N6-methyladenosine modification of circMARK2 enhances cytoplasmic export and stabilizes LIN28B, contributing to the progression of Wilms tumor.

BACKGROUND: The potential involvement of circular RNAs (circRNAs) and N6-methyladenosine (m6A) modification in the progression of Wilms tumor (WT) has not been fully elucidated. This study investigates the regulatory mechanisms and clinical significance of m6A-modified circMARK2 and its role in WT progression. METHODS: We identified dysregulated circRNAs through deep sequencing and validated their expression by qRT-PCR in WT tissues. The biological functions of circMARK2 were assessed using clone formation, transwell migration, and orthotopic animal models. To dissect the underlying mechanisms, we employed RNA immunoprecipitation, RNA pull-down, dual-luciferase reporter assays, Western blotting, and immunofluorescence and immunohistochemical staining. RESULTS: CircMARK2, upregulated in WT tissues, was found to be m6A-modified and promoted cytoplasmic export. It facilitated WT progression by stabilizing LIN28B mRNA through the circMARK2/IGF2BP2 interaction. In vitro and in vivo studies demonstrated that circMARK2 enhances the malignant behavior of WT cells. Clinically, higher circMARK2 levels in tumor tissues of WT patients were linked to increased tumor aggressiveness and reduced survival rates. CONCLUSIONS: Our study provides the first comprehensive evidence that m6A-modified circMARK2 contributes to WT progression by enhancing LIN28B mRNA stability, promoting cellular aggressiveness. CircMARK2 emerges as a potential biomarker for prognosis and a promising target for therapeutic intervention in WT, underscoring the clinical relevance of m6A modification in pediatric renal cancer.

Single-cell multiomics reveals ENL mutation perturbs kidney developmental trajectory by rewiring gene regulatory landscape.

How disruptions to normal cell differentiation link to tumorigenesis remains incompletely understood. Wilms tumor, an embryonal tumor associated with disrupted organogenesis, often harbors mutations in epigenetic regulators, but their role in kidney development remains unexplored. Here, we show at single-cell resolution that a Wilms tumor-associated mutation in the histone acetylation reader ENL disrupts kidney differentiation in mice by rewiring the gene regulatory landscape. Mutant ENL promotes nephron progenitor commitment while restricting their differentiation by dysregulating transcription factors such as Hox clusters. It also induces abnormal progenitors that lose kidney-associated chromatin identity. Furthermore, mutant ENL alters the transcriptome and chromatin accessibility of stromal progenitors, resulting in hyperactivation of Wnt signaling. The impacts of mutant ENL on both nephron and stroma lineages lead to profound kidney developmental defects and postnatal mortality in mice. Notably, a small molecule inhibiting mutant ENL's histone acetylation binding activity largely reverses these defects. This study provides insights into how mutations in epigenetic regulators disrupt kidney development and suggests a potential therapeutic approach.

DROSHA Regulates Mesenchymal Gene Expression in Wilms Tumor.

Wilms tumor, the most common pediatric kidney cancer, resembles embryonic renal progenitors. Currently, there are no ways to therapeutically target Wilms tumor driver mutations, such as in the microRNA processing gene DROSHA. In this study, we used a "multiomics" approach to define the effects of DROSHA mutation in Wilms tumor. We categorized Wilms tumor mutations into four mutational subclasses with unique transcriptional effects: microRNA processing, MYCN activation, chromatin remodeling, and kidney developmental factors. In particular, we find that DROSHA mutations are correlated with de-repressing microRNA target genes that regulate differentiation and proliferation and a self-renewing, mesenchymal state. We model these findings by inhibiting DROSHA expression in a Wilms tumor cell line, which led to upregulation of the cell cycle regulator cyclin D2 (CCND2). Furthermore, we observed that DROSHA mutations in Wilms tumor and DROSHA silencing in vitro were associated with a mesenchymal state with aberrations in redox metabolism. Accordingly, we demonstrate that Wilms tumor cells lacking microRNAs are sensitized to ferroptotic cell death through inhibition of glutathione peroxidase 4, the enzyme that detoxifies lipid peroxides. Implications: This study reveals genotype-transcriptome relationships in Wilms tumor and points to ferroptosis as a potentially therapeutic vulnerability in one subset of Wilms tumor.

