Molecular and Cellular Biology最新文献

Transcription factor 12 (TCF12) is a known oncogene in many cancers. However, whether TCF12 can regulate malignant phenotypes and angiogenesis in osteosarcoma is not elucidated. In this study, we demonstrated increased expression of TCF12 in osteosarcoma tissues and cell lines. High TCF12 expression was associated with metastasis and poor survival rate of osteosarcoma patients. Knockdown of TCF12 reduced the proliferation, migration, and invasion of osteosarcoma cells. TCF12 was found to bind to the promoter region of sphingosine kinase 1 (SPHK1) to induce transcriptional activation of SPHK1 expression and enhance the secretion of sphingosine-1-phosphate (S1P), which eventually resulted in the malignant phenotypes of osteosarcoma cells. In addition, S1P secreted by osteosarcoma cells promoted the angiogenesis of HUVECs by targeting S1PR4 on the cell membrane to activate the STAT3 signaling pathway. These findings suggest that TCF12 may induce transcriptional activation of SPHK1 to promote the synthesis and secretion of S1P. This process likely enhances the malignant phenotypes of osteosarcoma cells and induces angiogenesis via the S1PR4/STAT3 signaling pathway.

The human Origin Recognition Complex (ORC) is required not only for the initiation of DNA replication, but is also implicated in diverse cellular functions, including chromatin organization, centrosome biology, and cytokinesis. The smallest subunit of ORC, Orc6, is poorly conserved amongst eukaryotes. Recent studies from our laboratory have suggested that human Orc6 is not required for replication licensing, but is needed for S-phase progression. Further, ATR-dependent phosphorylation of Orc6 at T229 is implicated in DNA damage response during S-phase. In this study, we demonstrate that the CDK-dependent phosphorylation of Orc6 at T195 occurs during mitosis. While the phosphorylation at T195 does not seem to be required to exit mitosis, cells expressing the phosphomimetic T195E mutant of Orc6 impede S-phase progression. Moreover, the phosphorylated form of Orc6 associates with ORC more robustly, and Orc6 shows enhanced association with the ORC outside of G1, supporting the view that Orc6 may prevent the role of Orc1-5 in licensing outside of G1. Finally, Orc6 and the phosphorylated Orc6 localize to the nucleolar organizing centers and regulate ribosome biogenesis. Our results suggest that phosphorylated Orc6 at T195 prevents replication.

Myocardial infarction (MI) seriously threatens the health of elderly people, and reducing myocardial injury is of great significance for the treatment of MI. LncRNA-TTN-AS1 shows protective effects on cardiomyocyte injury, while the role of TTN-AS1 in MI remains unknown. CCK8, flow cytometry, and JC-1 staining assessed cell viability, apoptosis and mitochondrial membrane potential (MMP), respectively. Cellular reactive oxygen species (ROS) and secreted lactate dehydrogenase (LDH) levels were measured. The interactions between ELF5, TTN-AS1, PCBP2 and CDK6 were explored using ChIP, luciferase reporter assay, RIP, and pull-down. The severity of MI in mice was evaluated using TTC, H&E, and TUNEL staining. The data revealed that OGD/R significantly induced ROS, mitochondrial injury and apoptosis in AC16 cells, while overexpression of ELF5 or TTN-AS1 reversed these phenomena. ELF5 transcriptionally activated TTN-AS1 through binding with its promoter. TTN-AS1 increased CDK6 stability via recruiting PCBP2. CDK6 knockdown abolished the inhibitory effects of TTN-AS1 overexpression on OGD/R-induced myocardial injury. Furthermore, overexpression of TTN-AS1 or ELF5 alleviated MI progression in mice by upregulating CDK6. Collectively, TTN-AS1 transcriptionally regulated by ELF5 alleviated myocardial apoptosis and injury during MI via recruiting PCBP2 to increase CDK6 stability, which shed new lights on exploring new strategies against MI.

