Immunoexpression Patterns of Megalin, Cubilin, Caveolin-1, Gipc1 and Dab2IP in the Embryonic and Postnatal Development of the Kidneys in Yotari (Dab1-/-) Mice.

Our study examines the immunoexpression patterns of Megalin, Cubilin, Caveolin-1, Gipc1 and Dab2IP in the embryonic development (E) and postnatal (P) mouse kidney, with a focus on differentiating patterns between wild-type (wt) and yotari, Dab1-/- (yot) mice. Immunofluorescence revealed raised immunoexpression of receptors Megalin and Cubilin at the ampulla/collecting ducts and convoluted tubules across all developmental stages, with the most prominent immunoexpression observed in the convoluted tubules and the parietal epithelium of the Bowman's capsule. Quantitative analysis showed a higher percentage of Megalin and Cubilin in wt compared to yot mice at E13.5. Co-expression of Megalin and Cubilin was observed at the apical membrane of convoluted tubules and the parietal layer of the Bowman's capsule. The staining intensity of Megalin varied across developmental stages, with the strongest reactivity observed at the ampulla and collecting ducts at embryonic day (E) 13.5 in wt mice. In contrast, Caveolin-1 exhibited high immunoexpression in the metanephric mesenchyme, blood vessels, and the border area between the metanephric mesenchyme and renal vesicle, with a decrease in immunoexpression as development progressed. Gipc1 showed diffuse cytoplasmic staining in metanephric mesenchyme, convoluted tubules and collecting ducts, with significant differences in immunoexpression between wild-type and yot mice at both investigated embryonic time points. Dab2IP immunofluorescent staining was most prominent in renal vesicle/glomeruli and ampulla/collecting ducts at E13.5, with mild staining intensity observed in the distal convoluted tubules postnatally. Our findings elucidate distinct immunoexpression of patterns and potential parts of these proteins in the development and function of the kidney, highlighting the importance of further investigation into their regulatory mechanisms.

CircFAM114A2 Suppresses Cell Proliferation, Migration, and Invasion of Colorectal Cancer Through Sponging miR-647 to Upregulate DAB2IP Expression.

BACKGROUND: Increasing evidence had proved that some circular RNA (circRNA) exerted critical roles in tumors progression by functioning as "microRNAs (miRNAs) sponges" to regulate their targeted genes. METHODS: circFAM114A2 and miR-647 expression was measured in CRC tissues and cells by quantitative real-time polymerase chain reaction (qRT-PCR), and the prognostic value of circFAM114A2 evaluated by Kaplan-Meier survival curve. Subsequently, wounding healing and transwell assays were performed to assess cell proliferation, migration, and invasion. RNA pull-down and dual-luciferase reporter assays were used to confirm the interactions between circFAM114A2, miR-647, and DAB2IP. RESULTS: CircFAM114A2 was notably downregulated in CRC tissues and cells, and low circFAM114A2 expression indicated the poor prognosis of CRC patients. Next, overexpression of circFAM114A2 suppressed CRC cells proliferation, migration, and invasion in vitro and impede CRC tumor growth in vivo. Mechanically, circFAM114A2 competitively bound to miR-647 and upregulated its target gene DAB2IP expression in CRC cells. CONCLUSION: Our results indicated that circFAM114A2/miR-647/DAP2IP axis played an important role in CRC progression, suggesting that circFAM114A2 might be a novel therapeutic target in patients with CRC.

circ-Erbb2ip from adipose-derived mesenchymal stem cell-derived exosomes promotes wound healing in diabetic mice by inducing the miR-670-5p/Nrf1 axis.