Effect of miR-590-3p/DKK1 Axis on the Progression of Wilms' Tumour.

BACKGROUND: Wilms' tumour is the most prevalent abdominal malignancy in children. This study focused on the mechanism of the miR-590-3p/Dickkopf 1 (DKK1) axis in Wilms' tumour. METHODS: The mRNA levels of miR-590-3p and DKK1 in 49 pairs of Wilms' tumour pathological specimens and normal tissues were determined using real-time quantitative polymerase chain reaction (RT-qPCR). Wilms' tumour cells' invasion ability and proliferative ability were assessed using a Transwell assay and Cell Counting Kit-8 (CCK-8) assay, respectively. Dual-luciferase assay was performed to evaluate the potential relationship between miR-590-3p and DKK1 in Wilms tumour. Furthermore, a mouse transplanted tumour model was constructed to explore the function of miR-590-3p inhibitor on Wilms' tumour growth in vivo. RESULTS: DKK1 emerged as a target gene of miR-590-3p in Wilms' tumour. DKK1 expression was downregulated (p < 0.01), but miR-590-3p was overexpressed (p < 0.01) in Wilms' tumour tissues compared to normal tissues. miR-590-3p overexpression accelerated Wilms' tumour invasive ability and cell proliferation (p < 0.01). Additionally, DKK1 partially reversed miR-590-3p-induced proliferation (p < 0.05) and invasion ability (p < 0.01). Furthermore, downregulation of miR-590-3p restrained the growth rate of transplanted tumours in nude mice (p < 0.01). CONCLUSIONS: Through the regulation of DKK1, miR-590-3p accelerated the invasion and proliferation of Wilms' tumour. The study suggests that the miR-590-3p/DKK1 axis represents a novel mechanism in Wilms' tumour.

Adjuvant Wilms' tumour 1-specific dendritic cell immunotherapy complementing conventional therapy for paediatric patients with high-grade glioma and diffuse intrinsic pontine glioma: protocol of a monocentric phase I/II clinical trial in Belgium.

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) and paediatric high-grade glioma (pHGG) are aggressive glial tumours, for which conventional treatment modalities fall short. Dendritic cell (DC)-based immunotherapy is being investigated as a promising and safe adjuvant therapy. The Wilms' tumour protein (WT1) is a potent target for this type of antigen-specific immunotherapy and is overexpressed in DIPG and pHGG. Based on this, we designed a non-randomised phase I/II trial, assessing the feasibility and safety of WT1 mRNA-loaded DC (WT1/DC) immunotherapy in combination with conventional treatment in pHGG and DIPG. METHODS AND ANALYSIS: 10 paediatric patients with newly diagnosed or pretreated HGG or DIPG were treated according to the trial protocol. The trial protocol consists of leukapheresis of mononuclear cells, the manufacturing of autologous WT1/DC vaccines and the combination of WT1/DC-vaccine immunotherapy with conventional antiglioma treatment. In newly diagnosed patients, this comprises chemoradiation (oral temozolomide 90 mg/m2 daily+radiotherapy 54 Gy in 1.8 Gy fractions) followed by three induction WT1/DC vaccines (8-10×106 cells/vaccine) given on a weekly basis and a chemoimmunotherapy booster phase consisting of six 28-day cycles of oral temozolomide (150-200 mg/m2 on days 1-5) and a WT1/DC vaccine on day 21. In pretreated patients, the induction and booster phase are combined with best possible antiglioma treatment at hand. Primary objectives are to assess the feasibility of the production of mRNA-electroporated WT1/DC vaccines in this patient population and to assess the safety and feasibility of combining conventional antiglioma treatment with the proposed immunotherapy. Secondary objectives are to investigate in vivo immunogenicity of WT1/DC vaccination and to assess disease-specific and general quality of life. ETHICS AND DISSEMINATION: The ethics committee of the Antwerp University Hospital and the University of Antwerp granted ethics approval. Results of the clinical trial will be shared through publication in a peer-reviewed journal and presentations at conferences. TRIAL REGISTRATION NUMBER: NCT04911621.

