Assessing antiviral treatment efficacy and risk factors for severe COVID-19 in kidney transplant recipients during the Omicron subvariant-dominant period: a retrospective study.

BACKGROUND: Kidney transplant recipients (KTRs) are at risk of severe coronavirus disease 2019 (COVID-19), and even now that Omicron subvariants have become dominant, cases of severe disease are certain to occur. The aims of this retrospective study were to evaluate the efficacy of antiviral treatment for COVID-19 and to identify risk factors for severe disease in KTRs during Omicron subvariant-dominant periods. METHODS: A total of 65 KTRs diagnosed with COVID-19 who received antiviral treatment between July 2022 and September 2023 were analyzed. Mild cases received oral molnupiravir (MP) as outpatient therapy, while moderate or worse cases received intravenous remdesivir (RDV) as inpatient therapy. In principle, mycophenolate mofetil was withdrawn and switched to everolimus. We investigated the efficacy of antiviral treatment and compared the clinical parameters of mild/moderate and severe/critical cases to identify risk factors for severe COVID-19. RESULTS: Among 65 cases, 49 were mild, 6 were moderate, 9 were severe, and 1 was of critical severity. MP was administered to 57 cases; 49 (86%) improved and 8 (14%) progressed. RDV was administered to 16 cases; 14 (87%) improved and 2 (13%) progressed. Seventeen (26%) cases required hospitalization, and none died. Comparisons of the severe/critical group (n = 10) with the mild/moderate group (n = 55) demonstrated that the severe/critical group had a significantly higher median age (64 vs. 53 years, respectively; p = 0.0252), prevalence of diabetes (70% vs. 22%, respectively; p = 0.0047) and overweight/obesity (40% vs. 11%, respectively; p = 0.0393), as well as a significantly longer median time from symptom onset to initial antiviral therapy (3 days vs. 1 day, respectively; p = 0.0026). Multivariate analysis showed that a longer time from symptom onset to initial antiviral treatment was an independent risk factor for severe COVID-19 (p = 0.0196, odds ratio 1.625, 95% confidence interval 1.081-2.441). CONCLUSION: These findings suggest that a longer time from symptom onset to initial antiviral treatment is associated with a higher risk of severe COVID-19 in KTRs. Initiating antiviral treatment as early as possible is crucial for preventing severe outcomes; this represents a valuable insight into COVID-19 management in KTRs.

Potential new therapy of Rapalink-1, a new generation mammalian target of rapamycin inhibitor, against sunitinib-resistant renal cell carcinoma.

Sunitinib, a multitargeted receptor tyrosine kinase inhibitor including vascular endothelial growth factor, has been widely used as a first-line treatment against metastatic renal cell carcinoma (mRCC). However, mRCC often acquires resistance to sunitinib, rendering it difficult to treat with this agent. Recently, Rapalink-1, a drug that links rapamycin and the mTOR kinase inhibitor MLN0128, has been developed with excellent therapeutic effects against breast cancer cells carrying mTOR resistance mutations. The aim of the present study was to evaluate the in vitro and in vivo therapeutic efficacy of Rapalink-1 against renal cell carcinoma (RCC) compared to temsirolimus, which is commonly used as a small molecule inhibitor of mTOR and is a derivative of rapamycin. In comparison with temsirolimus, Rapalink-1 showed significantly greater effects against proliferation, migration, invasion and cFolony formation in sunitinib-naïve RCC cells. Inhibition was achieved through suppression of the phosphorylation of substrates in the mTOR signal pathway, such as p70S6K, eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1) and AKT. In addition, Rapalink-1 had greater tumor suppressive effects than temsirolimus against the sunitinib-resistant 786-o cell line (SU-R 786-o), which we had previously established, as well as 3 additional SU-R cell lines established here. RNA sequencing showed that Rapalink-1 suppressed not only the mTOR signaling pathway but also a part of the MAPK signaling pathway, the ErbB signaling pathway and ABC transporters that were associated with resistance to several drugs. Our study suggests the possibility of a new treatment option for patients with RCC that is either sunitinib-sensitive or sunitinib-resistant.

