Membrane-bound Inhibitor Research Articles

Hepatic Bone Morphogenetic Protein and Activin Membrane-Bound Inhibitor Levels Decline in Hepatitis C but Are Not Associated with Progression of Hepatocellular Carcinoma.

Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is an antagonist of transforming growth factor (TGF)-β type 1 signaling. BAMBI functions as an anti-fibrotic protein and exerts pro- as well as anti-cancerogenic activities. Our study aimed to correlate hepatocyte BAMBI protein levels in hepatocellular carcinoma (HCC) with T stage, lymph node invasion, vessel invasion, grading, tumor size and Union for International Cancer Control (UICC) stage, as well as with liver inflammation and fibrosis stages. Hepatocyte BAMBI protein expression was assessed by immunohistochemistry in HCC tissues of 320 patients and non-tumor tissues of 51 patients. In the HCC tissues of the whole cohort and sex-specific analysis, BAMBI protein was not related to T stage, vessel invasion, lymph node invasion, histologic grade, UICC stage and tumor size. Accordingly, BAMBI was not associated with overall survival, recurrence-free and metastasis-free survival. BAMBI protein levels in tumor and non-tumor tissues were not related to inflammation and fibrosis grade. BAMBI protein levels in HCC tissues and non-tumor tissues from HCC patients, which were analyzed by immunoblot in a small cohort and by immunohistochemistry in the tissues of patients described above, were similar. Notably, BAMBI protein was low-abundant in HCC tissues of hepatitis C virus (HCV) compared to hepatitis B virus (HBV)-infected patients with comparable disease severity. Immunoblot analysis revealed reduced BAMBI protein in non-tumor tissues of patients with HCV in comparison to patients with HBV and normal human liver tissues. In summary, this analysis showed that hepatocyte BAMBI protein levels of patients with HCC are related to HCV infection rather than the severity of the underlying liver disease and cancer staging.

MicroRNA-19a-3p augments TGF-β1-induced cardiac fibroblast activation via targeting BAMBI.

The main pathogenic factor leading to cardiac remodeling and heart failure is myocardial fibrosis. Recent research indicates that microRNAs are essential for the progress of cardiac fibrosis. Myocardial fibrosis is considered to be alleviated through the bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), which does this by blocking the transforming growth factor β1 (TGF-β1) signaling pathway. Here, this study sought to elucidate the post-transcriptional regulation of miR-19a-3p on BAMBI and its role in TGF-β1-induced cardiac fibroblast activation. Transverse aortic constriction (TAC) caused both myocardial interstitial and perivascular collagen deposition. RT-PCR showed that miR-19a-3p was upregulated in the myocardial tissue of cardiac fibrosis, and TGF-β1 induced an increase of miR-19a-3p expression in cardiac fibroblasts. The dual-luciferase reporter test and qRT-PCR confirmed that miR-19a-3p directly combined with BAMBI mRNA 3'UTR, thus reduced BAMBI expression, which diminished the capability of BAMBI to inhibit TGF-β1. Furthermore, miR-19a-3p mimic increased the activation of TGF-β1/SMAD2/3 pathway signaling, which supported cardiac fibroblast activation, which blocked by overexpression of BAMBI. These findings imply that miR-19a-3p enhances the activation of TGF-β1/SMAD2/3 by inhibiting BAMBI, further boosting the activation of cardiac fibroblasts, and may thus offer a novel strategy to tackling myocardial fibrosis.

Elevated expression of complement factor I in lung cancer cells associates with shorter survival–Potentially via non-canonical mechanism

