Identification of targets of tumor suppressor microRNA-34a using a reporter library system

miRNAs play critical roles in various biological processes by targeting specific mRNAs. Current approaches to identifying miRNA targets are insufficient for elucidation of a miRNA regulatory network. Here, we created a cell-based screening system using a luciferase reporter library composed of 4,891 full-length cDNAs, each of which was integrated into the 3′ UTR of a luciferase gene. Using this reporter library system, we conducted a screening for targets of miR-34a, a tumor-suppressor miRNA. We identified both previously characterized and previously uncharacterized targets. miR-34a overexpression in MDA-MB-231 breast cancer cells repressed the expression of these previously unrecognized targets. Among these targets, GFRA3 is crucial for MDA-MB-231 cell growth, and its expression correlated with the overall survival of patients with breast cancer. Furthermore, GFRA3 was found to be directly regulated by miR-34a via its coding region. These data show that this system is useful for elucidating miRNA functions and networks

Functional analysis of RRAS2 pathogenic variants with a Noonan-like phenotype

Introduction: RRAS2, a member of the R-Ras subfamily of Ras-like low-molecular-weight GTPases, is considered to regulate cell proliferation and differentiation via the RAS/MAPK signaling pathway. Seven RRAS2 pathogenic variants have been reported in patients with Noonan syndrome; however, few functional analyses have been conducted. Herein, we report two patients who presented with a Noonan-like phenotype with recurrent and novel RRAS2 pathogenic variants (p.Gly23Val and p.Gly24Glu, respectively) and the results of their functional analysis.Materials and methods: Wild-type (WT) and mutant RRAS2 genes were transiently expressed in Human Embryonic Kidney293 cells. Expression of RRAS2 and phosphorylation of ERK1/2 were confirmed by Western blotting, and the RAS signaling pathway activity was measured using a reporter assay system with the serum response element-luciferase construct. WT and p.Gly23Val RRAS2 were expressed in Drosophila eye using the glass multiple reporter-Gal4 driver. Mutant mRNA microinjection into zebrafish embryos was performed, and the embryo jaws were observed.Results: No obvious differences in the expression of proteins WT, p.Gly23Val, and p.Gly24Glu were observed. The luciferase reporter assay showed that the activity of p.Gly23Val was 2.45 ± 0.95-fold higher than WT, and p.Gly24Glu was 3.06 ± 1.35-fold higher than WT. For transgenic flies, the p.Gly23Val expression resulted in no adults flies emerging, indicating lethality. For mutant mRNA-injected zebrafish embryos, an oval shape and delayed jaw development were observed compared with WT mRNA-injected embryos. These indicated hyperactivity of the RAS signaling pathway.Discussion: Recurrent and novel RRAS2 variants that we reported showed increased in vitro or in vivo RAS signaling pathway activity because of gain-of-function RRAS2 variants. Clinical features are similar to those previously reported, suggesting that RRAS2 gain-of-function variants cause this disease in patients

Sensitive and Rapid Detection of Edwardsiellosis in Fish by a Loop-Mediated Isothermal Amplification Method

Here we report a rapid and sensitive method (using loop-mediated isothermal amplification [LAMP]) for the diagnosis of edwardsiellosis, a fish disease caused by Edwardsiella tarda, in Japanese flounder. A set of four primers was designed, and conditions for the detection were optimized for the detection of E. tarda in 45 min at 65°C. No amplification of the target hemolysin gene was detected in other related bacteria. When the LAMP primers were used, detection of edwardsiellosis in infected Japanese flounder kidney, and spleen and seawater cultures was possible. We have developed a rapid and sensitive diagnostic protocol for edwardsiellosis detection in fish. This is the first report of the application of LAMP for the diagnosis of a fish pathogen

Two cases of pelvic hematoma after prostatic urethral lift surgery

Introduction There are few reports of pelvic hematoma after prostatic urethral lift. Here, we report two cases of pelvic hematoma in Japan. Case presentation The first case was a 71‐year‐old man with benign prostatic hyperplasia who underwent prostatic urethral lift. Although the procedure was uneventful, he experienced lower abdominal pain the day after the operation. CT revealed a hematoma in the right pelvis; however, it was manageable with conservative treatment. The second case was a 68‐year‐old man. The procedure was uneventful; however, 6 days after the operation, a subcutaneous hematoma appeared in the lower abdomen. CT revealed a hematoma in the left pelvis. We then performed pelvic hematoma removal surgery. Conclusions Pelvic hematomas after PUL may requires attention, particularly in men with the narrow pelvises. Appropriate compression of the prostate and a high lithotomy position procedure could effectively avoid the occurrence of pelvic hematomas

