Gene mutations in sporadic lymphangioleiomyomatosis and genotype-phenotype correlation analysis.

BACKGROUND: Sporadic lymphangioleiomyomatosis (S-LAM) is a rare neoplasm with heterogeneous clinical features that is conventionally considered to be related to TSC2. This study serves to elucidate the mutation landscape and potential correlation between S-LAM genomic profiles and clinical phenotypes. METHODS: Genomic profiles of 22 S-LAM patients were obtained by sequencing genomic DNA and cell-free DNA from various specimens using an NGS (next-generation sequencing)-based tumor-driver gene panel. Detected mutations were summarized. Symptoms, serum vascular endothelial growth factor D (VEGF-D) values, pulmonary function, and six-minute walk distance (6MWD) were compared among groups with different TSC2 status and genotypes to analyze genotype-phenotype correlations. RESULTS: 67 Variants in 43 genes were detected, with a TSC2 mutation detection rate of 68.2%. The TSC2 detection rate was similar in specimens obtained either through transbronchial lung biopsy (TBLB) or surgical lung biopsy (70.0% vs. 69.2%, p > 0.05). A novel mutation in VEZF1 (c.A659G) was detected in four participants and may represent a mild disease state. TSC2 mutation was significantly related to a shorter 6MWD (p < 0.05), and a higher percentage of VEGF-D over 800 pg/mL (p < 0.05); stop-gain mutation was significantly related to a higher prevalence of pneumothorax. CONCLUSIONS: Tumor-driver mutations in genes other than TSC2 may have a role in S-LAM, and TBLB specimens are practical alternatives for genomic analysis. TSC2 mutation detectability and types are related to the disease severity and phenotypes of S-LAM.

Circ_0006768 upregulation attenuates oxygen-glucose deprivation/reoxygenation-induced human brain microvascular endothelial cell injuries by upregulating VEZF1 via miR-222-3p inhibition.

Circular RNAs (circRNAs) have been widely implicated in multiple diseases, including ischemic stroke. This study aimed to explore the function and functional mechanism of circ_0006768 in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced brain injury models of ischemic stroke. Human brain microvascular endothelial cells (HBMECs) were induced by OGD/R to mimic ischemic stroke models in vitro. The expression of circ_0006768, microRNA-222-3p (miR-222-3p) and vascular endothelial zinc finger 1 (VEZF1) was detected by quantitative real-time PCR (qPCR). Cell viability, angiogenesis ability and cell migration were assessed by cell counting kit-8 (CCK-8) assay, tube formation assay and wound healing assay, respectively. The releases of pro-inflammatory factors were determined by commercial enzyme-linked immunosorbent assay (ELISA) kits. The protein levels of vascular endothelial growth factor A (VEGFA), N-cadherin and VEZF1 were detected by western blot. The putative relationship between miR-222-3p and circ_0006768 or VEZF1 was validated by dual-luciferase reporter assay, RNA Immunoprecipitation (RIP) assay and pull-down assay. Circ_0006768 was poorly expressed in ischemic stroke plasma and OGD/R-induced HBMECs. OGD/R inhibited cell viability, angiogenesis and cell migration and promoted the releases of pro-inflammatory factors, while circ_0006768 overexpression or miR-222-3p inhibition partially abolished the effects of OGD/R. MiR-222-3p was targeted by circ_0006768, and VEZF1 was a target of miR-222-3p. Circ_0006768 enriched the expression of VEZF1 via mediating miR-222-3p inhibition. Rescue experiments presented that the effects of circ_0006768 overexpression were reversed by miR-222-3p restoration or VEZF1 knockdown. Circ_0006768 overexpression attenuates OGD/R-induced HBMEC injuries by upregulating VEZF1 via miR-222-3p inhibition.

Transcriptional Repression of Raf Kinase Inhibitory Protein Gene by Metadherin during Cancer Progression.