The obligate intracellular pathogen Chlamydia trachomatis has unique metabolic requirements as it proceeds through its biphasic developmental cycle from within the inclusion within the host cell. In our previous study, we identified a host protein, PICALM, which localizes to the chlamydial inclusion. PICALM functions in many host pathways including the recycling of receptors, specific SNARE proteins, and molecules like transferrin, and maintaining cholesterol homeostasis. Hence, we hypothesized that PICALM functions to maintain the cholesterol content and to moderate trafficking from the endosomal recycling pathway to the inclusion, which controls chlamydial access to this pathway. In uninfected cells, siRNA knockdown of PICALM resulted in increased cholesterol within the Golgi and transferrin receptor (TfR) positive vesicles (recycling endosomes). PICALM knockdown in cells infected with C. trachomatis resulted in increased levels of Golgi-derived lipid and protein, TfR, transferrin, and Rab11-FIP1 localized to inclusions and a decrease of Golgi fragmentation at and Rab11 trafficking to the inclusion. Interestingly, chlamydial infection alone also increases cholesterol in TfR and Rab11-associated vesicles, and PICALM knockdown reverses this effect. Our data suggest that PICALM functions to balance or limit chlamydial access to multiple subcellular trafficking pathways to maintain the health of the host cell during chlamydial infection.

Spinocerebellar ataxias (SCAs) are autosomal dominant diseases characterized by cerebellar atrophy and ataxia. The SCA subtype SCA34 is caused by specific mutations in the gene ELOVL4, which encodes a fatty acid (FA) elongase that synthesizes ultra-long-chain (ULC; ≥C26) FAs. However, the pathogenesis and molecular mechanism that confers dominant inheritance remains unknown. Here, a cell-based assay demonstrated that each of the five known SCA34 mutants produced shorter ULC polyunsaturated FA-containing phosphatidylcholines (ULC-PCs) than wild-type protein, in the following order of severity: Q180P and T233M > W246G > I171T and L168F. Next, we generated knock-in mouse embryonic stem cells that contained heterozygous Q180P, heterozygous W246G, or homozygous W246G mutations. Neuronal differentiation-dependent production of ULC-PCs was reduced in heterozygous Q180P and homozygous W246G cells relative to control cells, and we observed shortening of the FA moiety in all mutant cells. This FA shortening was consistent with our prediction that amino acid residues substituted by SCA34 mutations are located in the transmembrane helices that interact with the ω-end region of the FA moiety of the substrate acyl-CoA. Hence, reduced levels and shortening of ULC-PCs in neurons may cause SCA34, and incomplete elongation of ULC polyunsaturated acyl-CoAs by mutated ELOVL4 may induce dominant inheritance.

Cancer-associated fibroblasts (CAFs)-derived exosomes have emerged as a key driver of ovarian cancer (OVCA) tumor progression. The mechanisms behind the specific circular RNA (circRNA) activity encapsulated by CAF-generated exosomes (CAF-exo) requires to be elucidated. Herein, this study selected specific circRNA (hsa_circIFNGR2) molecules and aimed to clarify novel function of CAF-derived exosomal circIFNGR2 on growth, and metastasis of OVCA cells. In this study, we clarified that the exosomes of CAFs originating from human ovarian cancer hindered tumor cell proliferation, metastasis and EMT in vitro. Interestingly, CAFs directly transferred exosomes into OVCA cells to enrich intracellular circIFNGR2 levels. Biologically, activation of exosomal circIFNGR2 blocked cell proliferation, metastasis and EMT. Mechanistically, enhanced circIFNGR2 activated the miR-378/ST5 axis and directly inhibited the malignant evolution of tumor cells. Furthermore, rescue experiments evidenced that circIFNGR2 and ST5 were two essential participants in OVCA, concretely manifested in the co-culture of OVCA cells with exosomes that reversed the effects of intracellular circIFNGR2 and ST5 depletion. Finally, we observed that CAF-exo treatment hindered tumor growth and increased the size and number of metastatic nodules in mice. Our study revealed a previously unknown regulatory pathway whereby CAFs-derived exosomes delivered circIFNGR2 and inhibited the malignant progression of OVCA by circIFNGR2/miR-378/ST5 axis.

