Expression Of Transforming Growth Factor-beta Research Articles

Pan-cancer analysis revealing the multidimensional expression and prognostic and immunologic roles of TGFB1 in cancer.

This study aimed to perform an integrated pan-cancer analysis to characterize the expression patterns, prognostic value, genetic alterations, and immunologic roles of transforming growth factor beta 1 (TGFB1) across diverse human cancer types. Bioinformatics analyses were conducted using multiple public databases including The Cancer Genome Atlas, Genotype-Tissue Expression, Clinical Proteomic Tumor Analysis Consortium, TIMER2, GEPIA2, cBioPortal, StringDB, and others. Differential expression, survival, immune correlation, and protein interaction network analyses were performed. TGFB1 was overexpressed in several tumor types compared with that in normal tissues. High TGFB1 expression was associated with an advanced stage and poorer prognosis in certain cancers. TGFB1 mutations occurred in 1.3% of 10,967 cases surveyed. TGFB1 expression correlated with tumor-infiltrating immune cells and immunotherapy-related genes. This comprehensive multi-omics analysis revealed the complex expression and prognostic landscape of TGFB1 across cancers. TGFB1 is emerging as a potential immunotherapeutic target in certain contexts. Further research should elucidate its multifaceted tumor-promoting and tumor-suppressive mechanisms. Our pan-cancer analysis provides new insights into TGFB1 as a prognostic biomarker and immunotherapeutic target in human cancers, and our findings may guide future preclinical and clinical investigations of TGFB1-directed therapies.

Anti-proliferative activity and apoptotic induction of tannins extracted from Quercus infectoria on oral cancer KB cell lines

The present study aimed to evaluate the phytochemicals in Quercus infectoria ethanolic extract and to test the cytotoxic effect on an oral cancer cell line KERATIN-HeLa cells (KB cells). The cytotoxic potential of tannins extracted from Q. infectoria was tested by cell cycle analysis, transforming growth factor (TGF)-beta expression, matrix metalloproteinase (MMP) by fluorescent activated cell sorte, Caspase 3, and Caspase 9 expression by ELISA in KB cell lines treated with the extract. Specific protein (Bcl-2) and the gene expression in the KB cell line upon tannin treatment were detected by western blot and RT-PCR technique. The Caspase 3 and 9 enzymatic activity was carried out using the ELISA method. Virtual screening and molecular docking analysis were conducted to know the binding affinity against the targets matrix metalloproteinase-2 (MMP-2), NF-kB, and RhoA, which are targets of oral cancer cells. Preliminary screening of Q. infectoria indicates it primarily contains tannins and glycosides. The IC50 value of the ethanolic extract was determined to be 76.82 μg/ml. Analysis of the cell cycle revealed that the extract induced dose-dependent arrest in the G0/G1 phase. It is also revealed that KB cell treatment with the extract led to downregulation of the anti-apoptotic protein Bcl-2. Moreover, TGF beta expression was downregulated, and Caspase 3 and Caspase 9 were up-regulated in a dose-dependent manner. In addition, the extract induced mitochondrial membrane potential and MMP induction. Virtual screening and molecular docking revealed that tannins have a favorable binding affinity against MMP-2, NF-kB p65, and RhoA. The study identified that “6-O-digalloyl-1,2,3,4 tetra-O-galloyl-β-D-glucose,” a gallotannin in Q. infectoria, is responsible for the anti-cancer activity. The results of our study found that Q. infectoria extracts have anti-proliferative and apoptotic induction activity, which can help in novel drug discovery and development to alleviate cancer.

Dysregulation of Immunity in Pulmonary Fibrosis is Associated with Increased Myeloid-specific Triggering Receptor-1 and Transforming Growth Factor-beta1 Expression.

Fibrosing pneumonia (FP) is classified into usual interstitial pneumonia (UIP) and nonspecific interstitial pneumonia (NSIP), each having its own etiology and prognosis. Both types of FP are progressive and chronic conditions with distinct etiologies. Cytokines and inflammatory mediators play critical roles in the pathogenesis of FP. Among them, the role of transforming growth factor beta-1 (TGF-β1) and modulators triggering fibrosis are not well understood. In this study, the expression of triggering receptor expressed on myeloid cells-1 (TREM-1) as a stimulator for the production of TGF-β1 and also CD4+CD25+Foxp3+ regulatory cells were investigted in FP patients. Sixteen UIP, 14 NSIP and 4 pulmonary fibrosis following Mycobacterium tuberculosis (TB) infection patients, were compared with 12 healthy controls. The frequency of blood CD14+TGF-β1+ and CD14+TREM1+-gated monocytes and CD4+CD25+Foxp3+ regulatory T cells (Treg), as well as the plasma levels of TGF-β1 and IL‑10 were measured. Fibrosis patients compared to healthy controls had a greater frequency of CD14+TGF-β1+ [15.9 (0.2-88.2) vs. 0.6 (0.2-11.0)] and CD14+TREM1+ [21.1 (2.3-91.2) vs. 10.3 (3.1-28.6)]-gated monocytes, and CD4+CD25+Foxp3+ [1.2 (0.3-3.6) vs. 0.2 (0.1-0.4)]-gated lymphocytes. Plasma TGF-β1 were also significantly increased in patients with fibrosis compared to healthy controls [9316.2 (±5554.4) vs. 3787.5 (±2255.6)]. These results confirm the importance of TGF-β1 and TREM1 in pulmonary fibrosis. It seems that this reciprocal cycle in healthy people is modulated by the production of IL‑10 by Treg cells,thus limiting fibrosis, as observed in patients following TB infection. Further investigations are recommended to evaluate possible immunomodulatory mechanisms defects in pulmonary fibrosis.