Raf kinase inhibitory protein (RKIP), also known as a phosphatidylethanolamine-binding protein 1 (PEBP1), functions as a tumor suppressor and regulates several signaling pathways, including ERK and NF-κκB. RKIP is severely downregulated in human malignant cancers, indicating a functional association with cancer metastasis and poor prognosis. The transcription regulation of RKIP gene in human cancers is not well understood. In this study, we suggested a possible transcription mechanism for the regulation of RKIP in human cancer cells. We found that Metadherin (MTDH) significantly repressed the transcriptional activity of RKIP gene. An analysis of publicly available datasets showed that the knockdown of MTDH in breast and endometrial cancer cell lines induced the expression RKIP. In addition, the results obtained from qRT-PCR and ChIP analyses showed that MTDH considerably inhibited RKIP expression. In addition, the RKIP transcript levels in MTDH-knockdown or MTDH-overexpressing MCF-7 cells were likely correlated to the protein levels, suggesting that MTDH regulates RKIP expression. In conclusion, we suggest that MTDH is a novel factor that controls the RKIP transcription, which is essential for cancer progression.

The transcription factor Vezf1 represses the expression of the antiangiogenic factor Cited2 in endothelial cells.

Formation of the vasculature by angiogenesis is critical for proper development, but angiogenesis also contributes to the pathogenesis of various disorders, including cancer and cardiovascular diseases. Vascular endothelial zinc finger 1 (Vezf1), is a Krüppel-like zinc finger protein that plays a vital role in vascular development. However, the mechanism by which Vezf1 regulates this process is not fully understood. Here, we show that Vezf1-/- mouse embryonic stem cells (ESC) have significantly increased expression of a stem cell factor, Cbp/p300-interacting transactivator 2 (Cited2). Compared with WT ESCs, Vezf1-/- ESCs inefficiently differentiated into endothelial cells (ECs), which exhibited defects in the tube-formation assay. These defects were due to reduced activation of EC-specific genes concomitant with lower enrichment of histone 3 acetylation at Lys27 (H3K27) at their promoters. We hypothesized that overexpression of Cited2 in Vezf1-/- cells sequesters P300/CBP away from the promoters of proangiogenic genes and thereby contributes to defective angiogenesis in these cells. This idea was supported by the observation that shRNA-mediated depletion of Cited2 significantly reduces the angiogenic defects in the Vezf1-/- ECs. In contrast to previous studies that have focused on the role of Vezf1 as a transcriptional activator of proangiogenic genes, our findings have revealed a role for Vezf1 in modulating the expression of the antiangiogenic factor Cited2. Vezf1 previously has been characterized as an insulator protein, and our results now provide insights into the mechanism, indicating that Vezf1 can block inappropriate, nonspecific interactions of promoters with cis-located enhancers, preventing aberrant promoter activation.

Characterization of Small Molecules Inhibiting the Pro-Angiogenic Activity of the Zinc Finger Transcription Factor Vezf1.

Discovery of inhibitors for endothelial-related transcription factors can contribute to the development of anti-angiogenic therapies that treat various diseases, including cancer. The role of transcription factor Vezf1 in vascular development and regulation of angiogenesis has been defined by several earlier studies. Through construction of a computational model for Vezf1, work here has identified a novel small molecule drug capable of inhibiting Vezf1 from binding to its cognate DNA binding site. Using structure-based design and virtual screening of the NCI Diversity Compound Library, 12 shortlisted compounds were tested for their ability to interfere with the binding of Vezf1 to DNA using electrophoretic gel mobility shift assays. We identified one compound, T4, which has an IC50 of 20 µM. Using murine endothelial cells, MSS31, we tested the effect of T4 on endothelial cell viability and angiogenesis by using tube formation assay. Our data show that addition of T4 in cell culture medium does not affect cell viability at concentrations lower or equal to its IC 50 but strongly inhibits the network formation by MSS31 in the tube formation assays. Given its potential efficacy, this inhibitor has significant therapeutic potential in several human diseases.

VEZF1 loss-of-function mutation underlying familial dilated cardiomyopathy.