While numerous membrane-bound complement inhibitors protect the body's cells from innate immunity's autoaggression, soluble inhibitors like complement factor I (FI) are rarely produced outside the liver. Previously, we reported the expression of FI in non-small cell lung cancer (NSCLC) cell lines. Now, we assessed the content of FI in cancer biopsies from lung cancer patients and associated the results with clinicopathological characteristics and clinical outcomes. Immunohistochemical staining intensity did not correlate with age, smoking status, tumor size, stage, differentiation grade, and T cell infiltrates, but was associated with progression-free survival (PFS), overall survival (OS) and disease-specific survival (DSS). Multivariate Cox analysis of low vs. high FI content revealed HR 0.55, 95 % CI 0.32-0.95, p=0.031 for PFS, HR 0.51, 95 % CI 0.25-1.02, p=0.055 for OS, and HR 0.32, 95 % CI 0.12-0.84, p=0.021 for DSS. Unfavorable prognosis might stem from the non-canonical role of FI, as the staining pattern did not correlate with C4d - the product of FI-supported degradation of active complement component C4b. To elucidate that, we engineered three human NSCLC cell lines naturally expressing FI with CRISPR/Cas9 technology, and compared the transcriptome of FI-deficient and FI-sufficient clones in each cell line. RNA sequencing revealed differentially expressed genes engaged in intracellular signaling pathways controlling proliferation, apoptosis, and responsiveness to growth factors. Moreover, in vitro colony-formation assays showed that FI-deficient cells formed smaller foci than FI-sufficient NSCLC cells, but their size increased when purified FI protein was added to the medium. We postulate that a non-canonical activity of FI influences cellular physiology and contributes to the poor prognosis of lung cancer patients.

Epitranscriptional regulation of TGF-β pseudoreceptor BAMBI by m6A/YTHDF2 drives extrinsic radioresistance.

Activation of TGF-β signaling serves as an extrinsic resistance mechanism that limits the potential for radiotherapy. Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) antagonizes TGF-β signaling and is implicated in cancer progression. However, the molecular mechanisms of BAMBI regulation in immune cells and its impact on antitumor immunity after radiation have not been established. Here, we show that ionizing radiation (IR) specifically reduces BAMBI expression in immunosuppressive myeloid-derived suppressor cells (MDSCs) in both murine models and humans. Mechanistically, YTH N6-methyladenosine RNA-binding protein F2 (YTHDF2) directly binds and degrades Bambi transcripts in an N6-methyladenosine-dependent (m6A-dependent) manner, and this relies on NF-κB signaling. BAMBI suppresses the tumor-infiltrating capacity and suppression function of MDSCs via inhibiting TGF-β signaling. Adeno-associated viral delivery of Bambi (AAV-Bambi) to the tumor microenvironment boosts the antitumor effects of radiotherapy and radioimmunotherapy combinations. Intriguingly, combination of AAV-Bambi and IR not only improves local tumor control, but also suppresses distant metastasis, further supporting its clinical translation potential. Our findings uncover a surprising role of BAMBI in myeloid cells, unveiling a potential therapeutic strategy for overcoming extrinsic radioresistance.

A friend in a forest of radiation-immune interactions: BAMBI improves antitumor effects by limiting radioresistance.

Radiation therapy (RT) remains one of the most effective and utilized oncologic treatments available. While it can directly yield tumor cell death, its impact on the immune microenvironment is more complex, promoting either an antitumor response or, conversely, a tumor-promoting state. TGF-β, induced by RT, yields a more immunosuppressive environment, including potentially blunting response to immune-checkpoint blockade. In this issue of the JCI, Wang and colleagues demonstrate that RT reduced expression of bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), a TGF-β pseudoreceptor. Limiting this effect, or increasing BAMBI, improved RT-induced tumor cell killing, tumor response, and antitumor immune effects. This realization points to a pathway of potential clinical translation.

BMP and activin receptor membrane bound inhibitor: BAMBI has multiple roles in gene expression and diseases (Review).

BMP and activin membrane-bound inhibitor (BAMBI) is a transmembrane glycoprotein, known as a pseudo-receptor for TGFβ, as, while its extracellular domain is similar to that of type I TGFβ receptors, its intracellular structure is shorter and lacks a serine/threonine phosphokinase signaling motif. BAMBI can regulate numerous biological phenomena, including glucose and lipid metabolism, inflammatory responses, and cell proliferation and differentiation. Furthermore, abnormal expression of BAMBI at the mRNA and protein levels contributes to various human pathologies, including obesity and cancer. In the present review, the structure of BAMBI is briefly introduced and its associated signaling pathways and physiological functions are described. Understanding of BAMBI structure and function may contribute to knowledge regarding the occurrence of diseases, including obesity and diabetes, among others. The present review provides a theoretical foundation for the development of BAMBI as a potential biomarker or therapeutic target.