BACKGROUND: To investigate the mechanism of exosomes (Exo) secretion by hypoxic pretreated adipose-derived mesenchymal stem cells (ADSCs) promoting skin wound healing in diabetic (DM) mice. METHODS: High-throughput sequencing was used to investigate abnormal expression of circRNA in hypoxic pretreatment ADSCs exosome (HExo) and ADSCs exosome (Exo). Bioinformatics analysis and luciferase reporting analysis were used to clarify the interacted relationship among circRNA, miRNA and mRNA. EPCs cells were employ to analysis the ROS, inflammatory cytokines expression, angiogenic differentiation function under hypoxic condition by using immunofluorescence, ELISA detection and tube forming experiment. DM ulceration mice model were constructed and the therapeutic effect of Exo were detected using immunohistochemistry, immunofluorescence. RESULTS: The result show that HExo have more treatment effect than Exo in promotes cutaneous wound healing of DM mice. High-throughput sequencing found that circ-Erbb2ip play a role in HExo mediated tissues repair. Downregulation circ-Erbb2ip decreased the therapeutic effect of HExo to wound healing in diabetic mice. Bioinformatics analysis and luciferase reporting analysis confirmed that both miR-670-5p and Nrf1 were downstream targets of circ-Erbb2ip. Downregulation of Nrf1 or overexpression of miR-670-5p reversed the protective effect of circ-Erbb2ip to EPCs after exposure to high glucose microenvironment. Upregulation circ-Erbb2ip increased the therapeutic effect of Exo to wound healing in diabetic mice by increased angiogenesis and decreased ROS, inflammatory cytokines expression. CONCLUSION: In conclusion, ADSC-Exos containing circ-Erbb2ip promotes wound healing by targeting miR-670-5p/Nrf1 pathway, and their effects in promoting soft tissue wound healing warrant further study.

DAB2IP associates with hereditary angioedema: Insights into the role of VEGF signaling in HAE pathophysiology.

BACKGROUND: In the recent years, there was an important improvement in the understanding of the pathogenesis of hereditary angioedema (HAE). Notwithstanding, in a large portion of patients with unknown mutation (HAE-UNK) the genetic cause remains to be identified. OBJECTIVES: To identify new genetic targets associated with HAE, a large Argentine family with HAE-UNK spanning 3 generations was studied. METHODS: Whole exome sequencing was performed on affected family members to identify potential genetic variants associated with HAE-UNK. In silico analyses and experimental studies were applied to assess the role of the identified gene variant. RESULTS: A missense variant (p.D239N) in DAB2IP was identified. The variant occurred in the C2-domain, the region interacting with vascular endothelial growth factor receptor 2 (VEGFR2). It was found to be rare, and predicted to have a detrimental effect on the functionality of DAB2IP. Protein structure modeling predicted changes in the mutant p.D239N protein structure, impacting protein stability. The p.D239N variant affected the subcellular localization of VEGFR2. Cells transfected with the DAB2IP-239N transcript exhibited an intracellular distribution, and VEGFR2 remained associated with the cell membrane. The altered localization pattern indicated reduced colocalization of the mutant protein with VEGFR2, suggesting a diminished ability of VEGFR2 binding. CONCLUSIONS: The study identified a novel missense variant (p.D239N) in DAB2IP in a family with HAE-UNK and highlighted the role of dysregulated VEGF-mediated signaling in altered endothelial permeability. DAB2IP loss-of-function pathogenic variants lead to the impairment of the endothelial VEGF/VEGFR2 ligand system and represent a new pathophysiologic cause of HAE-UNK.

miR-31-5p suppresses myocardial hypertrophy by targeting Nfatc2ip.

Cardiac hypertrophy, worldwide known as an adaptive functional compensatory state of myocardial stress, is mainly believed to proceed to severe heart diseases, even to sudden death. Emerging studies have explored the microRNA alteration during hypertrophy. However, the mechanisms of microRNAs involved in cardiac hypertrophy are still uncertain. We studied young rats to establish abdominal aorta coarctation (AAC) for 4 weeks. With the significant downregulated cardiac function and upregulated hypertrophic biomarkers, AAC-induced rats showed enlarged myocardiocytes and alterations in microRNAs, especially downregulated miR-31-5p. miR-31-5p targets the 3'UTR of Nfatc2ip and inhibits myocardial hypertrophy in vitro and in vivo. Furthermore, we verified that Nfatc2ip is necessary and sufficient for cardiac hypertrophy in neonatal rat cardiomyocytes. Moreover, we found miR-31-5p inhibited the colocalization of Nfatc2ip and hypertrophic gene ß-Mhc. Luciferase assay and ChiP-qPCR test demonstrated that Nfatc2ip binded to the core-promoter of ß-Mhc and enhanced its transcriptional activity. Above all, our study found a new pathway, mir-31-5p/Nfatc2ip/ß-Mhc, which is involved in cardiac hypertrophy, suggesting a potential target for intervention of cardiac hypertrophy.

