記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Kidney360  

Key PointsSystemically administered anti-microRNA (miR)-21-serinol nucleic acid (SNA) accumulates in the kidneys and suppresses renal cyst growth.Anti-miR-21-SNA treatment alters mitochondrial metabolism, apoptotic signaling, and inhibits kidney tissue fibrosis.In human autosomal dominant polycystic kidney disease kidney cells, anti-miR-21-SNA treatment reduces cyst size and intracellular cAMP content and increases Ca2⁺ concentration.BackgroundHereditary cystic kidney diseases are ciliopathies characterized by functional defects in the primary cilia of renal tubules. Abnormalities in the primary cilia enhance cell proliferation signals and cause cyst enlargement. The most common type is autosomal dominant polycystic kidney disease (ADPKD), but other diseases, such as nephronophthisis, have been discovered to be more common than previously considered. In ADPKD, several microRNAs are reportedly aberrantly expressed and involved in disease pathogenesis. Among these, we focused on microRNA (miR)-21, which is upregulated in response to cAMP signaling. In this study, we aimed to newly generate an anti-miR-21 oligonucleotide synthesized from serinol nucleic acid (anti-miR-21-SNA) to improve anti-microRNA activity and investigate its effects on cyst growth in vivo and in vitro.MethodsWe evaluated the effectiveness of anti-miR-21 treatment using an serinol nucleic acid-based antisense oligonucleotide in a mouse model of cystic kidney disease and human ADPKD cells.ResultsOur study revealed that anti-miR-21-SNA effectively prevented cyst growth in vivo and in vitro. In the mouse model of cystic kidney disease, we systemically administered anti-miR-21-SNA and observed its accumulation primarily in the kidneys, suggesting effective drug delivery. Anti-miR-21-SNA treatment reduced kidney size and blood urea nitrogen levels without inducing hepatotoxicity. Mechanistically, molecules related to mitochondrial metabolism, apoptosis, and fibrosis pathways were involved. In vitro, anti-miR-21-SNA treatment of primary cultured kidney cells from an ADPKD patient reduced cyst volume and intracellular cAMP content and increased Ca²⁺ concentration, supporting the efficacy of this treatment.ConclusionsOur results showed that anti-miR-21-SNA treatment represents a potential therapeutic strategy for cystic kidney disease.

DOI： 10.34067/KID.0000000771

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Biochemistry  

Anaplastic lymphoma kinase (ALK) is a well-known oncogene involved in various malignancies such as anaplastic large cell lymphoma, lung cancer and neuroblastoma. Several substrates for fused ALK have been identified and their biological functions have been described. However, the lack of a comprehensive identification of ALK substrates limits our understanding of the biological roles of receptor ALK. Thus, this study aimed to identify novel ALK substrates and characterize their biological functions. We screened the interactors of the kinase domain of receptor ALK using proximity-dependent biotin identification and identified 43 interactors. We narrowed down the candidates by evaluating whether these interactors were downstream of ALK in a neuroblastoma cell line, NB-1. Amongst these, we identified amyloid beta precursor protein-binding family B member 1 (APBB1) as an ALK downstream molecule involved in NB-1 cell viability. Finally, we assessed the kinase-substrate relationship between ALK and APBB1 and found that ALK phosphorylated multiple tyrosine residues in APBB1 both in-cell and in-tube assays, with tyrosine 269 as a major target. In conclusion, we successfully identified a new substrate for receptor ALK. Our results may help further elucidate the molecular mechanism of ALK downstream signalling in neuroblastoma.