Adipose tissue depot specific expression and regulation of fibrosis-related genes and proteins in experimental obesity.

Transforming growth factor beta (Tgfb) is a well-studied pro-fibrotic cytokine, which upregulates cellular communication network factor 2 (Ccn2), collagen, and actin alpha 2, smooth muscle (Acta2) expression. Obesity induces adipose tissue fibrosis, which contributes to metabolic diseases. This work aimed to analyze the expression of Tgfb, Ccn2, collagen1a1 (Col1a1), Acta2 and BMP and activin membrane-bound inhibitor (Bambi), which is a negative regulator of Tgfb signaling, in different adipose tissue depots of mice fed a standard chow, mice fed a high fat diet (HFD) and ob/ob mice. Principally, these genes were low expressed in brown adipose tissues and this difference was less evident for the ob/ob mice. Ccn2 and Bambi protein as well as mRNA expression, and collagen1a1 mRNA were not induced in the adipose tissues upon HFD feeding whereas Tgfb and Acta2 mRNA increased in the white fat depots. Immunoblot analysis showed that Acta2 protein was higher in subcutaneous and perirenal fat of these mice. In the ob/ob mice, Ccn2 mRNA and Ccn2 protein were upregulated in the fat depots. Here, Tgfb, Acta2 and Col1a1 mRNA levels and serum Tgfb protein were increased. Acta2 protein was, however, not higher in subcutaneous and perirenal fat of these mice. Col6a1 mRNA was shown before to be higher in obese fat tissues. Current analysis proved the Col6a1 protein was induced in subcutaneous fat of HFD fed mice. Notably, Col6a1 was reduced in perirenal fat of ob/ob mice in comparison to the respective controls. 3T3-L1 cells express Ccn2 and Bambi protein, whose levels were not changed by fatty acids, leptin, lipopolysaccharide, tumor necrosis factor and interleukin-6. All of these factors led to higher Tgfb in 3T3-L1 adipocyte media but did not increase its mRNA levels. Free fatty acids induced necrosis whereas apoptosis did not occur in any of the in vitro incubations excluding cell death as a main reason for higher Tgfb in cell media. In summary, Tgfb mRNA is consistently induced in white fat tissues in obesity but this is not paralleled by a clear increase of its target genes. Moreover, discrepancies between mRNA and protein expression of Acta2 were observed. Adipocytes seemingly do not contribute to higher Tgfb mRNA levels in obesity. These cells release more Tgfb proteinwhen challenged with obesity-related metabolites connecting metabolic dysfunction and fibrosis.

MAP-2:CD55 chimeric construct effectively modulates complement activation.

The complement system is a complex, tightly regulated protein cascade involved in pathogen defense and the pathogenesis of several diseases. Thus, the development of complement modulators has risen as a potential treatment for complement-driven inflammatory pathologies. The enzymatically inactive MAP-2 has been reported to inhibit the lectin pathway by competing with its homologous serine protease MASP-2. The membrane-bound complement inhibitor CD55 acts on the C3/C5 convertase level. Here, we fused MAP-2 to the four N-terminal domains of CD55 generating a targeted chimeric inhibitor to modulate complement activation at two different levels of the complement cascade. Its biological properties were compared in vitro with the parent molecules. While MAP-2 and CD55 alone showed a minor inhibition of the three complement pathways when co-incubated with serum (IC50MAP-2+CD55 1-4 = 60.98, 36.10, and 97.01 nM on the classical, lectin, and alternative pathways, respectively), MAP-2:CD551-4 demonstrated a potent inhibitory activity (IC50MAP-2:CD55 1-4 = 2.94, 1.76, and 12.86 nM, respectively). This inhibitory activity was substantially enhanced when pre-complexes were formed with the lectin pathway recognition molecule mannose-binding lectin (IC50MAP-2:CD55 1-4 = 0.14 nM). MAP-2:CD551-4 was also effective at protecting sensitized sheep erythrocytes in a classical hemolytic assay (CH50 = 13.35 nM). Finally, the chimeric inhibitor reduced neutrophil activation in full blood after stimulation with Aspergillus fumigatus conidia, as well as phagocytosis of conidia by isolated activated neutrophils. Our results demonstrate that MAP-2:CD551-4 is a potent complement inhibitor reinforcing the idea that engineered fusion proteins are a promising design strategy for identifying and developing drug candidates to treat complement-mediated diseases.