Regulation of ITGA3 by the dual-stranded microRNA-199 family as a potential prognostic marker in bladder cancer.

This corrects the article DOI: 10.1038/bjc.2017.43.

Regulation of ITGA3 by the dual-stranded microRNA-199 family as a potential prognostic marker in bladder cancer.

BACKGROUND: Based on the microRNA (miRNA) signature of bladder cancer (BC) by deep sequencing, we recently found that several double-stranded mature miRNAs derived from the same pre-miRNAs were sufficiently expressed and acted as tumour suppressors by regulating common target genes in BC. Our deep-sequencing signature of BC showed that all miR-199 family members (miR-199a-3p/-5p and miR-199b-3p/-5p) were also downregulated. We hypothesised that these miRNAs may function as tumour suppressors by regulating common target genes. METHODS: Functional assays of BC cells were performed using transfection of mature miRNA. In silico analyses and luciferase reporter analyses were applied to identify target genes of these miRNAs. The overall survival of patients with BC in The Cancer Genome Atlas (TCGA) database was evaluated by the Kaplan-Meier method. RESULTS: Restoration of these miRNAs significantly inhibited cell migration and invasion in BC cells. Integrin α3 (ITGA3) was directly regulated by these miRNAs. The Cancer Genome Atlas database showed that patients with low pre-miR-199 family (miR-199a-1/-2 and miR-199b) expression exhibited significantly poorer overall survival compared with patients with high pre-miR-199 family expression. CONCLUSIONS: miR-199 family miRNAs functioned as tumour suppressors in BC cells by targeting ITGA3 and might be good prognostic markers for predicting survival in patients with BC.

Dietary milk fat globule membrane improves endurance capacity in mice.

Milk fat globule membrane (MFGM) comprises carbohydrates, membrane-specific proteins, glycoproteins, phospholipids, and sphingolipids. We evaluated the effects of MFGM consumption over a 12-wk period on endurance capacity and energy metabolism in BALB/c mice. Long-term MFGM intake combined with regular exercise improved endurance capacity, as evidenced by swimming time until fatigue, in a dose-dependent manner. The effect of dietary MFGM plus exercise was accompanied by higher oxygen consumption and lower respiratory quotient, as determined by indirect calorimetry. MFGM intake combined with exercise increased plasma levels of free fatty acids after swimming. After chronic intake of MFGM combined with exercise, the triglyceride content in the gastrocnemius muscle increased significantly. Mice given MFGM combined with exercise had higher mRNA levels of peroxisome proliferator-activated receptor-γ coactivator 1α (Pgc1α) and CPT-1b in the soleus muscle at rest, suggesting that increased lipid metabolism in skeletal muscle contributes, in part, to improved endurance capacity. MFGM treatment with cyclic equibiaxial stretch consisting of 10% elongation at 0.5 Hz with 1 h on and 5 h off increased the Pgc1α mRNA expression of differentiating C2C12 myoblasts in a dose-dependent manner. Supplementation with sphingomyelin increased endurance capacity in mice and Pgc1α mRNA expression in the soleus muscle in vivo and in differentiating myoblasts in vitro. These results indicate that dietary MFGM combined with exercise improves endurance performance via increased lipid metabolism and that sphingomyelin may be one of the components responsible for the beneficial effects of dietary MFGM.

Hard x-ray telescopes to be onboard ASTRO-H.

The new Japanese x-ray astronomy satellite, ASTRO-H, will carry two identical hard x-ray telescopes (HXTs), which cover the energy range of 5 to 80 keV. The HXT mirrors employ tightly nested, conically approximated thin-foil Wolter-I optics, and the mirror surfaces are coated with Pt/C depth-graded multilayers to enhance the hard x-ray effective area by means of Bragg reflection. The HXT comprises foils 120-450 mm in diameter and 200 mm in length, with a focal length of 12 m. To obtain a large effective area, 213 aluminum foils 0.2 mm in thickness are tightly nested confocally. The requirements for HXT are a total effective area of >300 cm2 at 30 keV and an angular resolution of <1.7' in half-power diameter (HPD). Fabrication of two HXTs has been completed, and the x-ray performance of each HXT was measured at a synchrotron radiation facility, SPring-8 BL20B2 in Japan. Angular resolutions (HPD) of 1.9' and 1.8' at 30 keV were obtained for the full telescopes of HXT-1 and HXT-2, respectively. The total effective area of the two HXTs at 30 keV is 349 cm2.