PSII-A-11 Suppressing the Cxcl12-Cxcr4 Chemokine Axis at the Fetal-Maternal Interface During Implantation Results in Altered Expression of Inflammatory Cytokines in Ovine Placenta at Midgestation

Abstract A proper balance of pro and anti-inflammatory cytokines at the fetal-maternal interface is crucial for successful embryo implantation and placental formation (placentation) during early gestation. However, mechanisms controlling this inflammatory profile are not well characterized. The C-X-C motif chemokine ligand 12 (CXCL12) and its receptor CXCR4 are expressed by fetal and maternal placental tissues and implicated in cytokine synthesis; roles of this axis during placentation are unclear. Using an in vivo sheep model, we demonstrated that suppressing CXCL12-CXCR4 signaling at the fetal-maternal interface during implantation results in dysregulated production of inflammatory cytokines in the placenta. The objective of the current study was to determine if the disrupted cytokine profile from suppressing CXCL12-CXCR4 signaling early in gestation would result in altered placental inflammatory profile at midgestation. On day 12 post-breeding, osmotic pumps were surgically installed in 37 ewes and delivered either CXCR4 inhibitor (AMD3100) at 1X dose, (n=8), 1.5X, (n=8), or 3X, (n=8) or saline (n=13) into the uterine lumen ipsilateral to the corpus luteum for 14 days. On days 90 and 135, placental tissues were collected, and maternal and fetal placenta components separated and analyzed. Gene expression for select cytokines were analyzed in the placenta using Real-time qPCR. On day 90, mRNA expression was significantly altered by AMD3100 treatment for tumor necrosis factor (TNF), interleukin 12 (IL-12), and transforming growth factor beta (TGFB) in fetal placenta. Expression of TGFB in fetal placenta was also altered by AMD3100 treatment compared to control on day 135 whereas IL-12 expression was significantly altered in maternal placenta on day 135. In conclusion, modulating CXCL12-induced actions is a novel approach to manipulating the fetal-maternal environment when most pregnancy losses occur. Increasing our understanding of CXCL12-induced actions controlling the placental inflammatory environment will hopefully reveal methods to improve reproductive success and fetal health in livestock.

Abstract 6172: Artificial intelligence-powered spatial analysis of tumor-infiltrating lymphocytes reveals immune-excluded phenotype is correlated with TGF-beta pathway related genomic aberrations

Abstract Aberrant transforming growth factor-beta(TGF-B) pathway in the tumor microenvironment has been highlighted as one of the core resistance pathways of immunotherapy, by excluding tumor-infiltrating lymphocytes (TIL) out of the tumor area. However, no studies have coupled immune phenotypes classified by spatial analysis of TIL in whole slide images (WSI) with TGF-B pathway analysis on a large-scale database. Here, we hypothesized that the immune-excluded phenotype classified by a deep-learning spatial analysis model, Lunit SCOPE IO, would be correlated with the aberrant TGF-B pathway in The Cancer Genome Atlas (TCGA) cohorts. Aberrant TGF-B pathway was measured by Trimmed Mean of M-values (TMM) normalized and transformed to log2 of counts-per-million of previously published gene sets of fibroblast-specific TGF-beta responsive gene signature, using edgeR packages from TCGA RNA-sequencing data (n=6,709) across the 23 cancer types. Lunit SCOPE IO was developed to identify immune phenotypes trained and validated from 3,166 multi-cancer H&E WSI with sections of 2.8e+9 mm2 tumor tissue containing 5.9e+6 TIL annotated by 52 board-certified pathologists. Lunit SCOPE IO classified immune phenotypes as immune-inflamed and -excluded according to the proportion of TIL density either highly conserved in cancer epithelium (CE) or cancer stroma (CS), respectively, and otherwise, classified as immune-desert with low TIL density in CE and CS. Aberrant TGF-B expression was highly enriched in multiple cancer types including pancreatic cancer, head and neck cancer, kidney clear cell carcinoma, lung squamous cell carcinoma, and breast cancer, in ascending order. TGF-B expression was increased in microsatellite-stable tumor samples (p = 7.4e-15) or samples with low tumor mutational burden (TMB, < 10/megabase, p = 4.9e-8), compared to those with microsatellite instability-high or high TMB, respectively. Interestingly, TGF-B expression was highly correlated with the proportion of cancer stroma in WSI (R = 0.315, p < 2.2e-16) and the proportion of immune-excluded phenotype (R = 0.115, p < 2.2e-16) across multiple cancer types. Tumor samples with SMAD4 mutations (n = 161, 2.4%) had significantly higher TGF-B expression (p = 0.0190), and a higher proportion of immune-excluded phenotype (p < 2.2e-16) in WSI, compared to wild-type SMAD4. Aberrant TGF-B pathway is clearly associated with increased proportion of cancer stroma, and excluded TIL, or immune-excluded phenotype in a large-scale pan-carcinoma analysis. Citation Format: Gahee Park, Sanghoon Song, Hyung-Gyo Cho, Soo Ick Cho, Wonkyung Jung, Lunit AI team, Sergio Pereira, Donggeun Yoo, Kyunghyun Paeng, Chan-Young Ock. Artificial intelligence-powered spatial analysis of tumor-infiltrating lymphocytes reveals immune-excluded phenotype is correlated with TGF-beta pathway related genomic aberrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6172.