Circular RNA circSP5 promotes liver metastasis of colorectal cancer via SP5-mediated BAMBI transcription.

Liver metastasis of colorectal cancer (CRC) is a major cause of cancer morbidity and mortality. Circular RNAs (circRNAs) have been widely reported to be implicated in cancer metastasis. This study aims to investigate the effect of circSP5 (has_circ_0057010) on liver metastasis of CRC. Quantitative real-time PCR (RT-qPCR) analysis was performed to detect gene expression. The level of proteins was measured by western blot. The migration and invasion of CRC cells were assessed by wound healing assay and transwell assay. In vivo assays were performed after the construction of the CRC xenograft model and CRC model with liver metastasis. Mechanism analyses were performed via RNA-binding protein immunoprecipitation (RIP), RNA pulldown, luciferase reporter, chromatin immunoprecipitation (ChIP), and DNA pulldown assays. We found that circSP5 is significantly overexpressed in CRC with liver metastasis and its depletion suppresses the progression of CRC with liver metastasis in vitro and in vivo. Moreover, circSP5 enhances the expression of Sp5 transcription factor (SP5) via competitively sponging microRNA (miR)-1249-3p and could regulate BMP and activin membrane-bound inhibitor (BAMBI) via transcriptional activation. CircSP5 promotes the migration, invasion, and epithelial-mesenchymal transition (EMT) of CRC cells via BAMBI. In sum, circSP5 promotes liver metastasis of CRC by up-regulating SP5-mediated BAMBI transcription.

Dynamics and Molecular Interactions of GPI-Anchored CD59.

CD59 is a GPI-anchored cell surface receptor that serves as a gatekeeper to controlling pore formation. It is the only membrane-bound inhibitor of the complement membrane attack complex (MAC), an immune pore that can damage human cells. While CD59 blocks MAC pores, the receptor is co-opted by bacterial pore-forming proteins to target human cells. Recent structures of CD59 in complexes with binding partners showed dramatic differences in the orientation of its ectodomain relative to the membrane. Here, we show how GPI-anchored CD59 can satisfy this diversity in binding modes. We present a PyLipID analysis of coarse-grain molecular dynamics simulations of a CD59-inhibited MAC to reveal residues of complement proteins (C6:Y285, C6:R407 C6:K412, C7:F224, C8β:F202, C8β:K326) that likely interact with lipids. Using modules of the MDAnalysis package to investigate atomistic simulations of GPI-anchored CD59, we discover properties of CD59 that encode the flexibility necessary to bind both complement proteins and bacterial virulence factors.

CSNK1D-mediated phosphorylation of HNRNPA2B1 induces miR-25-3p/miR-93-5p maturation to promote prostate cancer cell proliferation and migration through m6A-dependent manner.

It has been reported that heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1) is highly expressed in prostate cancer (PCa) and associated with poor prognosis of patients with PCa. Nevertheless, the specific mechanism underlying HNRNPA2B1 functions in PCa remains not clear. In our study, we proved that HNRNPA2B1 promoted the progression of PCa through in vitro and in vivo experiments. Further, we found that HNRNPA2B1 induced the maturation of miR-25-3p/miR-93-5p by recognizing primary miR-25/93 (pri-miR-25/93) through N6-methyladenosine (m6A)-dependent manner. In addition, both miR-93-5p and miR-25-3p were proven as tumor promoters in PCa. Interestingly, by mass spectrometry analysis and mechanical experiments, we found that casein kinase 1 delta (CSNK1D) could mediate the phosphorylation of HNRNPA2B1 to enhance its stability. Moreover, we further proved that miR-93-5p targeted BMP and activin membrane-bound inhibitor (BAMBI) mRNA to reduce its expression, thereby activating transforming growth factor β (TGF-β) pathway. At the same time, miR-25-3p targeted forkhead box O3 (FOXO3) to inactivate FOXO pathway. These results collectively indicated that CSNK1D stabilized HNRNPA2B1 facilitates the processing of miR-25-3p/miR-93-5p to regulate TGF-β and FOXO pathways, resulting in PCa progression. Our findings supported that HNRNPA2B1 might be a promising target for PCa treatment.