Mechanism of LncRNA FTX regulates nephroblastoma progression through MiR-215-5p/PI3K/AKT axis.

BACKGROUND: Nephroblastoma, also more commonly known as Wilms tumor (WT), is a common childhood malignancy that connects tumorigenesis and organ development in the kidney. OBJECTIVE: The current study focused on the effect of lncRNA FTX in nephroblastoma. STUDY DESIGN: Expression of lncRNA FTX in nephroblastoma tissues and cells was determined. The expression location of lncRNA FTX was detected by FISH. The binding of lncRNA FTX and miR-215-5p with Ago2 was verified by RIP. Following gain- and loss-of-function approaches, the crucial role of lncRNA FTX and miR-215-5p in nephroblastoma cell functions was measured with the involvement of the PI3K/AKT pathway. RESULTS: LncRNA FTX was elevated and miR-215-5p was declined in nephroblastoma. Silencing of lncRNA FTX or mimic of miR-215-5p inhibited the malignant properties of nephroblastoma cells. LncRNA FTX was localized in the cytoplasm and might bind miR-215-5p. LncRNA FTX promoted the malignant features of nephroblastoma cells by inhibiting miR-215-5p through activating of the PI3K/AKT pathway. CONCLUSIONS: LncRNA FTX is capable of accelerating nephroblastoma development in vitro by reducing miR-215-5p through activating of the PI3K/AKT pathway, indicating LncRNA FTX may possibly a future target for the diagnosis and treatment of nephroblastoma. SUMMARY FIGURE.

Multidimensional Transcriptomics Unveils RNF34 as a Prognostic Biomarker and Potential Indicator of Chemotherapy Sensitivity in Wilms' Tumour.

Nephroblastoma, colloquially known as Wilms' tumour (WT), is the predominant malignant renal neoplasm arising in the paediatric population. Modern therapeutic approaches for WT incorporate a synergistic combination of surgical intervention, radiotherapy, and chemotherapy, which substantially ameliorate the overall patient survival rate. Despite this, the optimal sequence of chemotherapy and surgical intervention remains a matter of contention, with each strategy presenting its own strengths and weaknesses that could influence clinical decision-making. To make some headway on this clinical dilemma, we deployed a multidimensional transcriptomics integration approach by analysing bulk RNA sequencing data with 136 samples, as well as single-nucleus RNA sequencing (snRNA-seq) and paired spatial transcriptome sequencing (stRNA) data from 32 WT specimens. Our findings identified a distinct elevation of RNF34 expression within WT samples, which correlated with unfavourable prognostic outcomes. Leveraging the Genomics of Drug Sensitivity in Cancer (GDSC), we simultaneously revealed that patients with high expression of RNF34 have higher sensitivity to commonly used chemotherapy drugs for WT. Furthermore, our analysis of snRNA and stRNA data unveiled a reduced proportion of RNF34 expression in neoplastic cells after chemotherapy. Moreover, stRNA data delineated a significant association between a higher proportion of RNF34 expression in cancer cells and adverse features such as anaplastic histology and tumour recurrence. Intriguingly, we also observed a close association between elevated RNF34 expression and a characteristic exhausted tumour immune microenvironment. Collectively, our findings underscore the pivotal role of RNF34 in the prognostic prediction potential and treatment sensitivity of WT. This comprehensive analysis can potentially inform and refine clinical decision-making for WT patients and guide future studies towards the development of optimized, rational therapeutic strategies.