DOI： 10.1093/jb/mvae055

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Clinical and Experimental Nephrology  

Background: Extracellular vesicles (EVs) have received considerable attention as ideal biomarkers for kidney diseases. Most reports have focused on urinary EVs, that are mainly derived from the cells in the urinary tract. However, the detection and the application of kidney-derived EVs in plasma remains uncertain. Methods: We examined the kidney-derived small EVs (sEVs) in plasma that were supposedly released from renal mesangial and glomerular endothelial cells, using clinical samples from healthy controls and patients with kidney transplants. Plasma from healthy controls underwent ultracentrifugation, followed by on-bead flow cytometry, targeting α8 integrin, an antigen-specific to mesangial cells. To confirm the presence of kidney-derived sEVs in peripheral blood, plasma from ABO-incompatible kidney transplant recipients was ultracentrifuged, followed by western blotting for donor blood type antigens. Results: Immunohistochemistry and immunoelectron microscopy confirmed α8 integrin expression in kidney mesangial cells and their sEVs. The CD9-α8 integrin double-positive sEVs were successfully detected using on-bead flow cytometry. Western blot analysis further revealed transplanted kidney-derived sEVs containing blood type B antigens in non-blood type B recipients, who had received kidneys from blood type B donors. Notably, a patient experiencing graft kidney loss exhibited diminished signals of sEVs containing donor blood type antigens. Conclusion: Our findings demonstrate the potential usefulness of kidney-derived sEVs in plasma in future research for kidney diseases.

DOI： 10.1007/s10157-024-02464-z

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：International Journal of Molecular Sciences  

MYCN amplification occurs in approximately 20–30% of neuroblastoma patients and correlates with poor prognosis. The TH-MYCN transgenic mouse model mimics the development of human high-risk neuroblastoma and provides strong evidence for the oncogenic function of MYCN. In this study, we identified mitotic dysregulation as a hallmark of tumor initiation in the pre-cancerous ganglia from TH-MYCN mice that persists through tumor progression. Single-cell quantitative-PCR of coeliac ganglia from 10-day-old TH-MYCN mice revealed overexpression of mitotic genes in a subpopulation of premalignant neuroblasts at a level similar to single cells derived from established tumors. Prophylactic treatment using antimitotic agents barasertib and vincristine significantly delayed the onset of tumor formation, reduced pre-malignant neuroblast hyperplasia, and prolonged survival in TH-MYCN mice. Analysis of human neuroblastoma tumor cohorts showed a strong correlation between dysregulated mitosis and features of MYCN amplification, such as MYC(N) transcriptional activity, poor overall survival, and other clinical predictors of aggressive disease. To explore the therapeutic potential of targeting mitotic dysregulation, we showed that genetic and chemical inhibition of mitosis led to selective cell death in neuroblastoma cell lines with MYCN over-expression. Moreover, combination therapy with antimitotic compounds and BCL2 inhibitors exploited mitotic stress induced by antimitotics and was synergistically toxic to neuroblastoma cell lines. These results collectively suggest that mitotic dysregulation is a key component of tumorigenesis in early neuroblasts, which can be inhibited by the combination of antimitotic compounds and pro-apoptotic compounds in MYCN-driven neuroblastoma.

DOI： 10.3390/ijms242115571

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BMC Biology  

Background: Combinatorial gene regulation by multiple microRNAs (miRNAs) is widespread and closely spaced target sites often act cooperatively to achieve stronger repression (“neighborhood” miRNA cotargeting). While miRNA cotarget sites are suggested to be more conserved and implicated in developmental control, the pathological significance of miRNA cotargeting remains elusive. Results: Here, we report the pathogenic impacts of combinatorial miRNA regulation on inflammation in systemic lupus erythematosus (SLE). In the SLE mouse model, we identified the downregulation of two miRNAs, miR-128 and miR-148a, by TLR7 stimulation in plasmacytoid dendritic cells. Functional analyses using human cell lines demonstrated that miR-128 and miR-148a additively target KLF4 via extensively overlapping target sites (“seed overlap” miRNA cotargeting) and suppress the inflammatory responses. At the transcriptome level, “seed overlap” miRNA cotargeting increases susceptibility to downregulation by two miRNAs, consistent with additive but not cooperative recruitment of two miRNAs. Systematic characterization further revealed that extensive “seed overlap” is a prevalent feature among broadly conserved miRNAs. Highly conserved target sites of broadly conserved miRNAs are largely divided into two classes—those conserved among eutherian mammals and from human to Coelacanth, and the latter, including KLF4-cotargeting sites, has a stronger association with both “seed overlap” and “neighborhood” miRNA cotargeting. Furthermore, a deeply conserved miRNA target class has a higher probability of haplo-insufficient genes. Conclusions: Our study collectively suggests the complexity of distinct modes of miRNA cotargeting and the importance of their perturbations in human diseases.