Transforming Growth Factor Beta1 Expression in Cancer- Associated Fibroblasts of Urinary Bladder Cancer: Crucial Applications and Deep Insights

Background: Urinary bladder carcinoma (UBC) is one of the most common malignancies in Egypt and all over the world. TGFB levels in plasma and urine were proved to connote predictive and prognostic attributes in UBC patients. Furthermore, Cancer associated fibroblasts (CAFs) are now recognized as a key player in carcinogenesis. Yet, TGFΒ1 expression in CAFs of UBC had not been elucidated. Moreover, TGFB1 targeted therapy is now emerging with potential benefits for TGFB1 expressing cancers.
 Aim of the study: we dedicated this study to explore potential implications of TGFB1 immunohistochemical expression in CAFs of UBC by correlating it to relevant clinical and pathological data.
 Material and methods: This retrospective study included 48 UBC specimens. Different tumor grades were presented in balanced groups. TGFB1 immunohistochemical expression was evaluated, categorized as low or high and compared in CAFs among different UBC grades, statistical analysis of the results was then followed.
 Results: TGFB1 expression in CAFs was significantly different among tumor histologic types (P=0.01), high tumor grade (P=<0.01), presence of muscle invasion (P=<0.001), higher tumor stage (P=0.01), presence of preceding bilharziasis (P=0.003), and necrosis (P=0.03). There was a highly significant difference between TGFB1 expression in both tumor cells and CAFs (P=0.002). Intense CAFs TGFB1 staining was also strikingly observed along the muscle invading frontside UBC cells further emphasizing the pivotal role of CAFs expressing TGFB1 in invasion.
 Conclusion: This study demonstrates significant predictive implications of TGFB1 in UBC, thus emphasizing its potential benefits in management and therapy.

Chorioamnionitis Causes Kidney Inflammation, Podocyte Damage, and Pro-fibrotic Changes in Fetal Lambs.

BackgroundPerinatal complications, such as prematurity and intrauterine growth restriction, are associated with increased risk of chronic kidney disease. Although often associated with reduced nephron endowment, there is also evidence of increased susceptibility for sclerotic changes and podocyte alterations. Preterm birth is frequently associated with chorioamnionitis, though studies regarding the effect of chorioamnionitis on the kidney are scarce. In this study, we aim to unravel the consequences of premature birth and/or perinatal inflammation on kidney development using an ovine model.MethodsIn a preterm sheep model, chorioamnionitis was induced by intra-amniotic injection of lipopolysaccharide (LPS) at either 2, 8, or 15 days prior to delivery. Control animals received intra-amniotic injections of sterile saline. All lambs were surgically delivered at 125 days’ gestation (full term is 150 days) and immediately euthanized for necropsy. Kidneys were harvested and processed for staining with myeloperoxidase (MPO), Wilms tumor-1 (WT1) and alpha-smooth muscle actine (aSMA). mRNA expression of tumor necrosis factor alpha (TNFA), Interleukin 10 (IL10), desmin (DES), Platelet derived growth factor beta (PDGFB), Platelet derived growth factor receptor beta (PDGFRB), synaptopodin (SYNPO), and transforming growth factor beta (TGFB) was measured using quantitative PCR.ResultsAnimals with extended (but not acute) LPS exposure had an inflammatory response in the kidney. MPO staining was significantly increased after 8 and 15 days (p = 0.003 and p = 0.008, respectively). Expression of TNFA (p = 0.016) and IL10 (p = 0.026) transcripts was increased, peaking on day 8 after LPS exposure. Glomerular aSMA and expression of TGFB was increased on day 8, suggesting pro-fibrotic mesangial activation, however, this was not confirmed with PDFGB or PDGFRB. The number of WT1 positive nuclei in the glomerulus, as well as expression of synaptopodin, decreased, indicating podocyte injury.ConclusionWe report that, in an ovine model of prematurity, LPS-induced chorioamnionitis leads to inflammation of the immature kidney. In addition, this process was associated with podocyte injury and there are markers to support pro-fibrotic changes to the glomerular mesangium. These data suggest a potential important role for antenatal inflammation in the development of preterm-associated kidney disease, which is frequent.

Influence of carrier materials and coatings on retinal pigment epithelium cultivation and functions