LncRNA LINC01339 Hinders the Development of Wilms' Tumor via MiR-135b-3p/ADH1C Axis.

Wilms' tumor is a malignant renal cancer that arises within the pediatric urinary system. This study intended to investigate how a novel long non-coding RNA LINC01339 functions in the pathogenesis of Wilms' tumor. An elevated miR-135b-3p expression as well as reduced levels of LINC01339 and ADH1C were observed in Wilms' tumor. LINC01339 mediated ADH1C expression by directly binding to miR-135b-3p. The enforced LINC01339 or ADH1C markedly hindered cell growth and migration in Wilms' tumor. The LINC01339 overexpression also repressed the growth of Wilms' tumors in vivo, whereas miR-135b-3p overexpression exerted the opposite effects on Wilms' tumor cells in vitro. Additionally, upregulating miR-135b-3p reversed LINC01339's effects on the cellular processes of Wilms' tumor cells, whereas ADH1C overexpression offset the cancer-promoting influence of miR-135b-3p upregulation on Wilms' tumor progression. Therefore, LINC01339 prevents Wilms' tumor progression by modulating the miR-135b-3p/ADH1C axis. Our findings substantiate that the LINC01339/miR-135 b-3p/ADH1C regulatory axis has potential to be a target for the treatment of Wilms' tumor.

Wilms' tumor gene 1: lessons from the interface between kidney development and cancer.

In 1990, mutations of the Wilms' tumor-1 gene (WT1), encoding a transcription factor in the embryonic kidney, were found in 10-15% of Wilms' tumors; germline WT1 mutations were associated with hereditary syndromes involving glomerular and reproductive tract dysplasia. For more than three decades, these discoveries prompted investigators to explore the embryonic role of WT1 and the mechanisms by which loss of WT1 leads to malignant transformation. Here, we discuss how alternative splicing of WT1 generates isoforms that act in a context-specific manner to activate or repress target gene transcription. WT1 also regulates posttranscriptional regulation, alters the epigenetic landscape, and activates miRNA expression. WT1 functions at multiple stages of kidney development, including the transition from resting stem cells to committed nephron progenitor, which it primes to respond to WNT9b signals from the ureteric bud. WT1 then drives nephrogenesis by activating WNT4 expression and directing the development of glomerular podocytes. We review the WT1 mutations that account for Denys-Drash syndrome, Frasier syndrome, and WAGR syndrome. Although the WT1 story began with Wilms' tumors, an understanding of the pathways that link aberrant kidney development to malignant transformation still has some important gaps. Loss of WT1 in nephrogenic rests may leave these premalignant clones with inadequate DNA repair enzymes and may disturb the epigenetic landscape. Yet none of these observations provide a complete picture of Wilms' tumor pathogenesis. It appears that the WT1 odyssey is unfinished and still holds a great deal of untilled ground to be explored.

TRIM28 inactivation in epithelial nephroblastoma is frequent and often associated with predisposing TRIM28 germline variants.