AIDA-1/ANKS1B Binds to the SynGAP Family RasGAPs with High Affinity and Specificity.

AIDA-1, encoded by ANKS1B, is an abundant postsynaptic scaffold protein essential for brain development. Mutations of ANKS1B are closely associated with various psychiatric disorders. However, very little is known regarding the molecular mechanisms underlying AIDA-1's involvements under physiological and pathophysiological conditions. Here, we discovered an interaction between AIDA-1 and the SynGAP family Ras-GTPase activating protein (GAP) via affinity purification using AIDA-1d as the bait. Biochemical studies showed that the PTB domain of AIDA-1 binds to an extended NPx[F/Y]-motif of the SynGAP family proteins with high affinities. The high-resolution crystal structure of AIDA-1 PTB domain in complex with the SynGAP NPxF-motif revealed the molecular mechanism governing the specific interaction between AIDA-1 and SynGAP. Our study not only explains why patients with ANKS1B or SYNGAP1 mutations share overlapping clinical phenotypes, but also allows identification of new AIDA-1 binding targets such as Ras and Rab interactors.

Terf2ip deficiency accelerates non-alcoholic steatohepatitis through regulating lipophagy and fatty acid oxidation via Sirt1/AMPK pathway.

BACKGROUND & AIMS: Our previous study has demonstrated that Telomeric repeat-binding factor 2-interacting protein 1(Terf2ip), played an important role in hepatic ischemia reperfusion injury. This study is aimed to explore the function and mechanism of Terf2ip in non-alcoholic steatohepatitis (NASH). METHODS: The expression of Terf2ip was detected in liver tissue samples obtained from patients diagnosed with NASH. Mice NASH models were constructed by fed with high-fat diet (HFD) or methionine/choline deficient diet (MCD) in Terf2ip knockout and wild type (WT) mice. To further investigate the role of Terf2ip in NASH, adeno-associated viruses (AAV)-Terf2ip was administrated to mice. RESULTS: We observed a significant down-regulation of Terf2ip levels in the livers of NASH patients and mice NASH models. Terf2ip deficiency was associated with an exacerbation of hepatic steatosis in mice under HFD or MCD. Additionally, Terf2ip deficiency impaired lipophagy and fatty acid oxidation (FAO) in NASH models. Mechanically, we discovered that Terf2ip bound to the promoter region of Sirt1 to regulate Sirt1/AMPK pathway activation. As a result, Terf2ip deficiency was shown to inhibit lipophagy through the AMPK pathway, while the activation of Sirt1 alleviated steatohepatitis in the livers of mice. Finally, re-expression of Terf2ip in hepatocyes alleviated liver steatosis, inflammation, and restored lipophagy. CONCLUSIONS: These results revealed that Terf2ip played a protective role in the progression of NASH through regulating lipophagy and FAO by binding to Sirt1 promoter. Our findings provided a potential therapeutic target for the treatment of NASH.

Dihydroartemisinin suppresses the tumorigenesis of esophageal carcinoma by elevating DAB2IP expression in a NFIC-dependent manner.

Dihydroartemisinin (DHA) has been identified to have the anticancer and anti-inflammatory activities. Disabled homolog 2 interacting protein (DAB2IP) is a well-recognized tumor suppressor. Both DHA and DAB2IP were proven to have suppressing effects on esophageal carcinoma (ESCA) tumorigenesis. However, whether DHA regulated ESCA cells via DAB2IP and its mechanism are still vague. Functional analyses were conducted using MTT, tube formation, sphere formation, and transwell assays in vitro as well as Tumor formation experiments in mice. Levels of genes and proteins were assayed by qRT-PCR and western blotting analyses. The interaction between DAB2IP and Nuclear Factor I C (NFIC) was confirmed using bioinformatics analysis and dual-luciferase reporter assay. DHA treatment suppressed ESCA cell angiogenesis, stemmess, migration, and invasion. DAB2IP level was decreased in ESCA tissues and cells, and DHA elevated DAB2IP expression in ESCA cells. Functionally, DAB2IP overexpression impaired ESCA cell angiogenesis, stemmess, migration and invasion. Mechanistically, NFIC had binding sites on the promoter region and directly targeted DAB2IP. DHA could up-regulate DAB2IP expression via NFIC. Moreover, NFIC was also decreased in ESCA tissues and cells, and its overexpression had anticancer activity in ESCA cells. In addition, DAB2IP knockdown reversed the anticancer effects of NFIC or DHA on ESCA cells. In further in vivo analysis, DHA also suppressed ESCA growth by regulating DAB2IP expression. DHA suppressed the tumorigenesis of ESCA by elevating DAB2IP expression in an NFIC-dependent manner, suggesting the potential clinical application of DHA in ESCA treatment.

