Genes to Cells最新文献

Lysosomes are acidic organelles that degrade a diverse range of substrates, and lysosome-associated membrane protein (LAMP)-1 and LAMP-2 are the major lysosomal membrane components. Three LAMP-2 splice variants have been identified, namely, LAMP-2A, LAMP-2B, and LAMP-2C. We previously demonstrated that when mouse LAMP-2C was stably expressed in HEK293 cells, a portion of it was present on the plasma membrane. LAMP-2C possesses a tyrosine-based motif that functions as a signal for lysosomal targeting and clathrin-mediated endocytosis (CME). However, whether cell surface LAMP-2C is indeed internalized via CME has not been clearly defined. If this occurs, it is unknown whether internalized LAMP-2C returns to the cell surface and/or moves to lysosomes from early endosomes. In this study, we found that cell surface LAMP-2C was internalized, and its internalization was impaired by knockdown of the clathrin heavy chain or the medium subunit of adaptor protein complex 2. Internalized LAMP-2C was transported to early endosomes, and a portion of the internalized LAMP-2C was recycled back to the plasma membrane. Furthermore, immunofluorescence and subcellular fractionation showed that the internalized LAMP-2C was transported to lysosomes. These results suggest that cell surface LAMP-2C is internalized by CME and that internalized LAMP-2C enters the recycling and lysosomal pathways.

Hepatocellular carcinoma (HCC) is a prevalent and deadly cancer worldwide, characterized by poor prognosis, multiple therapeutic challenges, and considerable heterogeneity among patients with diverse etiologies. This heterogeneity contributes to resistance to chemotherapies and molecularly targeted agents, posing a major therapeutic challenge. Therefore, there is an increasing need for treatment strategies targeting HCC across various biological processes. miR-192-5p has been reported to function as a tumor suppressor in HCC, but its target genes remain largely unknown. In this study, we aimed to identify novel target genes of miR-192-5p in HCC using RNA sequencing and 3′-untranslated region analysis. As a result, eight genes—EFEMP1, DLG5, PPP1CA, FAM234B, RPL4, SEC23B, ELOVL1, and CBFB—were identified as novel target genes of miR-192-5p, all of which were significantly upregulated in HCC tissues. Notably, three genes—CBFB, SEC23B, and RPL4—were also validated as novel targets of miR-194-5p, which clusters with miR-192-5p. These findings suggest that miR-192-5p exerts its tumor-suppressive function by inhibiting a novel gene network that may contribute to HCC progression. This study provides new insights into the molecular mechanisms underlying HCC heterogeneity and highlights miR-192-5p-regulated networks as potential therapeutic targets for HCC.

We created a novel replication-incompetent vector derived from the human parainfluenza virus type 2 (hPIV2). The vector lacks the hemagglutinin-neuraminidase (HN) gene (hPIV2/∆HN) that is essential for virus attachment and release. We also generated the stable packaging cell line expressing hPIV2 HN protein derived from Vero cells (Vero-HN). As SARS-CoV-2 vaccines, we created hPIV2/∆HN-based SARS-CoV-2 vaccines expressing the receptor binding domain of the spike protein (S-RBD) of SARS-CoV-2, and S-RBD with trimer formation domain (S-RBD-FD) with or without antigen 85B (Ag85B) having strong Th1-type cytokine inducing activity. Intratracheal administration of S-RBD-FD/hPIV2/∆HN and S-RBD-FD/Ag85B/hPIV2/∆HN induced the S-RBD-specific neutralizing antibodies in sera of transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2/TG), and also the sera neutralized the pseudo-typed lentivirus expressing the SARS-CoV-2 S protein. In particular, in an S-RBD-FD/Ag85B/hPIV2/∆HN-administered group, high levels of neutralizing antibodies were induced also in the bronchoalveolar lavage fluid (BALF) in the hACE2/TG mice. Further, hACE2/TG mice vaccinated with S-RBD-FD/hPIV2/∆HN or S-RBD-FD/Ag85B/hPIV2/∆HN did not exhibit weight loss and showed a high survival rate after authentic SARS-CoV-2 challenge. These results suggested that S-RBD-FD/Ag85B/hPIV2/∆HN is a vaccine candidate against SARS-CoV-2. Finally, hPIV2/∆HN or Ag85B/hPIV2/∆HN vector will be potentially applicable to vaccine development and/or human gene therapy for respiratory tract diseases.