Wilms tumors (WTs) are histologically diverse childhood cancers with variable contributions of blastema, stroma, and epithelia. A variety of cancer genes operate in WTs, including the tripartite-motif-containing-28 gene (TRIM28). Case reports and small case series suggest that TRIM28 mutations are associated with epithelial morphology and WT predisposition. Here, we systematically investigated the prevalence of TRIM28 inactivation and predisposing mutations in a cohort of 126 WTs with >2/3 epithelial cells, spanning 20 years of biobanking in the German SIOP93-01/GPOH and SIOP2001/GPOH studies. Overall, 44.4% (56/126) cases exhibited loss of TRIM28 by immunohistochemical staining. Of these, 48 could be further analyzed molecularly, revealing TRIM28 sequence variants in each case - either homozygous (~2/3) or heterozygous with epigenetic silencing of the second allele (~1/3). The majority (80%) of the mutations resulted in premature stops and frameshifts. In addition, we detected missense mutations and small deletions predicted to destabilize the protein through interference with folding of key structural elements such as the zinc-binding clusters of the RING, B-box-2, and PHD domains or the central coiled-coil region. TRIM28-mutant tumors otherwise lacked WT-typical IGF2 alterations or driver events, except for rare TP53 progression events that occurred with expected frequency. Expression profiling identified TRIM28-mutant tumors as a homogeneous subset of epithelial WTs that mostly present with stage I disease. There was a high prevalence of perilobar nephrogenic rests, putative precursor lesions, that carried the same biallelic TRIM28 alterations in 7/7 cases tested. Importantly, 46% of the TRIM28 mutations were present in blood cells or normal kidney tissue, suggesting germline events or somatic mosaicism, partly supported by family history. Given the high prevalence of predisposing variants in TRIM28-driven WT, we suggest that immunohistochemical testing of TRIM28 be integrated into diagnostic practice as the management of WT in predisposed children differs from that with sporadic tumors. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Altered binding affinity of SIX1-Q177R correlates with enhanced WNT5A and WNT pathway effector expression in Wilms tumor.

Wilms tumors present as an amalgam of varying proportions of tissues located within the developing kidney, one being the nephrogenic blastema comprising multipotent nephron progenitor cells (NPCs). The recurring missense mutation Q177R in NPC transcription factors SIX1 and SIX2 is most correlated with tumors of blastemal histology and is significantly associated with relapse. Yet, the transcriptional regulatory consequences of SIX1/2-Q177R that might promote tumor progression and recurrence have not been investigated extensively. Utilizing multiple Wilms tumor transcriptomic datasets, we identified upregulation of the gene encoding non-canonical WNT ligand WNT5A in addition to other WNT pathway effectors in SIX1/2-Q177R mutant tumors. SIX1 ChIP-seq datasets from Wilms tumors revealed shared binding sites for SIX1/SIX1-Q177R within a promoter of WNT5A and at putative distal cis-regulatory elements (CREs). We demonstrate colocalization of SIX1 and WNT5A in Wilms tumor tissue and utilize in vitro assays that support SIX1 and SIX1-Q177R activation of expression from the WNT5A CREs, as well as enhanced binding affinity within the WNT5A promoter that may promote the differential expression of WNT5A and other WNT pathway effectors associated with SIX1-Q177R tumors.

The association of miR-204 and mir-483 5p expression with clinicopathological features of Wilms tumor: Could this provide foresight?

BACKGROUND: Wilms tumor is the most common cancer of the kidney that occurs during childhood, and histologically, it mimics renal embryogenesis. With the development and improvement of up-to-date treatment protocols, the survival rates of Wilms tumor have increased. However, metastases or local relapses are still observed in 15% of patients. The search for reliable biomarkers to identify at-risk patients is ongoing to predict the variability in treatment success. Currently, the evaluation of clinical, histopathological and genetic features are common diagnostic methods; however, epigenetic features can be examined with microRNA expression analyses and might allow us to comment on the behavior of the tumor and treatment response. METHODS: In this study, we aimed to evaluate the relationship between microRNA-204 and microRNA-483-5p expression with clinicopathological data and the effect on Wilms tumor survival. For this purpose, the expression levels of RNU6B, microRNA-204 and microRNA-483-5p were evaluated in tumor and normal tissue by qreal time-polymerase chain reaction. We also investigated the relationship between microRNA expression levels with the clinicopathological and histological features of Wilms tumor. RESULTS AND CONCLUSION: The results of our study indicate that the relative expression levels of microRNA-204 in Wilms tumor tissues were significantly lower than that in adjacent normal tissues. By contrast, tumor tissue had a higher microRNA-483-5p expression than the corresponding normal tissues. A statistically significant difference between microRNA-204 expression level with age and the presence of anaplasia was observed. The upregulation of microRNA-483-5p was found to have a significant correlation with patients after preoperative chemotherapy and complete tumor necrosis. Taken together, our data suggest that microRNA-204 could play a critical role as a tumor suppressor, whereas microRNA-483-5p acts as an oncogene in Wilms tumor progression. More importantly, microRNA-204 might be a novel predictive biomarker for anaplastic histology and could be useful for developing therapeutic interventions targeting this marker.