Properties of retinal pigment epithelium (RPE) are relevant for the development of cell culture models concerning an exact reproduction of the ocular cell biology. Here, we want to investigate how different carrier materials and coatings influence proliferation, differentiation and functions of RPE in regard to development of a three-dimensional cell culture model based on primary porcine RPE. Human RPE cell line ARPE-19 and primary porcine RPE were used. Cells were cultivated on plates which were coated with collagen I, collagen IV, laminin or fibronectin, respectively, and cell numbers were assessed after different time periods via trypan blue staining. Also, the ARPE-19 were cultivated on polydimethylsiloxane (PDMS), alginate, gelatin methacrylate (GelMA), poly-N-isopropylacrylamide (PNIPAM) and cells number were assessed. Primary RPE were cultured on PDMS material. Supernatants were collected and analyzed via ELISA for their vascular endothelial growth factor (VEGF) and transforming growth factor β (TGF-β) content. After day 14 cells were lysed and retinal pigment epithelium-specific 65 kDa protein (RPE65) and bestrophin-1 (BEST1) expression was investigated via Western blot. Cellular functions were tested on collagen I, collagen IV, laminin and fibronectin with and without PDMS. Scratch assay was performed to detect wound healing 24 and 48 h after scratch application. Immunolabeling was used to highlight tight junctions in concert with Hoechst staining and phalloidin to label cell nuclei and actin filaments, respectively. Phagocytosis of fluorescently labeled latex beads opsonized with photoreceptor outer segments (POS) was assessed via fluorescence microscopy. Transepithelial electrical resistance was measured for detection of cellular barrier. Gene expression of RDH11 (retinol dehydrogenase 11), BEST1 (bestrophin 1) and TGFB1 (transforming growth factor beta 1) was investigated via real-time PCR. Only PDMS carrier material was appropriate for primary RPE and ARPE-19 cell cultivation. Coating of PDMS with laminin led to increased proliferation. In primary RPE, VEGF secretion was increased if PDMS was coated with laminin or fibronectin compared to uncoated PDMS. No significant changes in phagocytic ability and generation of tight junctions were detected between different coatings, but RPE65 expression was reduced on fibronectin coated PDMS. Laminin coating decreased TGF-β and increased BEST1 protein expression. Also, RPE on collagen IV showed highest TEER on transwell plates. The genes RDH11 and TGFB1 were decreased when coated with collagen IV without PDMS as well as coated PDMS. Laminin and collagen IV coating led to an increased wound healing. Cultivation of RPE and ARPE-1 on PDMS is a possible alternative for cell culture models whereas alginate, GelMA and PNIPAM were not suitable. Coating with laminin increased the proliferation, wound healing and VEGF secretion of the cells. The results suggest that laminin coated PDMS as carrier material is suitable for the development of 3D culture model systems.

Evaluating Different Low-intensity Extracorporeal Shockwave Therapy Intensities in the Treatment of Peyronie's Disease in a Rat Model

ABSTRACT Introduction Low-intensity extracorporeal shockwave therapy (Li-ESWT) is postulated to have physiologic effects including neo-angiogenesis, upregulation of vasoactive endothelial growth factors, nerve recovery, reduction of fibrotic changes, cavernosal tissue remodeling, and reduction in sympathetic tone. To date, randomized clinical trials have failed to demonstrate a change in penile curvature, with mixed results on erectile function, in Peyronie's disease (PD) patients. Objective To evaluate the treatment effect of Li-ESWT for amelioration of PD-associated fibrosis and PD-associated erectile dysfunction using a rat model of PD. Methods 32 adult male Sprague-Dawley rats were divided evenly into four groups: sham (S), control (T), low setting Li-ESWT (TL), and high setting Li-ESWT (TH). Normal saline was injected into the tunica albuginea (TA) of the sham group. Transforming growth factor beta-1 (TGF-B1) was injected into the TA of the remaining groups to induce Peyronies-like fibrosis. After 5 weeks of TGF-B1 induction, treatment with Li-ESWT shockwave (UroGold 100™) was initiated. Treatment was delivered biweekly for 3 weeks (6 total sessions). Device settings were 600 shocks at 3 Hz, at level 6 (0.093 mJ/mm2) for the Low group and at level 12 (0.125 mJ/mm2) for the High group. Two weeks after the end of treatment, erectile function was measured by ratio of intracavernosal pressure (ICP) to mean arterial pressure (MAP). Histological analysis by H&E and Trichrome staining was performed. Western blot analysis of Collagens I (COL1A1), III (COL3A1), elastin, alpha-smooth muscle actin (a-SMA), and TGF-B1 was performed. Results There was no significant difference demonstrated in erectile function between T control group and TL and TH groups (P > 0.05). However, there seems to be a trend in decreased amount of fibrosis upon examination of the prepared histologic sections. Animals in the TL and TH groups had decreased COL1A1, COL3A1, elastin, and TGF-B1 expression in the TA compared to the T group. a-SMA expression in the treatment groups was found to be increased compared to both S and T groups. Conclusions Our preliminary data shows that Li-ESWT treatments, at different intensities, may decrease fibrosis induced by TGF-B1 TA injections. Additionally, Li-ESWT was not found to have conclusive positive effects on erectile function. Disclosure Yes, this is sponsored by industry/sponsor: Sexual Medicine Society of North America Clarification Industry funding only - investigator initiated and executed study Any of the authors act as a consultant, employee or shareholder of an industry for: Endo Pharmaceuticals