Effects of BAMBI deficiency in the development of Systemic Lupus Erythematosus

Abstract Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies and immune complexes deposition in various organs. Follicular CD4+ T cells play a crucial role in this autoimmune syndrome by activating autoreactive germinal center B cells. The transforming growth factor β (TGBβ) inhibitor BAMBI (Bone Morphogenetic Protein and Activin Membrane-Bound Inhibitor) is known to control the differentiation of CD4+ T lymphocytes into either tolerogenic Tregs or pathogenic Th17 cells. BAMBI absence in mutant mice or its pharmacological inhibition with the B101.37 monoclonal antibody promotes an increase in Treg differentiation and activity and at the same time, a reduction in Th17 cell differentiation, resulting in the inhibition of autoimmune disease development. Based on these findings, in the present study we have analysed the effects of BAMBI deficiency in the development of SLE. We have employed the chronic graft-versus-host disease lupus model induced after the transference of 40–60 × 106 lymphocytes from B6 or B6-BAMBI-KO mice into either Bm12 or Bm12-BAMBI-KO recipients. As reported previously, Bm12 mice receiving lymphocytes from B6 mice produce high levels of circulating IgG anti-DNA and anti-nucleosomal autoantibodies and exhibit linear glomerular IgG deposits in their kidneys. In contrast, both Bm12 mice injected with allogeneic B6-BAMBI-KO lymphocytes and Bm12-BAMBI-KO recipients receiving lymphocytes from B6 donors do not produce significant levels of circulating autoantibodies and do not reveal immune complexes deposits. Taken together, these results indicate that the absence of BAMBI in either donor or recipient lymphocytes protects mice against the development of SLE.

Hypoxia-responsive PPARGC1A/BAMBI/ACSL5 axis promotes progression and resistance to lenvatinib in hepatocellular carcinoma.

Emerging evidence has indicated that peroxisome proliferator-activated receptor-gamma coactivator-1α (PPARGC1A) is involved in hepatocellular carcinoma (HCC). However, its detailed function and up- and downstream mechanisms are incompletely understood. In this study, we confirmed that PPAGC1A is lowly expressed in HCC and is associated with poor prognosis using large-scale public datasets and in-house cohorts. PPAGC1A was found to impair the progression and sensitivity of HCC to lenvatinib. Mechanistically, PPAGC1A repressed bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) by inhibiting WNT/β-catenin signaling. BAMBI mediated the function of PPARGC1A and regulated ACSL5 through TGF-β/SMAD signaling. PPARGC1A/BAMBI regulated ROS production and ferroptosis-related cell death by controlling ACSL5. PPARGC1A/BAMBI/ACSL5 axis was hypoxia-responsive. METTL3 and WTAP silenced PPARGC1A in an m6A-YTHDF2-dependent way under normoxia and hypoxia, respectively. Metformin restored PPARGC1A expression by reducing its m6A modification via inhibiting METTL3. In animal models and patient-derived organoids, consistent functional data of PPARGC1A/BAMBI/ACSL5 were observed. Conclusions: These findings provide new insights into the role of the aberrant PPARGC1A/BAMBI/ACSL5 axis in HCC. And the mechanism of PPARGC1A dysregulation was explained by m6A modification. Metformin may benefit HCC patients with PPARGC1A dysregulation.

The study on role of endothelial cell autophagy in rats with sepsis-induced acute kidney injury