Grid2 interacting protein is a potential biomarker related to immune infiltration in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is one of the three deadliest malignant tumors in the world, posing a severe hazard to human health. Nonetheless, the 5-year survival rate for advanced CRC remains unsatisfactory. Grid2 interacting protein (GRID2IP) is a Purkinje fiber postsynaptic scaffold protein implicated in a number of signal transduction pathways in the nervous system. Previous studies have shown that Grid2 is closely related to the occurrence and prognosis of gastric cancer and many other diseases. Therefore, we aim to identify the relationship between GRID2IP and the occurrence and prognosis of CRC. METHODS: Transcriptome data were retrieved from The Cancer Genome Atlas (TCGA) database to analyze the differential expression of GRID2IP in a variety of malignant tumors and then validate it by quantitative real time polymerase chain reaction(Q-PCR) and Western Blot in HT29 and SW480 cells. "DESeq2" package was used to analyze the differentially expressed genes (DEGs) between the high- and low-GRID2IP subgroups. In relation to DEGs, Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed. In addition, gene set enrichment analysis (GSEA) and single-sample gene set enrichment analysis (ssGSEA) were employed to examine DEGs-associated signaling pathways and GRID2IP-associated immune cell infiltration levels. Besides, overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) were compared between the two subgroups using a Kaplan-Meier analysis. In addition, a prognostic model for GRID2IP and clinical characteristics was developed using the univariate Cox regression method. The "pRRophetic" package was applied to predict the drug sensitivity of different subgroups. Moreover, we also performed single-cell analysis of GRID2IP using the TISCH database. RESULTS: GRID2IP is upregulated in CRC patients. The rise of GRID2IP inhibits the invasion of tumor-associated immune cells resulting in a lower immune score. In addition, high GRID2IP expression was associated with poor prognosis in different clinical subgroups. Analysis of single cells revealed that GRID2IP was predominantly expressed in immune cells, myofibroblasts, and cancerous cells. In terms of chemotherapy drug sensitivity, the subgroup with high GRID2IP expression was less sensitive to gemcitabine. CONCLUSIONS: Our results suggest that rising GRID2IP promotes tumor-associated immune cell infiltration and suggests adverse outcomes in CRC patients, which may be a useful biomarker for determining the prognosis of CRC and a potential target molecule for CRC therapy.

circSLCO1B7 suppresses the malignant progression of hepatocellular carcinoma via the miR-556-3p/DAB2IP axis.

Circular RNAs (circRNAs) are noncoding RNAs with a circular colsed structure that play an important role in the occurrence and development of cancers. The functional mechanism of circRNAs as ceRNAs in hepatocellular carcinoma (HCC) and its effect on the invasion and metastasis of HCC need to be further studied. Five pairs of HCC tissues were selected for high-throughput sequencing, and 19 circRNAs with differential expression were obtained. The expression of circSLCO1B7 was obviously downregulated in 50 pairs of tumor tissues and plasma of HCC patients, which was closely related to the TNM stage, lymph node metastasis and tumor size. Cell functional experiments showed that circSLCO1B7 could inhibit cell growth, migration, invasion and promote cell apoptosis. In the regulatory mechanism, circSLCO1B7 sponged miR-556-3p to regulate the expression of the downstream target gene DAB2IP and induced the Epithelial-mesenchymal transition (EMT) progression. Our results indicated that circSLCO1B7 significantly inhibits the metastasis of HCC via the miR-556-3p/DAB2IP axis. Thus, circSLCO1B7 is a good candidate as a therapeutic target.

DAB2IP stabilizes p27Kip1 via suppressing PI3K/AKT signaling in clear cell renal cell carcinoma.