Altered uterine angiogenesis in rats treated with a glyphosate-based herbicide

Glyphosate-based herbicides (GBHs) are the agrochemicals most used around the globe. However, they might have adverse effects on human and animal health. Previously, we showed that female rats neonatally exposed to GBHs exhibit altered expression of morphogenetic molecules and biomarkers of uterine development. We also observed a reduction in the size of implantation sites, altered expression of decidualization-related molecules, and increased post-implantation losses. Since decidualization comprises morphogenetic, biochemical and vascular changes, here we investigated the effects of neonatal GBH exposure on uterine angiogenesis in neonatal and pregnant rats. To achieve this, Wistar female rats were exposed to saline solution or GBH (2 mg glyphosate/kg-bw/day) on post-natal days (PND) 1, 3, 5 and 7. On PND8, uterine samples were collected for developmental studies. On PND90, the remaining females were mated and in the morning of gestational day (GD) 9, the implantation sites were collected. Angiogenesis-related molecules and cells involved in this process were identified and/or measured by immunohistochemistry or RT-PCR. On PND8, GBH-treated rats showed increased vascular endothelial growth factor (VEGF) expression and decreased Notch1, inducible nitric oxide synthase (iNOS) and Angiopoietin-2 (Ang2) mRNA levels. Vascular area, vessel diameter, endothelial cell proliferation, VEGF and Nestin protein expression, and VEGF, Notch1, iNOS and cyclooxygenase-2 (Cox-2) genes were downregulated in implantation sites of exposed females, while Ang2, VEGF receptor 1 and interleukin-10 (IL-10) were increased. Mast cells and macrophages were increased on PND8 and GD9 of treated rats. The increased Transforming growth factor-beta expression in the antimesometrial zone and IL-10 mRNA expression suggest that the M2 type is the predominant population of macrophages on implantation sites. In conclusion, neonatal GBH exposure alters the expression of angiogenesis-related molecules at neonatal uterine development and decidual reaction, suggesting altered vascular support. These alterations might contribute to the increased post-implantation losses observed in GBH-treated rats.

Development of the Diabetic Kidney Disease Mouse Model Culturing Embryos in α-Minimum Essential Medium In Vitro, and Feeding Barley Diet Attenuated the Pathology.

Diabetic kidney disease (DKD) is a critical complication associated with diabetes; however, there are only a few animal models that can be used to explore its pathogenesis. In the present study, we established a mouse model of DKD using a technique based on the Developmental Origins of Health and Disease theory, i.e., by manipulating the embryonic environment, and investigated whether a dietary intervention could ameliorate the model’s pathology. Two-cell embryos were cultured in vitro in α-minimum essential medium (MEM; MEM mice) or in standard potassium simplex-optimized medium (KSOM) as controls (KSOM mice) for 48 h, and the embryos were reintroduced into the mothers. The MEM and KSOM mice born were fed a high-fat, high-sugar diet for 58 days after they were 8 weeks old. Subsequently, half of the MEM mice and all KSOM mice were fed a diet containing rice powder (control diet), and the remaining MEM mice were fed a diet containing barley powder (barley diet) for 10 weeks. Glomerulosclerosis and pancreatic exhaustion were observed in MEM mice, but not in control KSOM mice. Renal arteriolar changes, including intimal thickening and increase in the rate of hyalinosis, were more pronounced in MEM mice fed a control diet than in KSOM mice. Immunostaining showed the higher expression of transforming growth factor beta (TGFB) in the proximal/distal renal tubules of MEM mice fed a control diet than in those of KSOM mice. Pathologies, such as glomerulosclerosis, renal arteriolar changes, and higher TGFB expression, were ameliorated by barley diet intake in MEM mice. These findings suggested that the MEM mouse is an effective DKD animal model that shows glomerulosclerosis and renal arteriolar changes, and barley intake can improve these pathologies in MEM mice.

Two naturally derived small molecules disrupt the sineoculis homeobox homolog 1-eyes absent homolog 1 (SIX1-EYA1) interaction to inhibit colorectal cancer cell growth.

Background:Emerging evidence indicates that the sineoculis homeobox homolog 1−eyes absent homolog 1 (SIX1–EYA1) transcriptional complex significantly contributes to the pathogenesis of multiple cancers by mediating the expression of genes involved in different biological processes, such as cell-cycle progression and metastasis. However, the roles of the SIX1–EYA1 transcriptional complex and its targets in colorectal cancer (CRC) are still being investigated. This study aimed to investigate the roles of SIX1–EYA1 in the pathogenesis of CRC, to screen inhibitors disrupting the SIX1–EYA1 interaction and to evaluate the efficiency of small molecules in the inhibition of CRC cell growth.Methods:Real-time quantitative polymerase chain reaction and western blotting were performed to examine gene and protein levels in CRC cells and clinical tissues (collected from CRC patients who underwent surgery in the Department of Integrated Traditional and Western Medicine, West China Hospital of Sichuan University, between 2016 and 2018, n = 24). In vivo immunoprecipitation and in vitro pulldown assays were carried out to determine SIX1–EYA1 interaction. Cell proliferation, cell survival, and cell invasion were determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, clonogenic assay, and Boyden chamber assay, respectively. The Amplified Luminescent Proximity Homogeneous Assay Screen (AlphaScreen) method was used to obtain small molecules that specifically disrupted SIX1–EYA1 interaction. CRC cells harboring different levels of SIX1/EYA1 were injected into nude mice to establish tumor xenografts, and small molecules were also injected into mice to evaluate their efficiency to inhibit tumor growth.Results:Both SIX1 and EYA1 were overexpressed in CRC cancerous tissues (for SIX1, 7.47 ± 3.54 vs.1.88 ± 0.35, t = 4.92, P = 0.008; for EYA1, 7.61 ± 2.03 vs. 2.22 ± 0.45, t = 6.73, P = 0.005). The SIX1/EYA1 complex could mediate the expression of two important genes including cyclin A1 (CCNA1) and transforming growth factor beta 1 (TGFB1) by binding to the myocyte enhancer factor 3 consensus. Knockdown of both SIX1 and EYA1 could decrease cell proliferation, cell invasion, tumor growth, and in vivo tumor growth (all P < 0.01). Two small molecules, NSC0191 and NSC0933, were obtained using AlphaScreen and they could significantly inhibit the SIX1–EYA1 interaction with a half-maximal inhibitory concentration (IC50) of 12.60 ± 1.15 μmol/L and 83.43 ± 7.24 μmol/L, respectively. Administration of these two compounds could significantly repress the expression of CCNA1 and TGFB1 and inhibit the growth of CRC cells in vitro and in vivo.Conclusions:Overexpression of the SIX1/EYA1 complex transactivated the expression of CCNA1 and TGFB1, causing the pathogenesis of CRC. Pharmacological inhibition of the SIX1–EYA1 interaction with NSC0191 and NSC0933 significantly inhibited CRC cell growth by affecting cell-cycle progression and metastasis.