Sepsis often causes acute kidney injury (AKI). Autophagy of renal tubular epithelial cells is considered a cytoprotective mechanism in septic AKI; however, the role of autophagy of renal endothelial cells is uninvestigated. The current study examined whether autophagy was induced by sepsis in renal endothelial cells and whether induction of autophagy in these cells attenuated the degree of AKI. Cecal ligation and puncture (CLP) was used as a model of sepsis in rats. Four experimental groups included: sham, CLP alone, CLP + rapamycin (RAPA), and CLP + dimethyl sulfoxide (DMSO), where RAPA was used as an activator of autophagy. CLP increased renal LC3-II protein levels with an additional transient increase by RAPA at 18 h. In addition, CLP induced autophagosome formation in renal endothelial cells had an additional increase induced by RAPA. Interestingly, the levels of bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), an endothelial cell-specific protein in the kidney, were also increased by CLP, albeit it was transiently downregulated by RAPA at 18 h. Serum thrombomodulin increased and renal vascular endothelial (VE)-cadherin decreased following CLP, and these changes were attenuated by RAPA. The renal cortex exhibited and inflammatory tissue damage after CLP, and RAPA alleviated these histopathological injuries. The current findings indicate that autophagy was induced by sepsis in renal endothelial cells, and upregulation of autophagy in these cells alleviated endothelial injury and AKI. In addition, BAMBI was induced by sepsis in the kidney, which may play a role in regulating endothelial stability in septic AKI.

Effects of BAMBI on luteinized follicular granulosa cell proliferation and steroid hormone production in sheep.

Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) regulates mammalian ovarian follicle growth and maturation; however, its effect on luteinized granulosa cells (LGCs) in sheep ovarian follicles remains unknown. Here we explored the regulatory role of LGC functions and steroid hormone synthesis by BAMBI. Multiple sequence alignment revealed that the sheep BAMBI gene sequence was relatively conserved. Sheep LGCs were strongly positive for BAMBI. LGC proliferation increased when BAMBI was silenced and decreased when BAMBI was overexpressed. After BAMBI overexpression, the expression of CASP3, CASP8, CASP9, and BAX significantly increased, whereas that of BCL2 and the ratio of BCL2/BAX expression decreased. The opposite was observed after BAMBI silencing. CDKN1A, CCND1, and CCND2 were downregulated with BAMBI overexpression and upregulated with BAMBI silencing. Expression of steroid hormone-related genes (CYP11A1, STAR, and 3BHSD), except CYP19A1, significantly increased after BAMBI overexpression. Moreover, estrogen and progesterone secretion increased after BAMBI overexpression and decreased after BAMBI interference. The effect of the exogenous addition of bone morphogenetic protein 2 (BMP2) on GCs was similar to that of BAMBI overexpression. In conclusion, BAMBI can regulate the proliferation and steroid hormone synthesis of sheep LGCs, and BMP2 can affect LGCs as an activator of BAMBI. These findings provide a basis for further research on the physiological role of BAMBI.

Expression and Function of BMP and Activin Membrane-Bound Inhibitor (BAMBI) in Chronic Liver Diseases and Hepatocellular Carcinoma.

BAMBI (bone morphogenetic protein and activin membrane-bound inhibitor) is a transmembrane pseudoreceptor structurally related to transforming growth factor (TGF)-β type 1 receptors (TGF-β1Rs). BAMBI lacks a kinase domain and functions as a TGF-β1R antagonist. Essential processes such as cell differentiation and proliferation are regulated by TGF-β1R signaling. TGF-β is the best-studied ligand of TGF-βRs and has an eminent role in inflammation and fibrogenesis. Liver fibrosis is the end stage of almost all chronic liver diseases, such as non-alcoholic fatty liver disease, and at the moment, there is no effective anti-fibrotic therapy available. Hepatic BAMBI is downregulated in rodent models of liver injury and in the fibrotic liver of patients, suggesting that low BAMBI has a role in liver fibrosis. Experimental evidence convincingly demonstrated that BAMBI overexpression is able to protect against liver fibrosis. Chronic liver diseases have a high risk of hepatocellular carcinoma (HCC), and BAMBI was shown to exert tumor-promoting as well as tumor-protective functions. This review article aims to summarize relevant studies on hepatic BAMBI expression and its role in chronic liver diseases and HCC.