Conditional deletion of CD98hc inhibits osteoclast development

The CD98 heavy chain (CD98hc) regulates virus-induced cell fusion and monocyte fusion, and is involved in amino acid transportation. Here, we examined the role that CD98hc plays in the formation of osteoclasts using CD98hcflox/floxLysM-cre peritoneal macrophages (CD98hc-defect macrophages). Peritoneal macrophages were stimulated with co-cultured with osteoblasts in the presence of 1,25(OH)2 vitamin D3, and thereafter stained with tartrate-resistant acid phosphatase staining solution. The multinucleated osteoclast formation was severely impaired in the peritoneal macrophages isolated from the CD98hc-defect mice compared with those from wild-type mice. CD98hc mediates integrin signaling and amino acid transport through the CD98 light chain (CD98lc). In integrin signaling, suppression of the M-CSF-RANKL-induced phosphorylation of ERK, Akt, JNK and p130Cas were observed at the triggering phase in the CD98h-defect peritoneal macrophages. Moreover, we showed that the general control non-derepressible (GCN) pathway, which was activated by amino acid starvation, was induced by the CD98hc-defect peritoneal macrophages stimulated with RANKL. These results indicate that CD98 plays two important roles in osteoclast formation through integrin signaling and amino acid transport

Targeted gene deletion of miRNAs in mice by TALEN system.

Mice are among the most valuable model animal species with an enormous amount of heritage in genetic modification studies. However, targeting genes in mice is sometimes difficult, especially for small genes, such as microRNAs (miRNAs) and targeting genes in repeat sequences. Here we optimized the application of TALEN system for mice and successfully obtained gene targeting technique in mice for intergenic region and series of microRNAs. Microinjection of synthesized RNA of TALEN targeting each gene in one cell stage of embryo was carried out and injected oocytes were transferred into pseudopregnant ICR female mice, producing a high success rate of the targeted deletion of miRNA genes. In our condition, TALEN RNA without poly(A) tail worked better than that of with poly(A) tail. This mutated allele in miRNA was transmitted to the next generation, suggesting the successful germ line transmission of this targeting method. Consistent with our notion of miRNAs maturation mechanism, in homozygous mutant mice of miR-10a, the non- mutated strand of miRNAs expression was completely diminished. This method will lead us to expand and accelerate our genetic research using mice in a high throughput way

Potential roles of DNA methylation in the initiation and establishment of replicative senescence revealed by array-based methylome and transcriptome analyses

<div><p>Cellular senescence is classified into two groups: replicative and premature senescence. Gene expression and epigenetic changes are reported to differ between these two groups and cell types. Normal human diploid fibroblast TIG-3 cells have often been used in cellular senescence research; however, their epigenetic profiles are still not fully understood. To elucidate how cellular senescence is epigenetically regulated in TIG-3 cells, we analyzed the gene expression and DNA methylation profiles of three types of senescent cells, namely, replicatively senescent, <i>ras</i>-induced senescent (RIS), and non-permissive temperature-induced senescent SVts8 cells, using gene expression and DNA methylation microarrays. The expression of genes involved in the cell cycle and immune response was commonly either down- or up-regulated in the three types of senescent cells, respectively. The altered DNA methylation patterns were observed in replicatively senescent cells, but not in prematurely senescent cells. Interestingly, hypomethylated CpG sites detected on non-CpG island regions (“open sea”) were enriched in immune response-related genes that had non-CpG island promoters. The integrated analysis of gene expression and methylation in replicatively senescent cells demonstrated that differentially expressed 867 genes, including cell cycle- and immune response-related genes, were associated with DNA methylation changes in CpG sites close to the transcription start sites (TSSs). Furthermore, several miRNAs regulated in part through DNA methylation were found to affect the expression of their targeted genes. Taken together, these results indicate that the epigenetic changes of DNA methylation regulate the expression of a certain portion of genes and partly contribute to the introduction and establishment of replicative senescence.</p></div

Podocyte-specific deletion of tubular sclerosis complex 2 promotes focal segmental glomerulosclerosis and progressive renal failure.

Obesity can initiate and accelerate the progression of kidney diseases. However, it remains unclear how obesity affects renal dysfunction. Here, we show that a newly generated podocyte-specific tubular sclerosis complex 2 (Tsc2) knockout mouse model (Tsc2Δpodocyte) develops proteinuria and dies due to end-stage renal dysfunction by 10 weeks of age. Tsc2Δpodocyte mice exhibit an increased glomerular size and focal segmental glomerulosclerosis, including podocyte foot process effacement, mesangial sclerosis and proteinaceous casts. Podocytes isolated from Tsc2Δpodocyte mice show nuclear factor, erythroid derived 2, like 2-mediated increased oxidative stress response on microarray analysis and their autophagic activity is lowered through the mammalian target of rapamycin (mTOR)-unc-51-like kinase 1 pathway. Rapamycin attenuated podocyte dysfunction and extends survival in Tsc2Δpodocyte mice. Additionally, mTOR complex 1 (mTORC1) activity is increased in podocytes of renal biopsy specimens obtained from obese patients with chronic kidney disease. Our work shows that mTORC1 hyperactivation in podocytes leads to severe renal dysfunction and that inhibition of mTORC1 activity in podocytes could be a key therapeutic target for obesity-related kidney diseases