in TEC. Mechanistically, SIRT2 reduced the phosphorylation, acetylation, and nuclear localization levels of SMAD2 and SMAD3, leading to inhibition of the TGF-ß signaling pathway. Further studies have revealed that that SIRT2 was able to directly interact with and deacetylate SMAD2 at lysine 451, promoting its ubiquitination and degradation. Notably, loss of SMAD specific E3 ubiquitin protein ligase 2 abolishes the ubiquitination and degradation of SMAD2 induced by SIRT2 in SMAD2. Regarding SMAD3, we have found that SIRT2 interact with and deacetylates SMAD3 at lysine 341 and 378 only in the presence of TGF-ß, thereby reducing its activation. This study provides initial indication of the anti-fibrotic role of SIRT2 in renal tubules and hepatocytes, suggesting its therapeutic potential for fibrosis.

The SMAD2/miR-4256/HDAC5/p16INK4a signaling axis contributes to gastric cancer progression.

The dysregulation of exosomal microRNAs (miRNAs) plays a crucial role in the development and progression of cancer. This study investigated the role of a newly identified serum exosomal miRNA miR-4256 in gastric cancer (GC) and the underlying mechanisms. The differentially expressed miRNAs were firstly identified in serum exosomes of GC patients and healthy individuals using next-generation sequencing and bioinformatics. Next, the expression of serum exosomal miR-4256 was analyzed in GC cells and GC tissues, and the role of miR-4256 in GC was investigated by in vitro and in vivo experiments. Then, the effect of miR-4256 on its downstream target genes HDAC5/p16INK4a was studied in GC cells, and the underlying mechanisms were evaluated using dual luciferase reporter assay and Chromatin Immunoprecipitation (ChIP). Additionally, the role of the miR-4256/HDAC5/p16INK4a axis in GC was studied using in vitro and in vivo experiments. Finally, the upstream regulators SMAD2/p300 that regulate miR-4256 expression and their role in GC were explored using in vitro experiments. miR-4256 was the most significantly upregulated miRNA and was overexpressed in GC cell lines and GC tissues; in vitro and in vivo results showed that miR-4256 promoted GC growth and progression. Mechanistically, miR-4256 enhanced HDAC5 expression by targeting the promoter of the HDAC5 gene in GC cells, and then restrained the expression of p16INK4a through the epigenetic modulation of HDAC5 at the p16INK4a promoter. Furthermore, miR-4256 overexpression was positively regulated by the SMAD2/p300 complex in GC cells. Our data indicate that miR-4256 functions as an oncogene in GC via the SMAD2/miR-4256/HDAC5/p16INK4a axis, which participates in GC progression and provides novel therapeutic and prognostic biomarkers for GC.

Activin A/ACVR2A axis inhibits epithelial-to-mesenchymal transition in colon cancer by activating SMAD2.

Colorectal cancer is one of the most common malignancies worldwide. Liver metastasis is the major direct cause of colorectal cancer-related deaths. Although radical resection is the most effective treatment for colorectal cancer liver metastasis, several patients are not eligible for surgery. Therefore, there is a need to develop novel treatments based on the understanding of the biological mechanisms underlying liver metastasis in colorectal cancer. This study demonstrated that activin A/ACVR2A inhibits colon cancer cell migration and invasion, as well as suppresses the epithelial-to-mesenchymal transition of mouse colon cancer cells. This finding has been further validated in animal experiments. Mechanistic studies revealed that activin A binds to Smad2 (instead of Smad3) and activates its transcription. Analysis of the paired clinical samples further confirmed that the expression levels of ACVR2A and SMAD2 were the highest in adjacent healthy tissues, followed by primary colon cancer tissues and liver metastasis tissues, suggesting that ACVR2A downregulation may promote colon cancer metastasis. Bioinformatics analysis and clinical studies demonstrated that ACVR2A downregulation was significantly associated with liver metastasis and poor disease-free and progression-free survival of patients with colon cancer. These results suggest that the activin A/ACVR2A axis promotes colon cancer metastasis by selectively activating SMAD2. Thus, targeting ACVR2A is a potential novel therapeutic strategy to prevent colon cancer metastasis.

Repurposing of carvedilol to alleviate bleomycin-induced lung fibrosis in rats: Repressing of TGF-ß1/α-SMA/Smad2/3 and STAT3 gene expressions.