Identification and Characterization of the Wilms Tumor Cancer Stem Cell.

A nephrogenic progenitor cell (NP) with cancer stem cell characteristics driving Wilms tumor (WT) using spatial transcriptomics, bulk and single cell RNA sequencing, and complementary in vitro and transplantation experiments is identified and characterized. NP from WT samples with NP from the developing human kidney is compared. Cells expressing SIX2 and CITED1 fulfill cancer stem cell criteria by reliably recapitulating WT in transplantation studies. It is shown that self-renewal versus differentiation in SIX2+CITED1+ cells is regulated by the interplay between integrins ITGß1 and ITGß4. The spatial transcriptomic analysis defines gene expression maps of SIX2+CITED1+ cells in WT samples and identifies the interactive gene networks involved in WT development. These studies define SIX2+CITED1+ cells as the nephrogenic-like cancer stem cells of WT and points to the renal developmental transcriptome changes as a possible driver in regulating WT formation and progression.

In vitro expansion of Wilms' tumor protein 1 epitope-specific primary T cells from healthy human peripheral blood mononuclear cells.

Wilms' tumor protein 1 (WT1) is a tumor-associated antigen overexpressed in various cancers. As a self-antigen, negative selection reduces the number of WT1-specific T cell receptors (TCRs). Here, we provide a protocol to generate WT137-45-specific TCRs using healthy human peripheral blood mononuclear cells. We describe the expansion of WT1-specific T cell clones by two consecutive in vitro stimulations with autologous WT137-45-pulsed dendritic cells and peripheral blood lymphocytes. We then detail the detection with human leukocyte antigen/WT137-45 tetramers.

Mechanism of action of DSP-7888 (adegramotide/nelatimotide) Emulsion, a peptide-based therapeutic cancer vaccine with the potential to turn up the heat on non-immunoreactive tumors

Background Wilms’ tumor 1 (WT1) is highly expressed in various solid tumors and hematologic malignancies. DSP-7888 (adegramotide/nelatimotide) Emulsion is an investigational therapeutic cancer vaccine comprising three synthetic epitopes derived from WT1. We evaluated the mechanism of action of DSP-7888 Emulsion, which is hypothesized to induce WT1-specific cytotoxic T lymphocytes (CTLs) and helper T lymphocytes (HTLs). Methods The ability of nelatimotide and adegramotide to induce WT1-specific CD8+ T cells and CD4+ T cells was assessed in human peripheral blood mononuclear cells (PBMCs). The ability of DSP-7888 Emulsion to induce WT1-specific CTLs in vivo was assessed using human leukocyte antigen-I (HLA-I) transgenic mice. To assess how adegramotide, the helper peptide in DSP-7888 Emulsion, enhances WT1-specific CTLs, HLA-I transgenic mice were administered DSP-7888 or nelatimotide-only Emulsion. Interferon-gamma secretion under antigen stimulation by splenocytes co-cultured with or without tumor cells was then quantified. The effects of combination treatment with DSP-7888 Emulsion and an anti–programmed cell death protein 1 (PD-1) antibody on tumor volume and the frequency of tumor-infiltrating WT1-specific T cells were assessed in HLA-I transgenic mice implanted with WT1 antigen-positive tumors. Result The peptides in DSP-7888 Emulsion were shown to induce WT1-specific CTLs and HTLs in both human PBMCs and HLA-I transgenic mice. Unlike splenocytes from nelatimotide-only Emulsion-treated mice, splenocytes from DSP-7888 Emulsion-treated mice exhibited high levels of interferon-gamma secretion, including when co-cultured with tumor cells (AU)

MicroRNA-143-3p Inhibits Wilms' Tumor Cell Growth By Targeting the Ras/Raf/MEK/ERK Pathway.