Genome-Wide Association Study Identifies Variants Associated with Clinical Outcomes in Pediatric Patients with Newly Diagnosed Acute Myeloid Leukemia

Genetic aberrations in leukemic blasts are associated with clinical outcomes in patients with AML. However, the role of inherited genetic variants with regards to variation in AML treatment outcome remains obscure. To that end, we conducted a genome wide association study (GWAS) on 400 newly diagnosed pediatric AML patients to identify single nucleotide polymorphism (SNPs) that may be associated with minimal residual disease (MRD1) after induction 1 chemotherapy, event-free survival (EFS) and overall survival (OS). In this study, we utilized whole-genome genotyping with Illumina's Omni2.5Exome-8 BeadChip array to investigate SNPs for association with clinical outcome in 2 pediatric AML cohorts: the multi-site AML02 [NCT00136084, n=167] and AML08 [NCT00703820, n=233] trials. Post GWAS quality control, 1,365,439 unphased SNP genotypes were selected for imputation using TOPMed Imputation Server. Following imputation and additional quality control filtering, 6,741,200 SNPs evaluated for association with clinical outcomes (MRD1, EFS and OS). To improve statistical rigor, two GWAS approaches were utilized: i) A one-stage GWAS with both clinical trial cohorts combined (n=400) and ii) A two-stage GWAS where we utilized a two-step iterative resampling procedure (PMID: 26377241) by splitting the cohort at a 70:30 ratio into discovery and replication cohorts, balanced by clinical trial, treatment arm and initial risk group at diagnosis (100 randomizations performed). For each of the 100 iterations of the two-stage GWAS, we used the discovery cohort to perform a genome-wide analysis, followed by a replication screen. SNPs replicated in at least 20 of 100 rounds of discovery-replication were prioritized. A comparison of the two approaches showed all significant SNPs in the two-stage GWAS were also captured in the one-stage GWAS. For EFS, a Bonferroni-adjusted genome-wide significant association was observed for a SNP in CSMD1 and this SNP was replicated in 83 out of 100 discovery-replication rounds for two-stage GWAS. For CSMD1 SNP, presence of variant A allele has significantly reduced EFS (HR=3.29, 95% CI (2.177-4.975), P=1.61x10-8, Figure 1A). CSMD1 is a membrane-bound complement inhibitor that has been shown to act as a putative tumor suppressor gene in other malignancies (PMIDs:27764775, 35623420). Another significant marker worth mentioning was an intronic SNP in CDKN2B-AS1 that associated with poor outcome [EFS: HR=2.37, 95% CI (1.700-3.317), P=4.40x10-7] and [OS: HR=2.87, 95% CI (1.954-4.225), P=8.06x10-8, Figure 1B]. This marker is in high linkage disequilibrium with 26 other SNPs within the CDKN2B and CDKN2B-AS1 cluster. The product of this gene is an RNA molecule that interacts with polycomb repressive complex-1 (PRC1) and -2 (PRC2) involved in silencing other genes within this cluster through epigenetic mechanisms (PMID: 20716771). CDKN2B-AS1 has been reported to play a role in tumorigenesis and chemoresistance in pediatric T-cell acute lymphoblastic leukemia and with the risk of chronic lymphocytic leukemia (PMIDs: 32976214/23770605). Though limited by numbers, this is one of the largest GWAS conducted in pediatric AML to identify SNPs associated with clinical outcomes. Our top results show that genetic variation in several genes of biological relevance may impact MRD1, EFS and OS. Future studies are aimed at looking at SNP/gene and association with gene expression collected from leukemic cells at time of diagnosis to establish AML-specific eQTLs and functional validation of the most promising candidates. Additionally, future analyses will include other subgroups such as cytogenetic risk group and validation of these findings in larger cohort of patients. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

BMP and activin membrane-bound inhibitor regulate connective tissue growth factor controlling mesothelioma cell proliferation

BackgroundMalignant mesothelioma (MM) is an aggressive mesothelial cell cancer type linked mainly to asbestos inhalation. MM characterizes by rapid progression and resistance to standard therapeutic modalities such as surgery, chemotherapy, and radiotherapy. Our previous studies have suggested that tumor cell-derived connective tissue growth factor (CTGF) regulates the proliferation of MM cells as well as the tumor growth in mouse xenograft models.MethodsIn this study, we knock downed the bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) and CTGF in MM cells and investigated the relationship between both and their impact on the cell cycle and cell proliferation.ResultsThe knockdown of CTGF or BAMBI reduced MM cell proliferation. In contrast to CTGF knockdown which decreased BAMBI, knockdown of BAMBI increased CTGF levels. Knockdown of either BAMBI or CTGF reduced expression of the cell cycle regulators; cyclin D3, cyclin-dependent kinase (CDK)2, and CDK4. Further, in silico analysis revealed that higher BAMBI expression was associated with shorter overall survival rates among MM patients.ConclusionsOur findings suggest that BAMBI is regulated by CTGF promoting mesothelioma growth by driving cell cycle progression. Therefore, the crosstalk between BAMBI and CTGF may be an effective therapeutic target for MM treatment.

