Lu Cai

Background: Intermittent hypoxia (IH), as a key pathogenic factor of obstructive sleep apnea syndrome (OSAS), can cause many diseases, such as increased inflammation and oxidative stress, diabetes, cardiovascular disease, and Alzheimer's disease (AD). The response of cells to hypoxia involves multiple levels of regulatory mechanisms, including transcriptional regulation of gene expression, regulation of mRNA stability, post‐transcriptional regulation, and post‐translational modification regulation. Aims: The regulation of miRNA and alternative splicing (AS) in neuronal response to intermittent hypoxia deserve further study. Materials & Methods: By establishing a mouse model of intermittent hypoxia, we conducted functional studies on key miRNAs and splicing factor using methods such as miRNA sequencing, bioinformatics, and molecular biology. Results: In the mouse hippocampus, intermittent hypoxia altered the expression of many miRNAs, with miR‐448‐3p and miR‐1264‐3p changing over the course of more than three time periods. Interestingly, the expression of Fam76b , the common target gene of these two miRNAs, also changed under intermittent hypoxia. Further studies showed that Fam76b may regulate the ratio of Nbr1 and Dph3 transcripts in response to hypoxia by affecting the localization of hnRNPA2B1 protein within cells. Discussion: Research into intermittent hypoxia‐induced disorders, including Alzheimer's disease and other neurodegenerative diseases, might benefit from a better understanding of the regulatory mechanisms of miRNA and alternative splicing in hypoxic response at the animal and cell levels. Conclusion: This study demonstrates that intermittent hypoxia alters the expression of miR‐448‐3p and miR‐1264‐3p, as well as the localization of the splicing factor hnRNPA2B1 in the cell nucleus. These findings enhance our understanding of the molecular mechanisms of neuronal responses to hypoxia and hold potential implications for treating hypoxia‐related diseases like Alzheimer's disease.

Hypoxia can cause a variety of diseases, including ischemic stroke and neurodegenerative diseases. Within a certain range of partial pressure of oxygen, cells can respond to changes in oxygen. Changes in oxygen concentration beyond a threshold will cause damage or even necrosis of tissues and organs, especially for the central nervous system. Therefore, it is very important to find appropriate measures to alleviate damage. MiRNAs can participate in the regulation of hypoxic responses in various types of cells. MiRNAs are involved in regulating hypoxic responses in many types of tissues by activating the hypoxia-inducible factor (HIF) to affect angiogenesis, glycolysis and other biological processes. By analyzing differentially expressed miRNAs in hypoxia and hypoxia-related studies, as well as the HT22 neuronal cell line under hypoxic stress, we found that the expression of miR-18a was changed in these models. MiR-18a could regulate glucose metabolism in HT22 cells under hypoxic stress by directly regulating the 3’UTR of the Hif1a gene. As a small molecule, miRNAs are easy to be designed into small nucleic acid drugs, so this study can provide a theoretical basis for the research and treatment of nervous system diseases caused by hypoxia. Graphical Abstract

Nucleosomes not only serve as the basic building blocks for eukaryotic chromatin but also regulate many biological processes, such as DNA replication, repair, and recombination. To modulate gene expression in vivo, the histone variant H2A.Z can be dynamically incorporated into the nucleosome. However, the assembly dynamics of H2A.Z-containing nucleosomes remain elusive. Here, we demonstrate that our previous chemical kinetic model for nucleosome assembly can be extended to H2A.Z-containing nucleosome assembly processes. The efficiency of H2A.Z-containing nucleosome assembly, like that of canonical nucleosome assembly, was also positively correlated with the total histone octamer concentration, reaction rate constant, and reaction time. We expanded the kinetic model to represent the competitive dynamics of H2A and H2A.Z in nucleosome assembly, thus providing a novel method through which to assess the competitive ability of histones to assemble nucleosomes. Based on this model, we confirmed that histone H2A has a higher competitive ability to assemble nucleosomes in vitro than histone H2A.Z. Our competitive kinetic model and experimental results also confirmed that in vitro H2A.Z-containing nucleosome assembly is governed by chemical kinetic principles.