Background: A previous study found that microRNA-143-3p (miR-143-3p) is a tumor suppressor in various types of human cancer. However, the roles and molecular mechanisms of miR-143-3p in the progression of Wilms' tumor (WT) remain to be clarified. The aim of the present study was to determine the expression and biological functions of miR-143-3p in WT. Material and Methods: The expression levels of miR-143-3p in primary WT tissues and adjacent tissues were determined using quantitative-reverse transcription polymerase chain reaction (qRT-PCR), and the association of the miR-143-3p expression level with various clinicopathological features of WT was investigated. Western blotting was used to evaluate the protein expression of the related signaling pathway. Results: The expression of miR-143-3p was significantly downregulated in WT tissues and its expression levels were closely associated with tumor stage and lymph node metastasis. Overexpression of miR-143-3p in SK-NEP-1 and G401 cell lines inhibited cell proliferation by G0/G1 cell cycle phase arrest and induction of apoptosis. Moreover, k-Ras, a unique oncogene, was confirmed as a direct target of miR-143-3p, and k-Ras messenger RNA (mRNA) expression was increased in WT tissues and inversely correlated with miR-143-3p. Knockdown of k-Ras by si-k-Ras could inhibit, whereas overexpression of k-Ras could promote. cell proliferation in WT cells. Meanwhile, overexpression of k-Ras reversed the inhibitory effects on WT cells induced by miR-143-3p mimics. Conclusion: Our findings indicate that miR-143-3p may be a potential novel prognostic biomarker and therapeutic target for WT.

WT1 regulates expression of DNA repair gene Neil3 during nephrogenesis.

Mammalian nephrons arise from a population of nephron progenitor cells (NPCs) expressing the master transcription factor Wilms tumor-1 (WT1), which is crucial for NPC proliferation, migration, and differentiation. In humans, biallelic loss of WT1 precludes nephrogenesis and leads to the formation of Wilms tumor precursor lesions. We hypothesize that WT1 normally primes the NPC for nephrogenesis by inducing expression of NPC-specific DNA repair genes that protect the genome. We analyzed transcript levels for a panel of DNA repair genes in embryonic day 17.5 (E17.5) versus adult mouse kidneys and noted seven genes that were increased >20-fold. We then isolated Cited1+ NPCs from E17.5 kidneys and found that only one gene, nei-like DNA glycosylase 3 (Neil3), was enriched. RNAscope in situ hybridization of E17.5 mouse kidneys showed increased Neil3 expression in the nephrogenic zone versus mature nephron structures. To determine whether Neil3 expression is WT1 dependent, we knocked down Wt1 in Cited1+ NPCs (60% knockdown efficiency) and noted a 58% reduction in Neil3 transcript levels. We showed that WT1 interacts with the Neil3 promoter and that activity of a Neil3 promoter-reporter vector was increased twofold in WT1+ versus WT1- cells. We propose that Neil3 is a WT1-dependent DNA repair gene expressed at high levels in Cited1+ NPCs, where it repairs mutational injury to the genome during nephrogenesis. NEIL3 is likely just one of many such lineage-specific repair mechanisms that respond to genomic injury during kidney development.NEW & NOTEWORTHY We studied the molecular events leading to Wilms tumors as a model for the repair of genomic injury. Specifically, we showed that WT1 activates DNA repair gene Neil3 in nephron progenitor cells. However, our observations offer a much broader principle, demonstrating that the embryonic kidney invests in lineage-specific expression of DNA repair enzymes. Thus, it is conceivable that failure of these mechanisms could lead to a variety of "sporadic" congenital renal malformations and human disease.

The potential key genes and pathways associated with Wilms tumor in quest of proper candidates for diagnostic and therapeutic purposes.