Idiopathic pulmonary fibrosis (IPF) is the most widely studied interstitial lung disease. IPF eventually leads to respiratory insufficiency, lung cancer, and death. Carvedilol (CAR) is a third-generation ß-adrenergic receptor antagonist with an α1-blocking effect. CAR demonstrates antifibrotic activities in various experimental models of organ fibrosis. AIMS: This work is designed to explore the possible alleviating effects of CAR on bleomycin (BLM)-induced lung fibrosis in rats. MAIN METHODS: The BLM rat model of lung fibrosis was achieved by intratracheal delivery of a single dose of 5 mg/kg of BLM. Seven days following BLM injection, either prednisolone or CAR was orally administered at doses of 10 mg/kg once daily for 21 days to the rats. The actions of CAR were evaluated by lung oxidant/antioxidant parameters, protein concentration and total leucocyte count (TLC) in bronchoalveolar lavage fluid (BALF), fibrosis regulator-related genes along with the coexistent lung histological changes. KEY FINDINGS: CAR effectively decreased lung malondialdehyde level, increased superoxide dismutase activity, declined both protein concentration and TLC in BALF, downregulated TGF-ß1/α-SMA/Smad2/3 and STAT3 gene expressions, and repaired the damaged lung tissues. SIGNIFICANCE: CAR conferred therapeutic potential against BLM-induced lung fibrosis in rats, at least in part, to its antioxidant, anti-inflammatory, and antifibrotic activities. CAR could be utilized as a prospective therapeutic option in patients with lung fibrosis in clinical practice.

The ß-catenin-LINC00183-miR-371b-5p-Smad2/LEF1 axis promotes adult T-cell lymphoblastic lymphoma progression and chemoresistance.

BACKGROUND: High-intensity chemotherapy regimens are often used in adult T-cell lymphoblastic lymphoma (T-LBL) patients. Nevertheless, the response rate remains unsatisfactory due to emergence of chemoresistance. Growing evidence has shown that long non-coding RNAs (lncRNAs) are involved in tumor progression and chemoresistance. Herein, we investigated the potential role of lncRNAs in T-LBLs. METHODS: RNAseq was used to screen and identify candidate lncRNAs associated with T-LBL progression and chemoresistance. Luciferase reporter assay was used to examine the binding of miR-371b-5p to the 3'UTR of Smad2 and LEF1, and the binding of TCF-4/LEF1 to the promoter of LINC00183. Chromatin immunoprecipitation assay was undertaken to analyze the connection between LEF1 and the LINC00183 promoter region. RNA immunoprecipitation assays were used to explore the mechanism whereby LINC00183 regulated miR-371b-5p. MTT and flow cytometry assays were used to measure apoptosis of T-LBL cells. RESULTS: LINC00183 was upregulated in T-LBL progression and chemoresistant tissues in both the Sun Yat-sen University Cancer Center dataset and the First Affiliated Hospital of Anhui Medical University dataset. High expression of LINC00183 was correlated with poorer overall survival and progression-free survival of T-LBL patients compared to those with low expression of LINC00183. Furthermore, miR-371b-5p was negatively regulated by LINC00183. In vivo and in vitro assays showed that LINC00183-mediated T-LBL chemoresistance depended on miR-371b-5p expression. The direct binding of miR-371b-5p to Smad2 and LEF1 was verified by luciferase assays. It was shown that TCF4/LEF1 could bind to the LINC00183 promoter site and increase its transcript level. Downregulation of miR-371b-5p led to increased expression of Smad2/LEF1, and in turn increased LINC00183 expression. Additionally, phospho-Smad2 promotes nuclear translocation of ß-catenin, LINC00183 downregulation decreased chemoresistance induced by ß-catenin and TGF-ß1 in T-LBL cells. CONCLUSION: We unraveled a ß-catenin-LINC00183-miR-371b-5p-Smad2/LEF1 feedback loop that promotes T-LBL progression and chemoresistance, indicating that LINC00183 may serve as a potential therapeutic target in T-LBLs.

Astragaloside IV suppresses the migration and EMT progression of cervical cancer cells by inhibiting macrophage M2 polarization through TGFß/Smad2/3 signaling.