Renal cell carcinoma (RCC) is the most lethal of the urologic malignancies. We previously discovered that DAB2IP, a novel Ras GTPase-activating protein, was frequently epigenetically silenced in RCC, and DAB2IP loss was correlated with the overall survival of RCC patients. In this study, we determined the biological functions of DAB2IP in clear cell RCC (ccRCC) and its potential mechanisms of action. Correlations between DAB2IP expression level and ccRCC tumor size and patient survival were analyzed, and the results showed that ccRCC patients with high DAB2IP mRNA level exhibited smaller tumor size and better survival than the patients with low DAB2IP. Compared to control, DAB2IP knockdown significantly increased cell proliferation, promoted cell cycle progression in G1/S phase, and decreased p27 expression. Mechanism studies demonstrated that loss of DAB2IP promoted p27 protein phosphorylation, cytosolic sequestration, and subsequently ubiquitination-mediated degradation in ccRCC cells. Further studies confirmed that the proline-rich domain in C terminal (CPR) of DAB2IP suppressed AKT phosphorylation and p27 phosphorylation on S10. Hence, DAB2IP is essential for p27 protein stabilization in ccRCC, which is at less partly mediated by PI3K/AKT signaling pathway.

The Cytokinins BAP and 2-iP Modulate Different Molecular Mechanisms on Shoot Proliferation and Root Development in Lemongrass (Cymbopogon citratus).

The known activities of cytokinins (CKs) are promoting shoot multiplication, root growth inhibition, and delaying senescence. 6-Benzylaminopurine (BAP) has been the most effective CK to induce shoot proliferation in cereal and grasses. Previously, we reported that in lemongrass (Cymbopogon citratus) micropropagation, BAP 10 µM induces high shoot proliferation, while the natural CK 6-(γ,γ-Dimethylallylamino)purine (2-iP) 10 µM shows less pronounced effects and developed rooting. To understand the molecular mechanisms involved, we perform a protein-protein interaction (PPI) network based on the genes of Brachypodium distachyon involved in shoot proliferation/repression, cell cycle, stem cell maintenance, auxin response factors, and CK signaling to analyze the molecular mechanisms in BAP versus 2-iP plants. A different pattern of gene expression was observed between BAP- versus 2-iP-treated plants. In shoots derived from BAP, we found upregulated genes that have already been demonstrated to be involved in de novo shoot proliferation development in several plant species; CK receptors (AHK3, ARR1), stem cell maintenance (STM, REV and CLV3), cell cycle regulation (CDKA-CYCD3 complex), as well as the auxin response factor (ARF5) and CK metabolism (CKX1). In contrast, in the 2-iP culture medium, there was an upregulation of genes involved in shoot repression (BRC1, MAX3), ARR4, a type A-response regulator (RR), and auxin metabolism (SHY2).

Exosomal ERBB2IP contributes to tumor growth via elevating PSAT1 expression in non-small cell lung carcinoma.

BACKGROUND: Both exosomes and circular RNAs (circRNAs) are involved in tumor growth. Hsa_circ_0001492 (circERBB2IP) has been reported to be overexpressed in plasma exosomes from patients with lung adenocarcinoma, but the biological role of exosomal circERBB2IP in non-small cell lung carcinoma (NSCLC) is indistinct. METHODS: Exosomes isolated from serums and medium samples were validated by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. Relative expression of circERBB2IP was detected by RT-qPCR. Loss-of-function was done to determine the effect of circERBB2IP on NSCLC cell proliferation and migration. Molecular mechanisms associated with circERBB2IP were predicted by bioinformatic analysis and validated by dual-luciferase reporter, RIP, and RNA pulldown assays. In vivo experiments were performed to identify the function of circERBB2IP in NSCLC. RESULTS: We discovered that circERBB2IP expression was correlated with TNM grade, lymph node metastasis and tumor size of NSCLC patients. Upregulation of circERBB2IP was observed in exosomes derived from NSCLC patient's serum and circERBB2IP might be a potential diagnostic biomarker for NSCLC. CircERBB2IP was transmitted between carcinoma cells through exosomes. Knockdown of circERBB2IP lowered cell growth in mouse models and restrained NSCLC cell proliferation and migration. CircERBB2IP could mediate PSAT1 expression via sponging miR-5195-3p. CONCLUSION: In conclusion, circERBB2IP may drive NSCLC growth by the miR-5195-3p/PSAT1 axis in NSCLC, shedding light on a diagnostic biomarker and therapeutic target for NSCLC.