To designate the probable most important differentially expressed genes and genetic pathways in Wilms tumor and assess their expression and diagnostic potential by RT-PCR and statistical analysis. Systematic review of the literature and various bioinformatics analysis was carried out to gather and narrow down data. The expression of end-resulting genes was compared in Wilms tumor and normal tissue samples using RT-PCR. Statistical tests reported the diagnostic accuracy of genes and their correlation with clinicopathological features. Four genes including CDH1, NCAM1, EGF, and IGF2 were designated. The panel combining them has 100% sensitivity and specificity in differentiating tumors from normal tissue. Eight pathways, most involved in cell-cell and cell-basal matrix junction interactions, were found to be associated with disease pathogenesis. The suggested genes should undergo further evaluation to be validated as diagnostic biomarkers. Further research on the eight proposed pathways is recommended.

Essential role of Wtip in mouse development and maintenance of the glomerular filtration barrier.

Wilms' tumor interacting protein (Wtip) has been implicated in cell junction assembly and cell differentiation and interacts with proteins in the podocyte slit diaphragm, where it regulates podocyte phenotype. To define Wtip expression and function in the kidney, we created a Wtip-deleted mouse model using ß-galactosidase-neomycin (ß-geo) gene trap technology. Wtip gene trap mice were embryonic lethal, suggesting additional developmental roles outside kidney function. Using ß-geo heterozygous and normal mice, Wtip expression was identified in the developing kidneys, heart, and eyes. In the kidney, expression was restricted to podocytes, which appeared initially at the capillary loop stage coinciding with terminal podocyte differentiation. Heterozygous mice had an expected lifespan and showed no evidence of proteinuria or glomerular pathology. However, heterozygous mice were more susceptible to glomerular injury than wild-type littermates and developed more significant and prolonged proteinuria in response to lipopolysaccharide or adriamycin. In normal human kidneys, WTIP expression patterns were consistent with observations in mice and were lost in glomeruli concurrent with loss of synaptopodin expression in disease. Mechanistically, we identified the Rho guanine nucleotide exchange factor 12 (ARHGEF12) as a binding partner for WTIP. ARHGEF12 was expressed in human podocytes and formed high-affinity interactions through their LIM- and PDZ-binding domains. Our findings suggest that Wtip is essential for early murine embryonic development and maintaining normal glomerular filtration barrier function, potentially regulating slit diaphragm and foot process function through Rho effector proteins.NEW & NOTEWORTHY This study characterized dynamic expression patterns of Wilms' tumor interacting protein (Wtip) and demonstrates the novel role of Wtip in murine development and maintenance of the glomerular filtration barrier.

MicroRNA-27a-5p Inhibits Proliferation, Migration, and Invasion and Promotes Apoptosis of Wilms' Tumor Cell by Targeting PBOV1.

Wilms' tumor is the most common type of renal tumor in children. MicroRNAs (miRNAs) are small noncoding RNAs that play crucial regulatory roles in tumorigenesis. We aimed to study the expression profile and function of miR-27a-5p in Wilms' tumor. miR-27a-5p expression was downregulated in human Wilms' tumor tissues. Functionally, overexpression of miR-27a-5p promoted cell apoptosis of Wilms' tumor cells. Furthermore, upregulated miR-27a-5p delayed xenograft Wilms' tumor tumorigenesis in vivo. Bioinformatics analysis predicted that miR-27a-5p directly targeted the 3'-untranslated region (3'-UTR) of PBOV1, and luciferase reporter assay confirmed the interaction between miR-27a-5p and PBOV1. The function of PBOV1 in Wilms' tumor was evaluated in vitro, and knockdown of PBOV1 dampened cell migration. In addition, overexpression of PBOV1 antagonized the tumor-suppressive effect of miR-27a-5p in Wilms' tumor cells. Collectively, our findings reveal the regulatory axis of miR-27a-5p/PBOV1 in Wilms' tumor, and miR-27a-5p might serve as a novel therapeutic target in Wilms' tumor.