Cooperation of long noncoding RNA LOC100909675 and transcriptional regulator CTCF modulates Cdk1 transcript to control astrocyte proliferation.

Astrocyte activation and proliferation contribute to glial scar formation during spinal cord injury (SCI), which limits nerve regeneration. The long noncoding RNAs (lncRNAs) are involved in astrocyte proliferation and act as novel epigenetic regulators. Here, we found that lncRNA-LOC100909675 (LOC9675) expression promptly increased after SCI and that reducing its expression decreased the proliferation and migration of the cultured spinal astrocytes. Depletion of LOC9675 reduced astrocyte proliferation and facilitated axonal regrowth after SCI. LOC9675 mainly localized in astrocytic nuclei. We used RNA-seq to analyze gene expression profile alterations in LOC9675-depleted astrocytes and identified the cyclin-dependent kinase 1 (Cdk1) gene as a hub candidate. Our RNA pull-down and RNA immunoprecipitation assays showed that LOC9675 directly interacted with the transcriptional regulator CCCTC-binding factor (CTCF). Dual-luciferase reporter and chromatin immunoprecipitation assays, together with downregulated/upregulated expression investigation, revealed that CTCF is a novel regulator of the Cdk1 gene. Interestingly, we found that with the simultaneous overexpression of CTCF and LOC9675 in astrocytes, the Cdk1 transcript was restored to the normal level. We then designed the deletion construct of LOC9675 by removing its interacting region with CTCF and found this effect disappeared. A transcription inhibition assay using actinomycin D revealed that LOC9675 could stabilize Cdk1 mRNA, while LOC9675 depletion or binding with CTCF reduced Cdk1 mRNA stability. These data suggest that the cooperation between CTCF and LOC9675 regulates Cdk1 transcription at a steady level, thereby strictly controlling astrocyte proliferation. This study provides a novel perspective on the regulation of the Cdk1 gene transcript by lncRNA LOC9675.

GCN5/KAT2A contributes to axon growth and neurogenesis.

Posttranslational modification (PTM) of tubulin proteins is involved in microtubule dynamics. Acetylation, an important alpha-tubulin PTM, which is regarded as a hallmark event of stable microtubules, often occurs in neurogenesis and axon outgrowth. GCN5/KAT2A is a well-known histone acetyltransferase and has also been reported to hold the activity of nonhistone acetyltransferases, such as acetylated tubulin (Ace-tubulin). In this study, we investigated the role of GCN5/KAT2A in axon growth and neurogenesis. E18 cortical neurons obtained from day 18 embryos of pregnant Sprague-Dawley (SD) rats were cultured and transfected with GCN5 siRNA or treated with the GCN5 inhibitor MB-3. Neural stem cells (NSCs) derived from the cerebral cortexes of E14 SD rats were cultured and differentiated. During differentiation, MB-3 was applied to investigate the effect of GCN5 dysfunction on neurogenesis. The axonal length and the ratio and distribution of acetylated and tyrosinated tubulin (Tyr-tubulin) were evaluated by immunostaining assay. The expression levels of Nestin, Tuj1, acetylated tubulin, and tyrosinated tubulin proteins were analyzed by Western blotting assays. In primary neurons, both GCN5 siRNA and MB-3 treatment reduced acetylated tubulin protein, changed the ratio of acetylated and tyrosinated tubulin, and decreased axonal length. During NSC differentiation, MB-3 application reduced axon outgrowth, decreased acetylated tubulin and altered the distribution of acetylated tubulin and tyrosinated tubulin. This study revealed for the first time that the acetyltransferase GCN5/KAT2A could contribute to axon outgrowth by altering the ratio and distribution of acetylated tubulin.

DNMT1/miR-130a/ZEB1 Regulatory Pathway Affects the Inflammatory Response in Lipopolysaccharide-Induced Sepsis.

Sepsis is a global health care issue that affects millions of people. DNA methyltransferase I (DNMT1)-mediated DNA methylation is involved in a number of human diseases by affecting many types of cellular progression events. However, the role and underlying molecular mechanism of DNMT1 in development of sepsis remain largely unknown. Lipopolysaccharide (LPS) induced lung fibrosis in the sepsis mouse model, and DNMT1 was upregulated in lung tissues of a sepsis mouse model compared with lung tissues from control mice. Then, this study demonstrated that LPS induced the production of interleukin (IL)-7 and tumor necrosis factor (TNF)-α and promoted DNMT1 expression in primary type II alveolar epithelial cells (AECII cells). Knockdown of DNMT1 inhibited IL-7 and TNF-α secretion in AECII cells exposed to LPS. Further study demonstrated that DNMT1 repressed the expression of miR-130a in AECII cells with or without LPS exposure. Next, this study demonstrated that miR-130a inhibited ZEB1 expression in AECII cells exposed to LPS. Ultimately, this study revealed the role of the DNMT1/miR-130a/ZEB1 regulatory pathway in AECII cells exposed to LPS. Overall, our data revealed that LPS induced the secretion of inflammatory factors by modulating the DNMT1/miR-130a/ZEB1 regulatory pathway in AECII cells, thus providing a novel theoretical basis that might be beneficial for establishment of diagnostic and therapeutic strategies for sepsis.