Association of germline variants in telomere maintenance genes (POT1, TERF2IP, ACD, and TERT) with spitzoid morphology in familial melanoma: A multi-center case series.

Background: Spitzoid morphology in familial melanoma has been associated with germline variants in POT1, a telomere maintenance gene (TMG), suggesting a link between telomere biology and spitzoid differentiation. Objective: To assess if familial melanoma cases associated with germline variants in TMG (POT1, ACD, TERF2IP, and TERT) commonly exhibit spitzoid morphology. Methods: In this case series, melanomas were classified as having spitzoid morphology if at least 3 of 4 dermatopathologists reported this finding in ≥25% of tumor cells. Logistic regression was used to calculate odds ratios (OR) of spitzoid morphology compared to familial melanomas from unmatched noncarriers that were previously reviewed by a National Cancer Institute dermatopathologist. Results: Spitzoid morphology was observed in 77% (23 of 30), 75% (3 of 4), 50% (2 of 4), and 50% (1 of 2) of melanomas from individuals with germline variants in POT1, TERF2IP, ACD, and TERT, respectively. Compared to noncarriers (n = 139 melanomas), POT1 carriers (OR = 225.1, 95% confidence interval: 51.7-980.5; P < .001) and individuals with TERF2IP, ACD, and TERT variants (OR = 82.4, 95% confidence interval: 21.3-494.6; P < .001) had increased odds of spitzoid morphology. Limitations: Findings may not be generalizable to nonfamilial melanoma cases. Conclusion: Spitzoid morphology in familial melanoma could suggest germline alteration of TMG.

The human RAP1 and GFAPɛ proteins increase γ-secretase activity in a yeast model system.

Alzheimer's disease (AD) is an age-related disorder that results in progressive cognitive impairment and memory loss. Deposition of amyloid ß (Aß) peptides in senile plaques is a hallmark of AD. γ-secretase produces Aß peptides, mostly as the soluble Aß40 with fewer insoluble Aß42 peptides. Rare, early-onset AD (EOAD) occurs in individuals under 60 years of age. Most EOAD cases are due to unknown genetic causes, but a subset is due to mutations in the genes encoding the amyloid precursor protein that is processed into Aß peptides or the presenilins (PS1 and PS2) that process APP. PS1 interacts with the epsilon isoform of glial fibrillary acidic protein (GFAPɛ), a protein found in the subventricular zone of the brain. We have found that GFAPɛ interacts with the telomere protection factor RAP1 (TERF2IP). RAP1 can also interact with PS1 alone or with GFAPɛ in vitro. Our data show that the nuclear protein RAP1 has an extratelomeric role in the cytoplasm through its interactions with GFAPɛ and PS1. GFAPɛ coprecipitated with RAP1 from human cell extracts. RAP1, GFAPɛ, and PS1 all colocalized in human SH-SY5Y cells. Using a genetic model of the γ-secretase complex in Saccharomyces cerevisiae, RAP1 increased γ-secretase activity, and this was potentiated by GFAPɛ. Our studies are the first to connect RAP1 with an age-related disorder.

DAB2IP-knocking down resulted in radio-resistance of breast cancer cells is associated with increased hypoxia and vasculogenic mimicry formation.