DOI： 10.1186/s12915-022-01447-4

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Jci Insight  

Monocarboxylates, such as lactate and pyruvate, are precursors for biosynthetic pathways, including those for glucose, lipids, and amino acids via the tricarboxylic acid (TCA) cycle and adjacent metabolic networks. The transportation of monocarboxylates across the cellular membrane is performed primarily by monocarboxylate transporters (MCTs), the membrane localization and stabilization of which are facilitated by the transmembrane protein basigin (BSG). Here, we demonstrate that the MCT/BSG axis sits at a crucial intersection of cellular metabolism. Abolishment of MCT1 in the plasma membrane was achieved by Bsg depletion, which led to gluconeogenesis impairment via preventing the influx of lactate and pyruvate into the cell, consequently suppressing the TCA cycle. This net anaplerosis suppression was compensated in part by the increased utilization of glycogenic amino acids (e.g., alanine and glutamine) into the TCA cycle and by activated ketogenesis through fatty acid β-oxidation. Complementary to these observations, hyperglycemia and hepatic steatosis induced by a high-fat diet were ameliorated in Bsg-deficient mice. Furthermore, Bsg deficiency significantly improved insulin resistance induced by a high-fat diet. Taken together, the plasma membrane–selective modulation of lactate and pyruvate transport through BSG inhibition could potentiate metabolic flexibility to treat metabolic diseases.

DOI： 10.1172/jci.insight.142464

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Nagoya Journal of Medical Science  

Midkine (MK), a heparin-binding growth factor, is associated with the poor prognosis of the pediatric tumor, neuroblastoma. MK would be a druggable target as many studies showed inhibition of its function in various cancers suppressed tumor developments. To establish the therapy targeting MK, identification of its binding partners, and elucidation of its intracellular signaling are needed. It was reported that exogenous MK induced phosphorylation of ribosomal protein S6 (RPS6) downstream of mTOR signaling. Using RPS6 phosphorylation as a marker of MK response, we searched for MK reactive cell lines. We found that MK cell lines expressing less MK tended to respond better to MK. Next, using an MK reactive neuroblastoma cell line, MK-knocked down SH-SY5Y cells, we employed a proximity-dependent biotin identification method, which was invented to evaluate protein-protein interactions by biotinylation. We confirmed that secreted MK fused to the biotin ligase BioID2 (MK-BioID2) was able to biotinylate proteins from the cells. Biotinylated proteins were identified by liquid chromatography-mass spectrometry analyses. Twenty five proteins were found to be overlapped after three independent experiments, among which insulin-like growth binding protein 2 (IGFBP2) was further analyzed. IGFBP2 was indeed detected with immunoblotting after streptavidin pull down of MK-BioID2 labeled cell extract of MK-knocked down SH-SY5Y cells. Our study suggests that the BioID2 method is useful to identify binding partners of growth factors.

