Pharmacological Inhibition of miR-130 Family Suppresses Bladder Tumor Growth by Targeting Various Oncogenic Pathways via PTPN1.

Previously, we have revealed that the miR-130 family (miR-130b, miR-301a, and miR-301b) functions as an oncomiR in bladder cancer. The pharmacological inhibition of the miR-130 family molecules by the seed-targeting strategy with an 8-mer tiny locked nucleic acid (LNA) inhibits the growth, migration, and invasion of bladder cancer cells by repressing stress fiber formation. Here, we searched for a functionally advanced target sequence with LNA for the miR-130 family with low cytotoxicity and found LNA #9 (A(L)^i^i^A(L)^T(L)^T(L)^G(L)^5(L)^A(L)^5(L)^T(L)^G) as a candidate LNA. LNA #9 inhibited cell growth in vitro and in an in vivo orthotopic bladder cancer model. Proteome-wide tyrosine phosphorylation analysis suggested that the miR-130 family upregulates a wide range of receptor tyrosine kinases (RTKs) signaling via the expression of phosphorylated Src (pSrcTyr416). SILAC-based proteome analysis and a luciferase assay identified protein tyrosine phosphatase non-receptor type 1 (PTPN1), which is implicated as a negative regulator of multiple signaling pathways downstream of RTKs as a target gene of the miR-130 family. The miR-130-targeted LNA increased and decreased PTPN1 and pSrcTyr416 expressions, respectively. PTPN1 knockdown led to increased tumor properties (cell growth, invasion, and migration) and increased pSrcTyr416 expression in bladder cancer cells, suggesting that the miR-130 family upregulates multiple RTK signaling by targeting PTPN1 and subsequent Src activation in bladder cancer. Thus, our newly designed miR-130 family targeting LNA could be a promising nucleic acid therapeutic agent for bladder cancer.

Crosslinked nanogel-based porous hydrogel as a functional scaffold for tongue muscle regeneration.

Surgical resection in tongue cancer can impair speech and swallowing, reducing quality of life. There is a need for biomaterials that can regenerate tongue muscle tissue defects. Ideally, such a biomaterial would allow controlled release of therapeutic proteins, support the survival and differentiation of therapeutic cells, and promote tongue muscle regeneration in vivo. The aim of the current study was to assess these factors in an acryloyl group-modified crosslinked nanogel, consisting of cholesterol-bearing pullulan hydrogel nanoparticles, to determine its potential as a regenerative therapeutic following tongue resection. The hydrogel demonstrated substantial porosity and underwent slow biodegradation. When loaded with a model protein, the gel enabled sustained protein release over two weeks in serum, with no initial burst release. Mouse myoblasts demonstrated adhesion to the hydrogel and cell survival was observed up to one week. Gel-encapsulated myoblasts demonstrated normal myotube differentiation. Myoblast-loaded gels were implanted in a tongue defect in mice, and there was a significant increase in newly-regenerated myofibers in gel-implanted animals. The developed biomaterial platform demonstrates significant potential as a regenerative treatment following tongue resection, as it facilitates both protein and cell-mediated therapy, and stimulates tongue muscle regeneration in vivo.

Phenotypic characterization of a new Grin1 mutant mouse generated by ENU mutagenesis.

In the RIKEN large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis project we screened mice with a dominant mutation that exhibited abnormal behavior in the open-field test, passive avoidance test and home-cage activity test. We tested 2045 progeny of C57BL/6J males treated with ENU and untreated DBA/2J females in the open-field test and isolated behavioral mutant M100174, which exhibited a significant increase in spontaneous locomotor activity. We identified a missense mutation in the Grin1 gene, which encodes NMDA receptor subunit 1, and designated the mutant gene Grin1(Rgsc174). This mutation results in an arginine to cysteine substitution in the C0 domain of the protein. Detailed analyses revealed that Grin1(Rgsc174) heterozygote exhibited increased novelty-seeking behavior and slight social isolation in comparison with the wild type. In contrast to other Grin1 mutant mice, this mutant exhibited no evidence of heightened anxiety. These results indicate that this is a unique behavioral Grin1 gene mutant mouse that differs from the known Grin1 mutant mice. The results of immunohistochemical and biochemical analyses suggested that impaired interaction between the glutamatergic pathway and dopaminergic pathway may underlie the behavioral phenotypes of the Grin1(Rgsc174) mutant.

Differential effects of PDE4 inhibitors on cortical neurons and T-lymphocytes.

Inhibitors of PDE4 (cAMP-specific phosphodiesterase) induce side effects, including nausea and emesis, that limit their therapeutic potential. We investigated the function of two catalytically active conformations of PDE4 (a low-affinity conformer detected by conventional cAMP hydrolytic activity and a high-affinity conformer detected by [(3)H]rolipram binding) in neuronal cells. We assessed enhancement of beta-adrenoceptor-mediated cAMP accumulation in cortical neurons in vitro by eleven PDE4 inhibitors with diverse biochemical profiles. The compounds tested have a wide inhibition range of PDE4 catalytic activity and [(3)H]rolipram binding. Inhibition potency for PDE4 catalytic activity and [(3)H]rolipram binding for each compound was different. Potency in augmentation of cAMP correlated significantly with the inhibitory effect on [(3)H]rolipram binding, but not with that against PDE4 catalytic activity. On the other hand, the inhibitory effect on proliferation of T-lymphocytes of the same PDE4 inhibitors correlated both with inhibition of PDE4 catalytic activity and with inhibition of [(3)H]rolipram binding. These findings indicate that the high affinity PDE4 conformer exists at a high level in cortical neurons and is important in the regulation of cAMP. Furthermore, the relative contributions of the two PDE4 conformers in cell function may cause different PDE4 inhibitor effects on cortical neurons and T-lymphocytes.

Excellent short-term results with steroid-free maintenance immunosuppression in low-risk simultaneous pancreas-kidney transplantation.

HYPOTHESIS: Steroid avoidance is possible in simultaneous pancreas-kidney transplantation with the use of newer immunosuppressive agents and induction therapy. DESIGN: A retrospective consecutive case review. SETTING: A university tertiary referral center. PATIENTS: Medical records of 40 consecutive patients who underwent pancreas-kidney transplantation from November 2000 to July 2002 were reviewed. INTERVENTION: The immunosuppression protocol used in this series of patients consisted of Thymoglobulin induction combined with mycophenolate mofetil, tacrolimus, and sirolimus for maintenance immunosuppression. Steroids were used as pretreatment only, given with Thymoglobulin, and were typically discontinued by postoperative week 1. MAIN OUTCOME MEASURES: Graft and patient survival rates, rejection rates of the kidney or pancreas, infection rates, and surgical complication rates. RESULTS: Patient, kidney, and pancreas survival rates were 95.0%, 92.5%, and 87.5%, respectively. Biopsy-proven pancreas rejection rates at 1 and 3 months' posttransplantation were 2.5%. Kidney rejection rates at 1 and 3 months were 2.5%. Steroids were given only to patients with documented transplant rejection. Surgical and medical complications were no different from earlier protocols. CONCLUSIONS: Immunosuppression protocols that do not include maintenance steroids have shown minimal rejection in the first 3 months and equivalent patient and graft survival rates compared with protocols that use steroids. The potential beneficial long-term impact of steroid avoidance will require further study.