Dilated cardiomyopathy (DCM), characteristic of left ventricular or biventricular dilation with systolic dysfunction, is the most common form of cardiomyopathy, and a leading cause of heart failure and sudden cardiac death. Aggregating evidence highlights the underlying genetic basis of DCM, and mutations in over 100 genes have been causally linked to DCM. Nevertheless, due to pronounced genetic heterogeneity, the genetic defects underpinning DCM in most cases remain obscure. Hence, this study was sought to identify novel genetic determinants of DCM. In this investigation, whole-exome sequencing and bioinformatics analyses were conducted in a family suffering from DCM, and a novel heterozygous mutation in the VEZF1 gene (coding for a zinc finger-containing transcription factor critical for cardiovascular development and structural remodeling), NM_007146.3: c.490A > T; p.(Lys164*), was identified. The nonsense mutation was validated by Sanger sequencing and segregated with autosome-dominant DCM in the family with complete penetrance. The mutation was neither detected in another cohort of 200 unrelated DCM patients nor observed in 400 unrelated healthy individuals nor retrieved in the Single Nucleotide Polymorphism database, the Human Gene Mutation Database and the Genome Aggregation Database. Biological analyses by utilizing a dual-luciferase reporter assay system revealed that the mutant VEZF1 protein failed to transactivate the promoters of MYH7 and ET1, two genes that have been associated with DCM. The findings indicate VEZF1 as a new gene responsible for DCM, which provides novel insight into the molecular pathogenesis of DCM, implying potential implications for personalized precisive medical management of the patients affected with DCM.

ETV2 and VEZF1 interaction and regulation of the hematoendothelial lineage during embryogenesis.

Ets variant 2 (Etv2), a member of the Ets factor family, has an essential role in the formation of endothelial and hematopoietic cell lineages during embryonic development. The functional role of ETS transcription factors is, in part, dependent on the interacting proteins. There are relatively few studies exploring the coordinated interplay between ETV2 and its interacting proteins that regulate mesodermal lineage determination. In order to identify novel ETV2 interacting partners, a yeast two-hybrid analysis was performed and the C2H2 zinc finger transcription factor VEZF1 (vascular endothelial zinc finger 1) was identified as a binding factor, which was specifically expressed within the endothelium during vascular development. To confirm this interaction, co-immunoprecipitation and GST pull down assays demonstrated the direct interaction between ETV2 and VEZF1. During embryoid body differentiation, Etv2 achieved its peak expression at day 3.0 followed by rapid downregulation, on the other hand Vezf1 expression increased through day 6 of EB differentiation. We have previously shown that ETV2 potently activated Flt1 gene transcription. Using a Flt1 promoter-luciferase reporter assay, we demonstrated that VEZF1 co-activated the Flt1 promoter. Electrophoretic mobility shift assay and Chromatin immunoprecipitation established VEZF1 binding to the Flt1 promoter. Vezf1 knockout embryonic stem cells had downregulation of hematoendothelial marker genes when undergoing embryoid body mediated mesodermal differentiation whereas overexpression of VEZF1 induced the expression of hematoendothelial genes during differentiation. These current studies provide insight into the co-regulation of the hemato-endothelial lineage development via a co-operative interaction between ETV2 and VEZF1.

TRIM29 regulates the SETBP1/SET/PP2A axis via transcription factor VEZF1 to promote progression of ovarian cancer.

Ovarian cancer (OC) is a common gynecological malignant tumor that seriously endangers the health of women worldwide. Tripartite motif containing 29 (TRIM29) is a TRIM family member that is frequently overexpressed in OC. However, the specific role of TRIM29 in OC remains obscure. To investigate the underlying molecular mechanism, a global proteomics analysis identified SET binding protein 1 (SETBP1) as a crucial target of TRIM29. Subsequently, the SETBP1/SET/Protein phosphatase 2 (PP2A) axis was confirmed to be required for the recovery of cancer stem cell (CSC)-like phenotype suppressed by TRIM29 knockdown. Mechanistically, TRIM29 facilitated SETBP1 transcriptional activation via the VEZF1 transcription factor. More importantly, TRIM29 promoted VEZF1 mRNA translation by recruiting RNA binding protein BICC1 to its 3'UTR. The clinical significance was established by the association of TRIM29 and SETBP1 expression with clinicopathological features in OC samples. The SETBP1/SET/PP2A axis driven by TRIM29 via transcription factor VEZF1 is at least one of the primary mechanisms underlying TRIM29 maintenance of the CSC-like characteristics in OC.