Ficus carica L. (Fig) promotes nerve regeneration in a mouse model of sciatic nerve crush.

Peripheral nerve injuries result in significant loss of motor and sensory function, and the slow rate of nerve regeneration can prolong recovery time. Thus, approaches that promote axonal regeneration are critical to improve the outcomes for patients with peripheral nerve injuries. In this study, we investigated the effects of Ficus carica L. (fig) and Vaccinium macrocarpon Ait. (cranberry), which are rich in phytochemicals with demonstrable and diverse medicinal properties, on nerve regeneration in a mouse model of sciatic nerve crush. Our investigation revealed that fig extract, but not cranberry extract, prevented the decline in muscle weight and nerve conduction velocity induced by nerve crush. The fig extract also mitigated motor function impairment, myelin thinning, and axon diameter reduction, indicating its potential to promote nerve regeneration. Furthermore, the fig extract enhanced macrophage infiltration into the nerve tissue, suggesting that it could ameliorate nerve injury by promoting tissue repair via increased macrophage infiltration. The study provides valuable insights into the potential of the fig extract as a novel agent promoting nerve regeneration. Further investigation into the mechanisms underlying the action of fig extracts is needed to translate these findings into clinical applications for patients with peripheral nerve injuries.

Combination treatment using percutaneous transluminal angioplasty and low-density lipoprotein apheresis in a patient with peripheral arterial disease and a history of chronic hemodialysis.

Peripheral arterial disease (PAD) is very common in dialysis patients, who tend to have diffuse calcification and severe peripheral arterial stenosis that make it difficult to treat limbs using only surgical or endovascular interventions. Better ways to treat this condition are therefore required and also follow-up studies to evaluate the effects of these treatments on the microcirculation. A 59-year-old man who had a cadaveric kidney transplant five years previously after 25 years of regular hemodialysis complained of pain at rest in his right lower limb and subsequently developed an intractable decubitus ulcer on his right fifth toe (Fontaine IV). Digital subtraction angiography revealed a severe obstruction of the right femoral artery and diffuse stenosis of the right superficial femoral artery. The patient underwent percutaneous transluminal angioplasty (PTA) and six sessions of low-density lipoprotein apheresis (LDL apheresis). At the end of these sessions his complaints were almost completely alleviated. The mean elevation in skin temperature after each session was (1.58 ± 0.99)°C [mean ± SD] over the right dorsalis pedis artery and (1.52 ± 0.88)°C at the tip of the right fifth toe. Ultrasound-measured blood flow rates in the right dorsalis pedis artery were 9.2 cm/s before PTA and 20.2 cm/s one month after PTA. Hemodialysis was resumed 3 days after the PTA due to contrast-induced nephropathy. The combination of PTA and LDL apheresis is useful for treating PAD in hemodialysis patients, with the changes in peripheral artery patency are able to be evaluated effectively by measuring skin temperature.

Tyrosine Is a Booster of Leucine-Induced Muscle Anabolic Response.

Leucine (Leu), an essential amino acid, is known to stimulate protein synthesis in the skeletal muscle via mTOR complex 1 (mTORC1) activation. However, the intrinsic contribution of other amino acids to Leu-mediated activation of mTORC1 signaling remains unexplored. This study aimed to identify amino acids that can promote mTORC1 activity in combination with Leu and to assess the effectiveness of these combinations in vitro and in vivo. We found that tyrosine (Tyr) enhanced Leu-induced phosphorylation of S6 kinase (S6K), an indicator of mTORC1 activity, although it exerted no such effect individually. This booster effect was observed in C2C12 cells, isolated murine muscle, and the skeletal muscles of mice orally administered the amino acids. To explore the molecular mechanisms underlying this Tyr-mediated booster effect, the expression of the intracellular Leu sensors, Sestrin1 and 2, was suppressed, and the cells were treated with Leu and Tyr. This suppression enabled Tyr alone to induce S6K phosphorylation and enhanced the booster effect, suggesting that Tyr possibly contributes to mTORC1 activation when Sestrin-GAP activity toward Rags 2 (GATOR2) is dissociated through Sestrin knockdown or the binding of Sestrins to Leu. Collectively, these results indicate that Tyr is a key regulator of Leu-mediated protein synthesis.

microRNA-99a-5p induces cellular senescence in gemcitabine-resistant bladder cancer by targeting SMARCD1.