Multiple tumor exacerbations and treatment procedures, such as extracellular matrix remodeling, metabolic reprogramming, immunological evasion, and resistance to chemotherapy and radiotherapy, are influenced by intratumoral hypoxia. It is becoming increasingly clear how hypoxia interacts with the extracellular matrix and how this affects the growth of cancer. We analyzed the published sequencing results of hypoxia-stressed mouse kidney tumor cells and found that the expression of miR-29b was significantly downregulated. There are several sites that are complementary to the miR-29 seed sequence in the 3’ non-coding regions (3’UTRs) of various extracellular matrix-related genes, including collagen IV. We analyzed the sequences of the 3’UTRs of different subunits of collagen IV in different species and constructed the corresponding phylogenetic trees. We found that the 3’UTRs of Col4a1 and Col4a4 may have been subjected to particular evolutionary pressures. By cloning the 3’UTRs of collagen IV subunits into the psiCHECK^TM-2 vector, we found that seven of the eight sites in the Col4a3–Col4a6 gene complementary to miR-29 were significantly repressed by miR-29a, b (except for the 7774–7781 of Col4a3 gene). The inhibitory efficiency of miR-29a, b on these seven sites was between 27% and 57%. The research on the regulation of miR-29 and extracellular matrix by hypoxia can provide a theoretical basis for tumor and fibrosis research and treatment.

As the elementary unit of eukaryotic chromatin, nucleosomes in vivo are highly dynamic in many biological processes, such as DNA replication, repair, recombination, or transcription, to allow the necessary factors to gain access to their substrate. The dynamic mechanism of nucleosome assembly and disassembly has not been well described thus far. We proposed a chemical kinetic model of nucleosome assembly and disassembly in vitro. In the model, the efficiency of nucleosome assembly was positively correlated with the total concentration of histone octamer, reaction rate constant and reaction time. All the corollaries of the model were well verified for the Widom 601 sequence and the six artificially synthesized DNA sequences, named CS1–CS6, by using the salt dialysis method in vitro. The reaction rate constant in the model may be used as a new parameter to evaluate the nucleosome reconstitution ability with DNAs. Nucleosome disassembly experiments for the Widom 601 sequence detected by Förster resonance energy transfer (FRET) and fluorescence thermal shift (FTS) assays demonstrated that nucleosome disassembly is the inverse process of assembly and can be described as three distinct stages: opening phase of the (H2A–H2B) dimer/(H3–H4)₂ tetramer interface, release phase of the H2A–H2B dimers from (H3–H4)₂ tetramer/DNA and removal phase of the (H3–H4)₂ tetramer from DNA. Our kinetic model of nucleosome assembly and disassembly allows to confirm that nucleosome assembly and disassembly in vitro are governed by chemical kinetic principles.

Exposure to specific doses of hypoxia can trigger endogenous neuroprotective and neuroplastic mechanisms of the central nervous system. These molecular mechanisms, together referred to as hypoxic preconditioning (HPC), remain poorly understood. In the present study, we applied RNA sequencing and bioinformatics analyses to study HPC in a whole-body HPC mouse model. The preconditioned (H4) and control (H0) groups showed 605 differentially expressed genes (DEGs), of which 263 were upregulated and 342 were downregulated. Gene Ontology enrichment analysis indicated that these DEGs were enriched in several biological processes, including metabolic stress and angiogenesis. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that the FOXO and Notch signaling pathways were involved in hypoxic tolerance and protection during HPC. Furthermore, 117 differential alternative splicing events (DASEs) were identified, with exon skipping being the dominant one (48.51%). Repeated exposure to systemic hypoxia promoted skipping of exon 7 in Edrf1 and exon 9 or 13 in Lrrc45. This study expands the understanding of the endogenous protective mechanisms of HPC and the DASEs that occur during HPC.