DOI： 10.18999/nagjms.83.3.495

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Cancer Science  

MYCN gene amplification is consistently associated with poor prognosis in patients with neuroblastoma, a pediatric tumor arising from the sympathetic nervous system. Conventional anticancer drugs, such as alkylating agents and platinum compounds, have been used for the treatment of high-risk patients with MYCN-amplified neuroblastoma, whereas molecule-targeting drugs have not yet been approved. Therefore, the development of a safe and effective therapeutic approach is highly desired. Although thymidylate synthase inhibitors are widely used for colorectal and gastric cancers, their usefulness in neuroblastoma has not been well studied. Here, we investigated the efficacies of approved antifolates, methotrexate, pemetrexed, and raltitrexed (RTX), on MYCN-amplified and nonamplified neuroblastoma cell lines. Cell growth-inhibitory assay revealed that RTX showed a superior inhibitory activity against MYCN-amplified cell lines. We found no significant differences in the protein expression levels of the antifolate transporter or thymidylate synthase, a primary target of RTX, among the cell lines. Because thymidine supplementation could rescue the RTX-induced cell growth suppression, the effect of RTX was mainly due to the reduction in dTTP synthesis. Interestingly, RTX treatments induced single-stranded DNA damage response in MYCN-amplified cells to a greater extent than in the nonamplified cells. We propose that the high DNA replication stress and elevated levels of DNA damage, which are a result of deregulated expression of MYCN target genes, could be the cause of increased sensitivity to RTX.

DOI： 10.1111/cas.14485

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：(一社)日本血液学会-東京事務局  

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Ebiomedicine  

Insertional mutagenesis is an important risk with all genetically modified cell therapies, including chimeric antigen receptor (CAR)-T cell therapy used for hematological malignancies. Here we describe a new tagmentation-assisted PCR (tag-PCR) system that can determine the integration sites of transgenes without using restriction enzyme digestion (which can potentially bias the detection) and allows library preparation in fewer steps than with other methods. Using this system, we compared the integration sites of CD19-specific CAR genes in final T cell products generated by retrovirus-based and lentivirus-based gene transfer and by the piggyBac transposon system. The piggyBac system demonstrated lower preference than the retroviral system for integration near transcriptional start sites and CpG islands and higher preference than the lentiviral system for integration into genomic safe harbors. Integration into or near proto-oncogenes was similar in all three systems. Tag-PCR mapping is a useful technique for assessing the risk of insertional mutagenesis.

DOI： 10.1016/j.ebiom.2018.07.008

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Oncotarget  

Neurocan (NCAN), a secreted chondroitin sulfate proteoglycan, is one of the major inhibitory molecules for axon regeneration in nervous injury. However, its role in cancer is not clear. Here we observed that high NCAN expression was closely associated with the unfavorable outcome of neuroblastoma (NB). NCAN was also highly and ubiquitously expressed in the early lesions and terminal tumor of TH-MYCN mice, a NB model. Interestingly, exogenous NCAN (i.e., overexpression, recombinant protein and conditioned medium) transformed adherent NB cells into spheres whose malignancies in vitro (anchorage-independent growth and chemoresistance) and in vivo (xenograft tumor growth) were potentiated. Both chondroitin sulfate sugar chains and NCAN's core protein were essential for the sphere formation. The CSG3 domain was essential in the moiety of NCAN. Our comprehensive microarray analysis and RT-qPCR of mRNA expression suggested that NCAN treatment promoted cell division, and urged cells to undifferentiated state. The knockdown of NCAN in tumor sphere cells cultured from TH-MYCN mice resulted in growth suppression in vitro and in vivo. Our findings suggest that NCAN, which stimulates NB cells to promote malignant phenotypes, is an extracellular molecule providing a growth advantage to cancer cells.

DOI： 10.18632/oncotarget.22435

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Oncotarget  

DOI： 10.18632/oncotarget.18464

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Oncoscience  

Neuroblastoma is the most common extracranial solid tumor in childhood, and develops mainly in the adrenal medulla but also in the sympathetic ganglia. Some cases of neuroblastoma show spontaneous regression. Particularly, stage 4S, where S stands for “special”, exhibits spontaneous regression despite multifocal tumors. Another interesting feature is that, with few exceptions, driver gene mutations are rare in neuroblastoma, while copy number alterations are common, including MYCN amplification, 17q gain, and others, suggesting the involvement of epigenetic regulation [1]. These features suggest that neuroblastoma tumorigenesis is closely related to the development of the sympathoadrenal lineage. However, the underlying mechanisms, even the origin of neuroblastoma, remain largely obscure. To elucidate those mechanisms, three important papers have been recently published [2-4].

DOI： 10.18632/oncoscience.360

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