Patients with advanced bladder cancer are generally treated with a combination of chemotherapeutics, including gemcitabine, but the effect is limited due to acquisition of drug resistance. Thus, in this study, we investigated the mechanism of gemcitabine resistance. First, gemcitabine-resistant cells were established and resistance confirmed in vitro and in vivo. Small RNA sequencing analyses were performed to search for miRNAs involved in gemcitabine resistance. miR-99a-5p, selected as a candidate miRNA, was downregulated compared to its parental cells. In gain-of-function studies, miR-99a-5p inhibited cell viabilities and restored sensitivity to gemcitabine. RNA sequencing analysis was performed to find the target gene of miR-99a-5p. SMARCD1 was selected as a candidate gene. Dual-luciferase reporter assays showed that miR-99a-5p directly regulated SMARCD1. Loss-of-function studies conducted with si-RNAs revealed suppression of cell functions and restoration of gemcitabine sensitivity. miR-99a-5p overexpression and SMARCD1 knockdown also suppressed gemcitabine-resistant cells in vivo. Furthermore, ß-galactosidase staining showed that miR-99a-5p induction and SMARCD1 suppression contributed to cellular senescence. In summary, tumor-suppressive miR-99a-5p induced cellular senescence in gemcitabine-resistant bladder cancer cells by targeting SMARCD1.

The cathepsin L gene is a direct target of FOXO1 in skeletal muscle.

FOXO1 (forkhead box O1), a forkhead-type transcription factor whose gene expression is up-regulated in the skeletal muscle during starvation, appears to be a key molecule of energy metabolism and skeletal muscle atrophy. Cathepsin L, a lysosomal proteinase whose expression is also up-regulated in the skeletal muscle during starvation, is induced in transgenic mice overexpressing FOXO1 relative to wild-type littermates. In the present study, we conducted in vivo and in vitro experiments focusing on FOXO1 regulation of Ctsl (cathepsin L gene; CTSL1 in humans) expression in the skeletal muscle. During fasting and refeeding of C57BL/6 mice, Ctsl was regulated in parallel with FOXO1 in the skeletal muscle. Fasting-induced Ctsl expression was attenuated in transgenic mice overexpressing a dominant-negative form of FOXO1 or in skeletal-muscle-specific Foxo1-knockout mice relative to respective wild-type controls. Using C2C12 mouse myoblasts overexpressing a constitutively active form of FOXO1, we showed that FOXO1 induces Ctsl expression. Moreover, we found FOXO1-binding sites in both the mouse Ctsl and human CTSL1 promoters. The luciferase reporter analysis revealed that the mouse Ctsl and human CTSL1 promoters are activated by FOXO1, which is abolished by mutations in the consensus FOXO1-binding sites. Gel mobility-shift and chromatin immunoprecipiation assays showed that FOXO1 is recruited and binds to the Ctsl promoter. The present study provides in vivo and in vitro evidence that Ctsl is a direct target of FOXO1 in the skeletal muscle, thereby suggesting a role for the FOXO1/cathepsin L pathway in fasting-induced skeletal muscle metabolic change and atrophy.

The Impact of Milk Fat Globule Membrane with Exercise on Age-Related Degeneration of Neuromuscular Junctions.