Clinical importance of screening differential gene set of monocytes based on single-cell sequencing and digital polymerase chain reaction technology for early diagnosis of sepsis

To verify the specific differentiated subsets of monocytes in sepsis, and to screen and construct the differential gene set of monocytes used for early diagnosis of sepsis. Patients with sepsis admitted to Guangdong Provincial People's Hospital from June 2020 to March 2021 were enrolled, and peripheral blood mononuclear cells (PBMC) were extracted. Single-cell sequencing technology and pseudo-time analysis were used to verify the differential subsets of monocytes. Bioinformatics methods were used to analyze the expression of genes in differential subsets of monocytes and screen out differential genes for the preliminary construction of a candidate differential gene set. The digital polymerase chain reaction (PCR) technology was used to verify the candidate differential genes in PBMC of sepsis patients and sepsis human myeloid leukemia mononuclear cells (THP-1) models, and the Venn diagram was used to construct the final differential gene set of monocytes. Gene Expression Omnibus (GEO) database was used to validate the differential gene set of monocytes. (1) The results of cell annotation and pseudo-time analysis showed that the differentiation of NEAT1+CD163+ monocyte occurred in the early stage of sepsis was significantly different from other subsets, which validated that NEAT1+CD163+ monocyte was the characteristic subset in the pathological process of sepsis. (2) Twenty-two differential genes related to sepsis were screened out from the gene expression of NEAT1+CD163+ monocyte. After further verification by digital PCR, basic leucine zipper ATF-like transcription factor (BATF), JUNB proto-oncogene, carcinoembryonic antigen-related cell adhesion molecule 4 (CEACAM4), chromosome 9 open reading frame 95 (C9orf95), G protein subunit alpha 15 (GNA15), complement C3a receptor 1 (C3AR1), transforming growth factor beta 1 (TGFB1) and mitochondrial carrier homolog 1 (MTCH1) were screened out to construct the final differential gene set of monocytes. (3) The external validation results showed that C9orf95 gene had no data in GSE154918 and GSE133822 from GEO, it was excluded during validation. In GSE154918, the expressions of BATF, JUNB, CEACAM4, GNA15, C3AR1, TGFB1, and MTCH1 in the sepsis group were significantly higher than those in the healthy control group (log2expression level: BATF was 12.78±0.08 vs. 11.39±0.35, JUNB was 16.88±0.07 vs. 16.04±0.03, CEACAM4 was 14.73±0.08 vs. 13.77±0.05, GNA15 was 13.16±0.06 vs. 12.30±0.04, C3AR1 was 14.62±0.13 vs. 12.87±0.05, TGFB1 was 16.95±0.05 vs. 16.57±0.36, MTCH1 was 14.80±0.02 vs. 14.61±0.15, all P < 0.05). In GSE133822, the expressions of BATF, CEACAM4, GNA15, and C3AR1 in the sepsis group were significantly higher than those in the health control group (log2expression level: BATF was 8.66±0.16 vs. 7.92±0.14, CEACAM4 was 9.20±0.16 vs. 8.36±0.20, GNA15 was 10.66±0.18 vs. 10.13±0.16, C3AR1 was 11.49±0.27 vs. 10.48±0.16, all P < 0.05), while the expressions of JUNB, TGFB1, and MTCH1 were not statistically different between two groups. The results of gene set variation analysis (GSVA) showed that the enrichment scores of monocytes differential gene set of sepsis group were significantly higher than those of the healthy control group in both GSE154918 (0.38±0.04 vs. -0.44±0.02) and GSE133822 (0.56±0.02 vs. 0.20±0.05, both P < 0.01). Receiver operator characteristic curve (ROC curve) analysis showed that the differential gene set of monocytes had a reliable diagnostic value for early sepsis with the area under ROC curve (AUC) of 0.993 [95% confidence interval (95%CI) was 0.980-1.000] in GSE154918 and 0.944 (95%CI was 0.873-1.000) in GSE133822. A differential gene set of monocytes (BATF, JUNB, CEACAM4, GNA15, C3AR1, TGFB1, and MTCH1) screened out by single-cell sequencing and digital PCR technology has a reliable diagnostic value for the early sepsis, and may provide a new idea for the early diagnosis of sepsis.

CB2 receptors modulate seizure-induced expression of pro-inflammatory cytokines in the hippocampus but not neocortex.