MiR-125a-3p aggravates ox-LDL-induced HUVEC injury through BAMBI.

Atherosclerosis (AS) is a chronic inflammatory disease characterized by the formation of atherosclerotic plaque in the intima of arteries. Among the known regulators of atherosclerosis, microRNAs (miRNAs) have been reported to play critical roles in lipoprotein homeostasis and plaque formation. But the roles of microRNA-125a-3p (miR-125a-3p) in the pathogenesis of AS remain unknown. Human umbilical vein endothelial cells (HUVECs) were treated with oxidized low-density lipoprotein (ox-LDL) to construct the vascular injury model of AS pathogenesis in vitro. miR-125a-3p and BMP and activin membrane-bound inhibitor (BAMBI) expression levels in HUVECs were then measured by quantitative real-time polymerase chain reaction and western blot. The viability and apoptosis of HUVECs were analyzed by Cell Counting Kit-8 assay, TUNEL assay, and flow cytometry, respectively. The relationship between BAMBI 3'-untranslated region and miR-125a-3p was validated by dual luciferase reporter gene assay. miR-125a-3p expression was raised in HUVECs induced with ox-LDL. In HUVECs, miR-125a-3p enhanced the effects of ox-LDL treatment on repressing the viability and promoting the apoptosis of cells. Additionally, BAMBI was confirmed as a direct target of miR-125a-3p and BAMBI overexpression reversed the effects of miR-125a-3p on HUVECs. miR-125a-3p aggravates the dysfunction of HUVECs induced by ox-LDL via BAMBI, which implies that miR-125a-3p is involved in the pathogenesis of AS.

Circ_0093887 regulated ox-LDL induced human aortic endothelial cells viability, apoptosis, and inflammation through modulating miR-758-3p/BAMBI axis in atherosclerosis.

Compelling evidence demonstrated that circular RNAs (circRNAs) were involved in the progression of atherosclerosis (AS). However, the role of circ_0093887 in the progression of AS is unclear. The purpose of this study was to explore the role and mechanism of circ_0093887 in oxidized-low density lipoprotein (ox-LDL)-induced human aortic endothelial cells (HAECs). HAECs were stimulated by ox-LDL to simulate AS-like injury in vitro. Circ_0093887, microRNA-758-3p (miR-758-3p), and BMP And Activin Membrane-Bound Inhibitor (BAMBI) levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). PCNA, Bax, Bcl-2, and BAMBI protein levels were detected by western blot. Cell viability and apoptosis were examined by Cell Counting Kit-8 (CCK-8) assay and flow cytometry. Tube formation assay was used to assess tube formation. The levels of inflammatory factors TNF-α and IL-1β were detected by corresponding ELISA kits. The relationship between miR-758-3p and circ_0093887 or BAMBI was tested via dual-luciferase reporter analysis and RNA immunoprecipitation. Oxidative stress related indexes (ROS and MDA) were detected by corresponding kits. The expression levels of circ_0093887 and BAMBI were prominently downregulated in ox-LDL-induced HAECs compared with control, whereas the expression of miR-758-3p was upregulated. Overexpression of circ_0093887 promoted HAECs viability and tube formation, and restrained cell apoptosis in ox-LDL-induced HAECs compared with untreated HAECs. Mechanistically, circ_0093887 regulated the expression of BAMBI through miR-758-3p. Further experiments showed that upregulation of miR-758-3p reversed changes in cell function induced by circ_0093887. In addition, reduced BAMBI salvaged miR-758-3p knockdown mediated effects on cell function. Circ_0093887 demonstrated its diagnostic and therapeutic value in AS by promoting the role of the miR-758-3p/BAMBI axis in the ox-LDL-induced endothelial injury of HAECs.