Morphological changes in neuromuscular junctions (NMJs), which are synapses formed between α-motor neurons and skeletal muscle fibers, are considered to be important in age-related motor dysfunction. We have previously shown that the intake of dietary milk fat globule membrane (MFGM) combined with exercise attenuates age-related NMJ alterations in the early phase of aging. However, it is unclear whether the effect of MFGM with exercise on age-related NMJ alterations persists into old age, and whether intervention from old age is still effective when age-related changes in NMJs have already occurred. In this study, 6- or 18-month-old mice were treated with a 1% MFGM diet and daily running wheel exercise until 23 or 24 months of age, respectively. MFGM treatment with exercise was effective in suppressing the progression of age-related NMJ alterations in old age, and even after age-related changes in NMJs had already occurred. Moreover, the effect of MFGM intake with exercise was not restricted to NMJs but extended to the structure and function of peripheral nerves. This study demonstrates that MFGM intake with exercise may be a novel approach for improving motor function in the elderly by suppressing age-related NMJ alterations.

Role of central leptin signaling in renal macrophage infiltration.

Monocytes/macrophages are key mediators of wound repair, tissue remodeling, and inflammation. However, the molecular mechanisms underlying macrophage recruitment to the site of inflammation is not fully understood. Leptin acts directly on the hypothalamus, thereby regulating food intake and energy expenditure. The leptin receptor, a single transmembrane protein that belongs to the gp130 family of cytokine receptor superfamily, is expressed not only in the hypothalamus but in a variety of peripheral tissues, suggesting the role of leptin as a pro-inflammatory adipocytokine in peripheral tissues. Here, we show that deficiency of leptin signaling reduces renal macrophage infiltration after unilateral ureteral obstruction (UUO). Bone marrow transplantation studies using leptin signaling-deficient db/db mice revealed that leptin signaling in bone marrow cells may not play a major role in the UUO-induced renal macrophage infiltration. Interestingly, central leptin administration reverses the otherwise reduced UUO-induced renal macrophage infiltration in leptin-deficient ob/ob mice. This is effectively abolished by central co-administration of SHU9119, a melanocortin-3 receptor/melanocortin-4 receptor antagonist. This study demonstrates that central leptin administration in ob/ ob mice accelerates renal macrophage infiltration through the melanocortin system, thereby suggesting that the central nervous system, which is inherent to integrate information from throughout the organism, is able to control peripheral inflammation.

Targeting NPL4 via drug repositioning using disulfiram for the treatment of clear cell renal cell carcinoma.

The alcohol-abuse drug disulfiram has antitumor effects against diverse cancer types via inhibition of the ubiquitin-proteasome protein nuclear protein localization protein 4 (NPL4). However, the antitumor effects of NPL4 and disulfiram in clear cell renal cell carcinoma (ccRCC) are unclear. Here, we evaluated the therapeutic potential of targeting the ubiquitin-proteasome pathway using disulfiram and RNA interference and investigated the mechanisms underlying disulfiram in ccRCC. According to data from The Cancer Genome Atlas, NPL4 mRNA expression was significantly upregulated in clinical ccRCC samples compared with that in normal kidney samples, and patients with high NPL4 expression had poor overall survival compared with patients with low NPL4 expression. Disulfiram and NPL4 siRNA inhibited ccRCC cell proliferation in vitro, and disulfiram inhibited ccRCC tumor growth in a xenograft model. Synergistic antiproliferative effects were observed for combination treatment with disulfiram and sunitinib in vitro and in vivo. In RCC cells from mice treated with disulfiram and/or sunitinib, several genes associated with serine biosynthesis and aldose reductase were downregulated in cells treated with disulfiram or sunitinib alone and further downregulated in cells treated with both disulfiram and sunitinib. These findings provided insights into the mechanisms of disulfiram and suggested novel therapeutic strategies for RCC treatment.

DOK7 Gene Therapy Enhances Neuromuscular Junction Innervation and Motor Function in Aged Mice.