We compared neuroinflammatory responses induced by nonconvulsive and convulsive seizures and analyzed the role that may be played by cannabinoid CB2 receptors in the neuroinflammatory response induced by generalized tonic-clonic seizures (GTCS). Using quantitative PCR, we analyzed expression of interleukin-1b, CCL2, interleukin-6, tumor necrosis factor (TNF), transforming growth factor beta 1 (TGFb1), fractalkine, and cannabinoid receptor type 2 in the neocortex, dorsal and ventral hippocampus, cortical leptomeninges, dura mater, and spleen in 3 and 6h after induction of GTCS by a high dose of pentylenetetrazole (PTZ, 70mg/kg) and absence-like activity by a low dose of PTZ (30mg/kg). The low dose of PTZ had no effect on the gene expression 3 and 6h after PTZ injection. In 3 and 6h after high PTZ dose, the expression of CCL2 and TNF increased in the neocortex. Both ventral and dorsal parts of the hippocampus responded to seizures by elevation of CCL2 expression 3h after PTZ. Cortical leptomeninges but not dura mater also had elevated CCL2 level and decreased TGFb1 expression 3h after GTCS. Activation of CB2 receptors by HU308 suppressed an inflammatory response only in the dorsal hippocampus but not neocortex. Suppression of CB2 receptors by AM630 potentiated expression of inflammatory cytokines also in the hippocampus but not in the neocortex. Thus, we showed that GTCS, but not the absence-like activity, provoke inflammatory response in the neocortex, dorsal and ventral hippocampus, and cortical leptomeninges. Modulation of CB2 receptors changes seizure-induced neuroinflammation only in the hippocampus but not neocortex.

Effect of Obstructive Sleep Apnea on Immunity in Cases of Chronic Rhinosinusitis With Nasal Polyps.

Objectives.Chronic rhinosinusitis with nasal polyps (CRSwNP) is a more severe inflammatory form of CRS that often coexists with obstructive sleep apnea (OSA). However, little is known about the relationship between OSA and the immune profile in patients with CRSwNP. We aimed to investigate the immune profile of patients with CRSwNP according to OSA severity.Methods.This study included 63 patients with CRSwNP and nine control subjects. Protein levels of inflammatory mediators were determined using multiplex immunoassays. All patients underwent standard polysomnography.Results.In patients with eosinophilic CRSwNP (ECRSwNP), interleukin (IL)-6 and chemokine [C-X-C motif] ligand (CXCL)-1 (type 1 immune-related markers) were upregulated in cases of moderate-to-severe OSA. Additionally, IL-4, IL-13, C-C motif chemokine (CCL)-11, CCL-24 (type 2 immune-related markers), and IL-17A (a type 3 immune-related marker) were present at elevated levels in patients with moderate-to-severe OSA. Although there were no significant differences in type 1, 2, or 3 immune-related markers among patients with non-eosinophilic CRSwNP (NECRSwNP) according to the severity of OSA, transforming growth factor-beta expression was higher in those with moderate-to-severe OSA. Furthermore, in ECRSwNP with moderate-to-severe OSA, associations were detected between serum markers and some upregulated inflammatory markers.Conclusion.OSA may increase the heterogeneity of the immune profile (types 1, 2, and 3) in patients with ECRSwNP, but not in those with NECRSwNP.

Prune-1 drives polarization of tumor-associated macrophages (TAMs) within the lung metastatic niche in triple-negative breast cancer

SummaryM2-tumor-associated macrophages (M2-TAMs) in the tumor microenvironment represent a prognostic indicator for poor outcome in triple-negative breast cancer (TNBC).Here we show that Prune-1 overexpression in human TNBC patients has positive correlation to lung metastasis and infiltrating M2-TAMs. Thus, we demonstrate that Prune-1 promotes lung metastasis in a genetically engineered mouse model of metastatic TNBC augmenting M2-polarization of TAMs within the tumor microenvironment. Thus, this occurs through TGF-β enhancement, IL-17F secretion, and extracellular vesicle protein content modulation.We also find murine inactivating gene variants in human TNBC patient cohorts that are involved in activation of the innate immune response, cell adhesion, apoptotic pathways, and DNA repair. Altogether, we indicate that the overexpression of Prune-1, IL-10, COL4A1, ILR1, and PDGFB, together with inactivating mutations of PDE9A, CD244, Sirpb1b, SV140, Iqca1, and PIP5K1B genes, might represent a route of metastatic lung dissemination that need future prognostic validations.

Decellularized extracellular matrix-rich hydrogel-silver nanoparticle mixture as a potential treatment for acute liver failure model.

Acute liver failure (ALF) occurs due to severe liver damage that triggers rapid loss of normal liver function. Here, we investigate the usefulness of an injectable liver extracellular matrix (LECM)-rich hydrogel generated from an optimized decellularization protocol incorporated with silver nanoparticles (AgNPs) as a promising therapy for ALF. First, we optimized a non-destructive protocol for rat liver decellularization to obtain ECM-rich well-preserved scaffold. Then, LECM hydrogel generated from two commonly used decellularization protocols were compared by LECM hydrogel obtained from our optimized protocol. The ALF model was induced by an intraperitoneal (IP) thioacetamide (TAA) injection followed by the IP injection of LECM hydrogel, collagen-AgNP mixture, or LECM hydrogel-AgNP mixture. LECM-rich scaffold and hydrogel were successfully obtained using our optimized decellularization protocol. Use of the LECM hydrogel-AgNP mixture to treat TAA-induced ALF greatly improved liver injury and histological liver regeneration. Interleukin-6 and transforming growth factor-beta expressions were significantly reduced, while albumin, hepatocyte growth factor, and Ki67-positive cells were highly expressed. Moreover, aspartate transaminase and alanine transaminase plasma levels and liver homogenate nitric oxide level were significantly lowered. In conclusion, the LECM hydrogel-AgNP mixture has potential efficient therapeutic and regenerative effects on TAA-induced liver injury.