We have established mouse heart organoids (mHOs) that are characterized by the presence of atrium- and ventricle-like structures that mimic entire embryonic hearts. However, maturation was not uniform, and little is known about their trabeculation status. Given the essential role of the trabeculae in cardiac morphogenesis, a new method that combines imaging analysis with a machine learning model was developed for quantifying the internal complexity in developing mHOs in a timely manner. We applied this method to screen for modified culture conditions and identified optimal treatment with valproic acid as a Notch activator and both bone morphogenetic protein 10 and Neuregulin 1 as downstream factors of Notch (a trabeculation-regulating signal) to promote mHO maturation. The established method using mouse fetal hearts as tests was suitable for comparing the internal complexity of both mHOs and mouse fetal hearts. The modified culture conditions improved the maturation uniformity as well as the internal structure of mHOs. Thus, this method can be applied to cardiac disorders with trabeculation problems and HOs developed by other methods. In addition, mHOs generated under these modified conditions may be an effective tool for studying the molecular mechanisms of heart development, including the signaling pathway of trabeculation related to these factors.

The RUNX3 gene is frequently involved in a variety of cancers and immunological diseases. Despite such widespread association with human diseases, the transcriptional regulation of RUNX3 remains elusive. Here we report the identification of an enhancer for Runx3, eR3(−18m/−28h), by employing a combination of in silico prediction and in vivo verification using zebrafish and mouse models. eR3 is active in CD8⁺CD103 (integrin α_E)⁺ cytotoxic T lymphocytes (CTLs) that reside in the intestinal epithelium via their interaction with the CD103 ligand, E-cadherin, on epithelial cells. Removal of eR3(−18m) specifically in CD8 T cells compromised the suppression of tumorigenesis in murine cancer models. In humans, single nucleotide polymorphisms (SNPs) in eR3(−28h) were overrepresented and were associated with weakened CTL activity in colorectal cancer patients. Together, our results indicate that eR3 plays a role in immune surveillance against gut-associated tumors by upregulating RUNX3 expression in specific CTLs.

The neuronal ELAV protein HuD is known to promote neuronal differentiation and stimulate cap-dependent translation, but the underlying mechanism remains incompletely understood. Here, we identify a functional domain within the HuD linker that governs interaction with the nuclear export factor TAP/NXF1 and stimulates translation. Using a series of deletion mutants, we show that TAP/NXF1 binding is functionally separable from cytoplasmic localization, and that loss of this interaction correlates with impaired translation and neurite outgrowth. These findings reveal a critical subdomain within HuD that recruits TAP/NXF1 to promote translation and neuronal differentiation, establishing a mechanistic link between RNA export factors and ELAV-mediated translational control.

Broccoli-derived 3,3′-diindolylmethane (DIM) exhibits anticancer effects. The compound also inhibits the growth of fission yeast cells. Reduction of mitochondrial translation alleviates the growth defects caused by DIM in fission yeast; however, the underlying molecular mechanisms remain unclear. In this study, we show that DIM-induced reactive oxygen species (ROS) colocalized with mitochondria. Deletion of tsf1⁺, which leads to reduced mitochondrial translation, suppressed this colocalization. Deletions of stress response genes, such as sty1⁺, pap1⁺, and atf1⁺, increased DIM sensitivity. Growth defects in the wild-type and sty1, pap1, and atf1 disruptants in the presence of DIM were suppressed by the ROS scavenger N-acetylcysteine. Moreover, the ROS scavenger Sod1, which is suggested to function in the mitochondrial intermembrane space and cytoplasm, was important for survival in the presence of DIM. Collectively, the study results suggest that DIM increases ROS levels in mitochondria and suppression of ROS increase in mitochondria via inhibition of mitochondrial translation is the mechanism by which DIM-induced growth defects in wild-type cells are suppressed. Overall, the study highlights the potential use of DIM as an anticancer drug to increase ROS generation in mitochondria in cancer cells.

Voltage-dependent calcium channels (VDCCs) have previously been thought to be functional in excitable cells, but recently accumulating evidence suggests that they are also functional in non-excitable cells such as microglia. In the present study, we investigated the role of the N-type VDCC (Cav2.2) in macrophages, a kind of non-excitable cell, in a mouse model of inflammatory bowel disease (IBD). The dextran sodium sulfate (DSS) colitis model, a widely used chemically induced model of IBD, was applied to Cav2.2 knockdown (KD) mice, where Cav2.2 expression in macrophages can be downregulated by the application of tamoxifen. After the 7 days of oral administration of 2.5% DSS, Cav2.2KD mice had a significantly higher disease activity index for colitis compared to wild-type (WT) mice, and histological examination of the colon from DSS-treated mice also suggested more severe intestinal damage in Cav2.2KD mice. Moreover, the densities of Iba1⁺ cells (macrophages/dendritic cells) in the colon were also elevated in Cav2.2KD mice. TNFα levels were significantly higher in Cav2.2KD mice as revealed by ELISA experiments. Collectively, these data suggest that the Cav2.2KD mice had more severe colonic inflammation compared to WT mice. Therefore, Cav2.2 in macrophages may play some roles in suppressing inflammation in the intestinal immune system.