Vezf1 regulates cardiac structure and contractile function.

BACKGROUND: Vascular endothelial zinc finger 1 (Vezf1) is a transcription factor previously shown to regulate vasculogenesis and angiogenesis. We aimed to investigate the role of Vezf1 in the postnatal heart. METHODS: The role of Vezf1 in regulating cardiac growth and contractile function was studied in zebrafish and in primary cardiomyocytes. FINDINGS: We find that expression of Vezf1 is decreased in diseased human myocardium and mouse hearts. Our experimental data shows that knockdown of zebrafish Vezf1 reduces cardiac growth and results in impaired ventricular contractile response to ß-adrenergic stimuli. However, Vezf1 knockdown is not associated with dysregulation of cardiomyocyte Ca2+ transient kinetics. Gene ontology enrichment analysis indicates that Vezf1 regulates cardiac muscle contraction and dilated cardiomyopathy related genes and we identify cardiomyocyte Myh7/ß-MHC as key target for Vezf1. We further identify a key role for an MCAT binding site in the Myh7 promoter regulating the response to Vezf1 knockdown and show that TEAD-1 is a binding partner of Vezf1. INTERPRETATION: We demonstrate a role for Vezf1 in regulation of compensatory cardiac growth and cardiomyocyte contractile function, which may be relevant in human cardiac disease.

Identification of a novel fusion transcript EWSR1-VEZF1 by anchored multiplex PCR in malignant peripheral nerve sheath tumor.

The aim of the study is to describe a novel genetic finding examining the molecular and pathological features of a case of malignant peripheral nerve sheath tumor occurring in the thigh of a 17-year-old male. Fusion gene detection using a next-generation sequencing-based anchored multiplex PCR technique (Archer FusionPlex Sarcoma Panel) was used to identify the novel fusion of EWSR1-VEZF1 from the frozen tumor sample. EWSR1-VEZF1 fusion is a novel molecular gene rearrangement involving exon 8 of the EWSR1 gene and exon 2 of the VEZF1 gene. Data were validated with gene sequencing and fluorescent in situ hybridization (FISH) analysis. This case report describes a novel rearrangement involving EWSR1 on chromosome 22 and VEZF1 on chromosome 17. The result obtained demonstrates the value of the next-generation sequencing-based anchored multiplex PCR technique (Archer FusionPlex Sarcoma Panel) both in diagnosis and patient care and might become a helpful diagnostic tool for this tumor type.

MiR-191 inhibit angiogenesis after acute ischemic stroke targeting VEZF1.

Acute ischemic stroke (AIS) is a major public health problem in China. Impaired angiogenesis plays crucial roles in the development of ischemic cerebral injury. Recent studies have identified that microRNAs (miRNAs) are important regulators of angiogenesis, but little is known the exact effects of angiogenesis-associated miRNAs in AIS. In the present study, we detected the expression levels of angiogenesis-associated miRNAs in AIS patients, middle cerebral artery occlusion (MCAO) rats, and oxygen-glucose deprivation/reoxygenation (OGD/R) human umbilical vein endothelial cells (HUVECs). MiR-191 was increased in the plasma of AIS patients, OGD/R HUVECs, and the plasma and brain of MCAO rats. Over-expression of miR-191 promoted apoptosis, but reduced the proliferation, migration, tube-forming and spheroid sprouting activity in HUVECs OGD/R model. Mechanically, vascular endothelial zinc finger 1 (VEZF1) was identified as the direct target of miR-191, and could be regulated by miR-191 at post-translational level. In vivo studies applying miR-191 antagomir demonstrated that inhibition of miR-191 reduced infarction volume in MCAO rats. In conclusion, our data reveal a novel role of miR-191 in promoting ischemic brain injury through inhibiting angiogenesis via targeting VEZF1. Therefore, miR-191 may serve as a biomarker or a therapeutic target for AIS.