Oncogenic Long Noncoding RNA DARS-AS1 in Childhood Acute Myeloid Leukemia by Binding to microRNA-425.

Objective:Acute myeloid leukemia (AML) represents a hematological cancer. The aim of the investigation was to probe the regulatory relevance of long non-coding RNA (lncRNA) aspartyl-tRNA synthetase anti-sense 1 (DARS-AS1)/microRNA-425 (miR-425)/transforming growth factor-beta 1 (TGFB1) to the development of AML.Methods:The DARS-AS1 expression in bone marrow tissues was first analyzed in healthy subjects and AML patients. Subsequently, AML cell lines with DARS-AS1 knockdown were constructed, followed by cell proliferation and apoptosis assays. Afterward, downstream miRNA of DARS-AS1 and target mRNA of the miRNA were analyzed by bioinformatics, and their binding relationships were verified. Functional rescue experiments were then implemented. Finally, activation of the Smad2/3 signaling in MV4-11 and BF-24 cells were detected by western blot.Results:DARS-AS1 was overexpressed in bone marrow tissues of AML patients and cells, and DARS-AS1 knockdown suppressed the proliferation of AML cells and induced apoptosis. DARS-AS1 bound to and negatively correlated with miR-425. Further results suggested that TGFB1 might be a target gene of miR-425 and could promote Smad2/3 phosphorylation and nuclear translocation. Finally, DARS-AS1 depletion could diminish the tumor volume in vivo.Conclusion:All in all, we highlighted here that DARS-AS1 enhanced the expression of TGFB1 through binding to miR-425 to modulate AML progression via the Smad2/3 pathway, which might perform as a therapeutic target for AML.

Application of “macromolecular crowding” in vitro to investigate the naphthoquinones shikonin, naphthazarin and related analogues for the treatment of dermal scars

Pathological scarring is an intractable problem for both patients and clinicians. A major obstacle for the development of scar remediation therapies is the paucity of suitable in vivo and in vitro models. The “Scar-in-a-jar” model was previously established by our colleagues based on the principle of “Macromolecular crowding”. This has been demonstrated to be an extracellular matrix-rich in vitro model offering a novel tool for studies related to the extracellular matrix. In the study reported herein, we have optimised this approach to model human dermal fibroblasts derived from hypertrophic tissues. This optimised in vitro model has been found to hold similar properties, such as increased collagen I, interleukins and transforming growth factor beta-1 expression, compared to that observed in hypertrophic scar tissue in vivo. In addition, Shikonin has been previously demonstrated to hold potential as a novel hypertrophic scar treatment due to its apoptosis-inducing property on hypertrophic scar fibroblasts. Other Shikonin analogues have also been reported to hold apoptosis-inducing properties in various cancer cell lines, however, the effects of these analogues on hypertrophic scar-related cells are unknown. We therefore evaluated the effects of Shikonin and its analogues on hypertrophic scar-derived human fibroblasts using the optimised “Macromolecular crowding” model. Our data indicates that Shikonin and Naphthazarin are the most effective molecules compared to related naphthoquinones. The data generated from the study offers a novel in vitro collagen-rich model of hypertrophic scar tissue. It also provides further evidences supporting the use of Shikonin and Naphthazarin as potential treatments for hypertrophic scars.

Marker of proliferation Ki-67 expression is associated with transforming growth factor beta 1 and can predict the prognosis of patients with hepatic B virus-related hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most frequent malignancy of the liver. Transforming growth factor beta 1 (TGFB1) and marker of proliferation Ki-67 (MKI67) regulate cell proliferation, differentiation, and growth. The association between MKI67 and TGFB1 expression and its clinical implications in HCC remain unknown.MethodsPublic databases were used to analyze TGFB1 and MKI67 expression in different pathologic grades/stages and tissue types of HCC. The association between MKI67 and TGFB1 expression was explored using pathway analysis and in a HepG2 cell line treated with TGFB1. Survival analysis was performed to evaluate the prognostic value of TGFB1 and MKI67 expression in patients with hepatitis B virus (HBV)-related HCC.ResultsWe identified that MKI67 expression was upregulated in liver cancer tissues. MKI67 and TGFB1 expression levels were different in various stages and tissue types of liver cancer. Furthermore, MKI67 expression was associated with TGFB1 expression in liver cancer tissues and HepG2 cells. Patients with HBV-related HCC and a higher level of MKI67 expression had a worse prognosis. Moreover, a nomogram was conducted to predict the clinical outcomes of patients with HBV-related HCC.ConclusionMKI67 expression level was associated with TGFB1 expression in liver cancer tissues and a HepG2 cell line. MKI67 expression level can predict the clinical outcomes of patients with HBV-related HCC.