PURPOSE: As a part of breast-conserving therapy (BCT), postoperative radiotherapy is one of the main means to improve the clinical efficacy of breast cancer (BCa). However, ionizing radiation (IR) may induce BCa cells to develop radioresistance, which causes tumor recurrence and metastasis after treatment. Recently, DOC-2/DAB2 interactive protein (DAB2IP) has been reported often down-regulated in a variety of cancers and is related to tumor tolerance to radiotherapy. In this study, BCa cell lines were introduced to study how DAB2IP deficient influenced BCa cell radiosensitivity in vitro and in vivo and discuss the possible mechanism. METHODS AND MATERIALS: Small RNA interference system (siRNA) was employed to decrease DAB2IP expression in two BCa cell lines, MDA-MB-231 and 4T1. Cells in response to IR or antineoplastics were detected by clone formation assay or MTT method, respectively. For in vivo studies, siDAB2IP or siControl cells were subcutaneously injected into the right flank of each female mouse. Sphere formation assay, soft agar colony anchoring assay and in vivo tumorigenesis assay were implemented to examine the stem cell-like features of BCa cells. Tube formation assay as well as immunofluorescence assay (IFA) were respectively applied to determine the angiogenesis of tumor cells in vitro and in vivo. The expression of a series of angiogenesis-related molecules was analyzed by qRT-PCR, western blot and IFA. RESULTS: It was observed that the downregulation of DAB2IP could significantly improve the clone formation ability of BCa cells, reduce their sensitivity to radiation and chemotherapy drugs, enhance their migration and invasion abilities and increase their stemness characteristics. It was also noted that either DAB2IP-knocking down or treated with the conditioned medium from DAB2IP-deficient BCa cells could promote the tube-forming ability of the endothelial cell. Similarly, in vivo studies showed that tumors developed from siDAB2IP BCa cells had higher tumor microvascular density (MVD) and more severe oxygen deficiency than that in DAB2IP- sufficient tumors. Meanwhile, Knock-down of DAB2IP inhibited vascular maturation and promoted the formation of vasculogenic mimicry (VM) in BCa tissues. Down-regulation of STAT3 could enhance siDAB2IP cells sensitivity to IR, accompanied by the decrease of VEGF expression. CONCLUSIONS: Our data support that loss of DAB2IP confers radio-resistance of BCa could be due to increased hypoxia, inhibited vascular maturation and promoted VM formation. STAT3 inhibition could be a potential way to overcome such DAB2IP-deficient induced tolerance in BCT.

The Genetic Background of Hearing Loss in Patients with EVA and Cochlear Malformation.

The most frequently observed congenital inner ear malformation is enlarged vestibular aqueduct (EVA). It is often accompanied with incomplete partition type 2 (IP2) of the cochlea and a dilated vestibule, which together constitute Mondini malformation. Pathogenic SLC26A4 variants are considered the major cause of inner ear malformation but the genetics still needs clarification. The aim of this study was to identify the cause of EVA in patients with hearing loss (HL). Genomic DNA was isolated from HL patients with radiologically confirmed bilateral EVA (n = 23) and analyzed by next generation sequencing using a custom HL gene panel encompassing 237 HL-related genes or a clinical exome. The presence and segregation of selected variants and the CEVA haplotype (in the 5' region of SLC26A4) was verified by Sanger sequencing. Minigene assay was used to evaluate the impact of novel synonymous variant on splicing. Genetic testing identified the cause of EVA in 17/23 individuals (74%). Two pathogenic variants in the SLC26A4 gene were identified as the cause of EVA in 8 of them (35%), and a CEVA haplotype was regarded as the cause of EVA in 6 of 7 patients (86%) who carried only one SLC26A4 genetic variant. In two individuals with a phenotype matching branchio-oto-renal (BOR) spectrum disorder, cochlear hypoplasia resulted from EYA1 pathogenic variants. In one patient, a novel variant in CHD7 was detected. Our study shows that SLC26A4, together with the CEVA haplotype, accounts for more than half of EVA cases. Syndromic forms of HL should also be considered in patients with EVA. We conclude that to better understand inner ear development and the pathogenesis of its malformations, there is a need to look for pathogenic variants in noncoding regions of known HL genes or to link them with novel candidate HL genes.

Analysis of SLC26A4, FOXI1, and KCNJ10 Gene Variants in Patients with Incomplete Partition of the Cochlea and Enlarged Vestibular Aqueduct (EVA) Anomalies