Cervical cancer (CC) is a gynecological malignant tumor worldwide. Astragaloside IV (AS-IV) has been found to exert antitumor effects on CC. In addition, M2-polarized macrophages, known as tumor-associated macrophages (TAMs), play an important role in promoting cancer cell growth and angiogenesis. Thus, we explored the association between the antitumor effect of AS-IV and macrophage polarization in CC. Flow cytometry, ELISA, and RT‒qPCR assays were applied to detect the levels of CD163, IL-10, TGFß, and CD206 in M2 macrophages with or without AS-IV treatment. In addition, conditioned medium (CM) was collected from these M2 macrophages, and CC cells were then cultured in various CMs. Wound healing and transwell assays were used to assess the migratory ability of CC cells. In this study, we found that AS-IV significantly inhibited M2 polarization of macrophages, as shown by decreased CD163, IL-10, TGFß, and CD206 expression. In addition, compared with CM from M2 macrophages, CM from AS-IV-treated M2 macrophages notably inhibited angiogenesis, migration, and epithelial-mesenchymal transition (EMT) in CC cells. Furthermore, compared with CM from M2 macrophages, CM from AS-IV-treated M2 macrophages markedly reduced p-Smad2 and p-Smad3 protein expression in CC cells, and these changes were reversed by TGF-ß treatment. Collectively, suppression of M2-like polarization of macrophages by AS-IV could prevent the migration and EMT of CC cells by inactivating TGF-ß/Smad2/3 signaling. These findings might provide some theoretical support for exploring novel treatments for CC.

TGF-ß1 induced activations of Smad2 and miRNAs inhibit SF-1- and LRH-1-dependent CYP19 expression in rat Leydig cells†.

P450 aromatase, encoded by the Cyp19 gene, catalyzes the synthesis of estrogen, which is crucial for mammalian germ cell differentiation. We have previously shown that transforming growth factor beta 1 (TGF-ß1) attenuated the accumulation of steroidogenic factor-1 (SF-1) and liver receptor homolog-1 (LRH-1) and eventually reduced the transcription of Cyp19 in rat Leydig cells (LCs). Here, we report that TGF-ß1 treatment-induced phosphorylation of Smad2 and decreased the expression levels of SF-1 and LRH-1 by elevating the expression levels of microRNA-21-3p and microRNA-339-5p in vivo and in vitro. Furthermore, both TGF-ß1 treatment and over-expression of Smad2 inhibited the SF-1 or LRH-1-regulated promoter activity of the Cyp19 gene, and p-Smad2 physically interacted with SF-1 and LRH-1. Our findings collectively suggest that TGF-ß1 may inhibit the expression of CYP19 in LCs mainly through two ways. On the one hand, TGF-ß1 acts through Smad2 to repress the accumulation of SF-1 and LRH-1 at post-transcriptional level by upregulating specific microRNAs. On the other hand, TGF-ß1 inhibits the transcriptional activity of Cyp19 through the interaction of p-Smad2 with SF-1/LRH-1.

GOLM1 Promotes Epithelial-Mesenchymal Transition by Activating TGFß1/Smad2 Signaling in Prostate Cancer.

Background: Prostate cancer (PC) is one of the most commonly diagnosed cancer in men worldwide. Epithelial-mesenchymal transition (EMT) is considered to play a crucial role in the development of the metastatic castration-resistant prostate cancer, which causes the majority of the death cases in PC. Golgi membrane protein 1 (GOLM1) is highly expressed in PC and has been identified as a driver factor for EMT in various cancers. However, its biological functions and underlying mechanisms remain ambiguous in PC. Method: GOLM1 expression level of PC was detected by Western blot and immunohistochemistry analyses. To investigate GOLM1 functions in cancer cells, we overexpressed and knocked down GOLM1 in different prostate cancer cell lines. Transwell assay and wound healing assay were used to determine the role of GOLM1 in cell EMT, such as migration and invasion abilities. TGF-ß1/Smad2 signaling pathway downstream of GOLM1 was detected by Western blot and Transwell assay. Result: GOLM1 expression is up-regulated in PC and correlated with a worse prognosis. GOLM1 promotes the abilities of migration and invasion in PC cell lines (DU145 and LNCaP). Furthermore, TGF-ß1/Smad2 signaling is positively regulated by GOLM1 to facilitate EMT in PC, whereas this role can be restored by TGF-ß1 after GOLM1 knockdown or be abrogated by p-Smad inhibitor SB431542. Conclusion: GOLM1 is significantly upregulated in PC and acts as a critical oncogene by promoting PC cell EMT process by activating TGF-ß1/Smad2 signaling pathway. Therefore, GOLM1 has the potential to be a biomarker for PC diagnosis and to predict the prognosis of PC patients. It is of great significance to seek effective and specific inhibitor of GOLM1 for PC treatment as well.