Numerous proteins are sumoylated in normally growing yeast and SUMO conjugation levels rise upon exposure to several stress conditions. We observe high levels of sumoylation also during early exponential growth and when nutrient-rich medium is used. However, we find that reduced sumoylation (∼75% less than normal) is remarkably well-tolerated, with no apparent growth defects under nonstress conditions or under osmotic, oxidative, or ethanol stresses. In contrast, strains with reduced activity of Ubc9, the sole SUMO conjugase, are temperature-sensitive, implicating sumoylation in the heat stress response, specifically. Aligned with this, a mild heat shock triggers increased sumoylation which requires functional levels of Ubc9, but likely also depends on decreased desumoylation, since heat shock reduces protein levels of Ulp1, the major SUMO protease. Furthermore, we find that a ubc9 mutant strain with only ∼5% of normal sumoylation levels shows a modest growth defect, has abnormal genomic distribution of RNA polymerase II (RNAPII), and displays a greatly expanded redistribution of RNAPII after heat shock. Together, our data implies that SUMO conjugations are largely dispensable under normal conditions, but a threshold level of Ubc9 activity is needed to maintain transcriptional control and to modulate the redistribution of RNAPII and promote survival when temperatures rise.

Glutamyl-prolyl-tRNA synthetase 1 (EPRS1) is known to associated with fibrosis through its catalytic activity to produce prolyl-tRNA. Although its catalytic inhibitor halofuginone (HF) has been known to inhibit the TGF-β pathway as well as to reduce prolyl-tRNA production for the control of fibrosis, the underlying mechanism how EPRS1 regulates the TGF-β pathway was not fully understood. Here, we show a noncatalytic function of EPRS1 in controlling the TGF-β pathway and hepatic stellate cell activation via its interaction with TGF-β receptor I (TβRI). Upon stimulation with TGF-β, EPRS1 is phosphorylated by TGF-β-activated kinase 1 (TAK1), leading to its dissociation from the multi-tRNA synthetase complex and subsequent binding with TβRI. This interaction increases the association of TβRI with SMAD2/3 while decreases that of TβRI with SMAD7. Accordingly, EPRS1 stabilizes TβRI by preventing the ubiquitin-mediated degradation of TβRI. HF disrupts the interaction between EPRS1 and TβRI, and reduces TβRI protein levels, leading to inhibition of the TGF-β pathway. In conclusion, this work suggests the novel function of EPRS1 involved in the development of fibrosis by regulating the TGF-β pathway and the antifibrotic effects of HF by controlling both of EPRS1 functions.

Recently, the diverse functions of microRNAs (miRNAs) in brain diseases have been demonstrated. We intended to uncover the functional role of microRNA-130b (miR-130b) in cerebral vasospasm (CVS) following subarachnoid hemorrhage (SAH). SAH was induced by injecting the autologous blood into the cisterna magna of Sprague Dawley rats. The cerebral vascular smooth muscle cells (cVSMCs) were extracted for in vitro experimentation. In vitro and in vivo assays were implemented with transfection of miR-130b mimic/inhibitor, sh-Kruppel-like factor 4 (KLF4), oe-KLF4 plasmids or p38/MAPK signaling pathway agonist (anisomycin), respectively, to elaborate the role of miR-130b in CVS following SAH. Elevated miR-130b and reduced KLF4 were found in SAH patients and rat models of SAH. KLF4 was the target gene of miR-130b. miR-130b promoted the proliferation and migration of cVSMCs through the Inhibition of KLF4. Besides, KLF4 inhibited the proliferation and migration of cVSMCs through blockage of the p38/MAPK pathway. Furthermore, in vivo assay confirmed the inhibitory effect of decreased miR-130b in CVS following SAH. In conclusion, miR-130b may activate the p38/MAPK signaling pathway through targeted inhibition of KLF4, thereby contributing to some extent to the development of cerebral vasospasm after SAH.