Pathogenic variants in the SLC26A4, FOXI1, and KCNJ10 genes are associated with hearing loss (HL) and specific inner ear abnormalities (DFNB4). In the present study, phenotype analyses, including clinical data collection, computed tomography (CT), and audiometric examination, were performed on deaf individuals from the Sakha Republic of Russia (Eastern Siberia). In cases with cochleovestibular malformations, molecular genetic analysis of the coding regions of the SLC26A4, FOXI1, and KCNJ10 genes associated with DFNB4 was completed. In six of the 165 patients (3.6%), CT scans revealed an incomplete partition of the cochlea (IP-1 and IP-2), in isolation or combined with an enlarged vestibular aqueduct (EVA) anomaly. Sequencing of the SLC26A4, FOXI1, and KCNJ10 genes was performed in these six patients. In the SLC26A4 gene, we identified four variants, namely c.85G>C p.(Glu29Gln), c.757A>G p.(Ile253Val), c.2027T>A p.(Leu676Gln), and c.2089+1G>A (IVS18+1G>A), which are known as pathogenic, as well as c.441G>A p.(Met147Ile), reported previously as a variant with uncertain significance. Using the AlphaFold algorithm, we found in silico evidence of the pathogenicity of this variant. We did not find any causative variants in the FOXI1 and KCNJ10 genes, nor did we find any evidence of digenic inheritance associated with double heterozygosity for these genes with monoallelic SLC26A4 variants. The contribution of biallelic SLC26A4 variants in patients with IP-1, IP-2, IP-2+EVA, and isolated EVA was 66.7% (DFNB4 in three patients, Pendred syndrome in one patient). Seventy-five percent of SLC26A4-biallelic patients had severe or profound HL. The morphology of the inner ear anomalies demonstrated that, among SLC26A4-biallelic patients, all types of incomplete partition of the cochlea are possible, from IP-1 and IP-2, to a normal cochlea. However, the dominant type of anomaly was IP-2+EVA (50.0%). This finding is very important for cochlear implantation, since the IP-2 anomaly does not have an increased risk of "gushers" and recurrent meningitis.

DAB2IP attenuates chemoresistance of triple-negative breast cancer through sequestration of RAC1 to prevent ß-catenin nuclear accumulation.

BACKGROUND: Although chemotherapy, the most widely used systemic treatment in triple-negative breast cancer (TNBC), markedly improved the patients' outcome, chemoresistance always occurs. This study purposed to explore new therapeutic strategies for the treatment of chemoresistance. METHODS AND RESULTS: The expression and prognostic value of DAB2IP were investigated in TNBC tissues and cell lines. Low DAB2IP expression predicted high mortality risk in TNBC. Inhibition of DAB2IP expression conferred cancer stem cell capacity and chemoresistance in TNBC cell lines. Using murine breast cancer (BC) xenograft models, we evaluated the association with DAB2IP and chemoresistance. DAB2IP inhibited TNBC tumourigenesis and chemoresistance in vivo. Further, we revealed that DAB2IP inhibited ß-catenin nuclear transport through competitive interaction with RAC1 and decreased ß-catenin accumulation in the cell nucleus. Finally, we found that the DNA methylation level was negatively associated with DAB2IP expression in TNBC. Inhibition of DNA methylation restored the DAB2IP expression and attenuated chemoresistance in TNBC. CONCLUSIONS: We revealed that DAB2IP attenuates chemoresistance of TNBC via inhibition of RAC1-mediated ß-catenin nuclear accumulation. Decitabine treatment results in re-expression of DAB2IP by inhibiting DNA methylation and could be a potential therapeutic strategy for chemoresistance in TNBC.

Cypermethrin inhibits proliferation of Sertoli cells through AR involving DAB2IP/PI3K/AKT signaling pathway in vitro.

Cypermethrin (CP) exhibits anti-androgenic effects through antagonism on androgen receptor (AR) activation. This study was to identify whether AR-mediated disabled 2 interacting protein (DAB2IP)/phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway was involved in CP-induced mouse Sertoli cells (TM4) proliferation disorder. Real-Time Cell Analysis-iCELLigence system was to measure cell proliferation. Bioinformatic analyses were performed to identify AR-regulated genes. Quantitative Real-Time PCR and western blot were to detect the genes and proteins levels in AR-mediated DAB2IP/PI3K/AKT pathway. Results showed CP suppressed TM4 proliferation and the expression of AR. Activation of AR restored the inhibition efficacy of CP on TM4 proliferation. AR regulated DAB2IP expression and phosphorylation levels of PI3K and AKT in CP-exposed TM4 cells. In addition, knockdown of DAB2IP alleviated the inhibition efficacy of CP on cell proliferation and phosphorylation of PI3K and AKT. Taken together, AR was a modulator in CP-induced inhibition of Sertoli cells proliferation by negatively regulating DAB2IP/PI3K/AKT signaling pathway. The study may provide a new insight for the mechanisms of male reproductive toxicity induced by CP.