Muscle denervation at the neuromuscular junction (NMJ), the essential synapse between motor neuron and skeletal muscle, is associated with age-related motor impairment. Therefore, improving muscle innervation at aged NMJs may be an effective therapeutic strategy for treating the impairment. We previously demonstrated that the muscle protein Dok-7 plays an essential role in NMJ formation, and, indeed, its forced expression in muscle enlarges NMJs. Moreover, therapeutic administration of an adeno-associated virus vector encoding human Dok-7 (DOK7 gene therapy) suppressed muscle denervation and enhanced motor activity in a mouse model of amyotrophic lateral sclerosis (ALS). Here, we show that DOK7 gene therapy significantly enhances motor function and muscle strength together with NMJ innervation in aged mice. Furthermore, the treated mice showed greatly increased compound muscle action potential (CMAP) amplitudes compared with the controls, suggesting enhanced neuromuscular transmission. Thus, therapies aimed at enhancing NMJ innervation have potential for treating age-related motor impairment.

Characterization of PHGDH expression in bladder cancer: potential targeting therapy with gemcitabine/cisplatin and the contribution of promoter DNA hypomethylation.

d-3-Phosphoglycerate dehydrogenase (PHGDH) conducts an important step in the synthesis of serine. Importantly, the PHGDH gene is often amplified in certain cancers. Our previous studies revealed that PHGDH gene amplification was associated with poor overall survival in clear cell renal cell carcinoma (ccRCC) and that metabolic reprogramming of serine synthesis through PHGDH recruitment allowed ccRCC cells to survive in unfavorable environments. There have been no investigations of the role of PHGDH expression in bladder cancer (BC). In this investigation, we examined the clinical importance of PHDGH in BC. Furthermore, we asked whether PHGDH expression could be exploited for BC therapy. Finally, we investigated the regulatory mechanisms that modulated the expression of PHGDH. Using data from The Cancer Genome Atlas, we found that patients with high-grade BC had significantly higher PHGDH expression levels than did those with low-grade BC. In addition, patients with high PHGDH expression did not survive as long as those with low expression. PHGDH downregulation by si-RNAs or an inhibitor in BC cell lines significantly inhibited proliferative ability and induced apoptosis. Furthermore, combined treatment using a PHGDH inhibitor and gemcitabine/cisplatin achieved synergistic tumor suppression compared to use of a single agent both in vitro as well as in vivo. Mechanistic analyses of PHGDH regulation showed that PHGDH expression might be associated with DNA copy number and hypomethylation in BC. These findings suggest novel therapeutic strategies could be used in BC. Finally, our data enhance our understanding of the role of PHGDH in BC.

Oncogenic effects of RAB27B through exosome independent function in renal cell carcinoma including sunitinib-resistant.

Exosomes are 40-100 nm nano-sized extracellular vesicles. They are released from many cell types and move into the extracellular space, thereby transferring their components to recipient cells. Exosomes are receiving increasing attention as novel structures participating in intracellular communication. RAB27B is one of the leading proteins involved in exosome secretion, and oncogenic effects have been reported in several cancers. In recent years, molecularly targeted agents typified by sunitinib are widely used for the treatment of metastatic or recurrent renal cell carcinoma (RCC). However, intrinsic or acquired resistance to sunitinib has become a major issue. The present study aimed to elucidate the role of RAB27B in RCC including sunitinib-resistant and its role in exosomes. Bioinformatic analyses revealed that high expression of RAB27B correlates with progression of RCC. The expression of RAB27B protein in RCC cell lines was significantly enhanced compared with that in normal kidney cell lines. Furthermore, RAB27B protein expression was enhanced in all of the tested sunitinib-resistant RCC cell lines compared to parental cells. Although no specific effect of RAB27B on exosomes was identified in RCC cells, loss-of-function studies demonstrated that knockdown of RAB27B suppressed cell proliferation, migration and invasive activities. Moreover, anti-tumor effects of RAB27B downregulation were also observed in sunitinib-resistant RCC cells. RNA sequence and pathway analysis suggested that the oncogenic effects of RAB27B might be associated with MAPK and VEGF signaling pathways. These results showed that RAB27B is a prognostic marker and a novel therapeutic target in sunitinib-sensitive and -resistant RCCs. Further analyses should improve our understanding of sunitinib resistance in RCC.

TRPM8-mediated cutaneous stimulation modulates motor neuron activity during treadmill stepping in mice.

Motor units are generally recruited from the smallest to the largest following the size principle, while cutaneous stimulation has the potential to affect spinal motor control. We aimed to examine the effects of stimulating transient receptor potential channel sub-family M8 (TRPM8) combined with exercise on the modulation of spinal motor neuron (MN) excitability. Mice were topically administrated 1.5% icilin on the hindlimbs, followed by treadmill stepping. Spinal cord sections were immunostained with antibodies against c-fos and choline acetyltransferase. Icilin stimulation did not change the number of c-fos+ MNs, but increased the average soma size of the c-fos+ MNs during low-speed treadmill stepping. Furthermore, icilin stimulation combined with stepping increased c-fos+ cholinergic interneurons near the central canal, which are thought to modulate MN excitability. These findings suggest that TRPM8-mediated cutaneous stimulation with low-load exercise promotes preferential recruitment of large MNs and is potentially useful as a new training method for rehabilitation.

Potential tumor­suppressive role of microRNA­99a­3p in sunitinib­resistant renal cell carcinoma cells through the regulation of RRM2.

Sunitinib is the most common primary molecular­targeted agent for metastatic clear cell renal cell carcinoma (ccRCC); however, intrinsic or acquired sunitinib resistance has become a significant problem in medical practice. The present study focused on microRNA (miR)­99a­3p, which was significantly downregulated in clinical sunitinib­resistant ccRCC tissues in previous screening analyses, and investigated the molecular network associated with it. The expression levels of miR­99a­3p and its candidate target genes were evaluated in RCC cells, including previously established sunitinib­resistant 786­o (SU­R­786­o) cells, and clinical ccRCC tissues, using reverse transcription­quantitative polymerase chain reaction. Gain­of­function studies demonstrated that miR­99a­3p significantly suppressed cell proliferation and colony formation in RCC cells, including the SU­R­786­o cells, by inducing apoptosis. Based on in silico analyses and RNA sequencing data, followed by luciferase reporter assays, ribonucleotide reductase regulatory subunit­M2 (RRM2) was identified as a direct target of miR­99a­3p in the SU­R­786­o cells. Loss­of­function studies using small interfering RNA against RRM2 revealed that cell proliferation and colony growth were significantly inhibited via induction of apoptosis, particularly in the SU­R­786­o cells. Furthermore, the RRM2 inhibitor Didox (3,4­dihydroxybenzohydroxamic acid) exhibited anticancer effects in the SU­R­786­o cells and other RCC cells. To the best of our knowledge, this is the first report demonstrating that miR­99a­3p directly regulates RRM2. Identifying novel genes targeted by tumor­suppressive miR­99a­3p in sunitinib­resistant RCC cells may improve our understanding of intrinsic or acquired resistance and facilitate the development of novel therapeutic strategies.

Tumor­suppressive microRNA­223 targets WDR62 directly in bladder cancer.

miRNA­223 (miR­223) has been reported to function not only as a tumor suppressor, but also as an oncogenic microRNA (miRNA or miR) in various cancer cells. Therefore, the functional role of miR­223 has not been elucidated to date, at least to the best of our knowledge. We previously performed the deep sequencing analysis of clinical bladder cancer (BC) specimens. It was revealed that miR­223 expression was significantly downregulated in BC, suggesting that miR­223 functions as a tumor suppressor miRNA in BC. The aim of this study was to investigate the functional roles of miR­223 and to identify its targets in BC. The expression levels of miR­223 were significantly decreased in our clinical BC specimens. The Cancer Genome Atlas (TCGA) database indicated that miR­223 expression was related to lymphovascular invasion and distant metastasis. The restoration of miR­223 expression significantly inhibited tumor aggressiveness and induced apoptosis via caspase­3/7 activation in BC cells. WD repeat domain 62 (WDR62), a candidate target of miR­223 according to in silico analyses, has been previously proposed to play a role in neurodevelopment. Direct binding between WDR62 and miR­223 was confirmed by luciferase assay. The TCGA database revealed positive associations between WDR62 mRNA expression and a higher tumor grade and stage in BC. The knockdown of WDR62 significantly inhibited tumor aggressiveness and induced the apoptosis of BC cells. On the whole, the findings of this study reveal a novel miR­223 target, oncogenic WDR62, and provided insight into the oncogenesis of BC.