LncRNA CALML3-AS1 modulated by m6A modification induces BTNL9 methylation to drive non-small-cell lung cancer progression.

Non-small cell lung cancer (NSCLC) is a common and lethal malignancy. The carcinogenic roles of lncRNA CALML3 antisense RNA 1 (CALML3-AS1) have been documented. However, the function and potential mechanisms of CALML3-AS1 in the progression of NSCLC need to be further explored. The molecule expression was assessed by qRT-PCR and Western blot. The subcellular localization of CALML3-AS1 was observed by fluorescence in situ hybridization (FISH). The malignant behaviors of NSCLC cells were evaluated by CCK-8, colony formation, EdU, wound healing and transwell assays. In vivo xenograft tumor and liver metastatic models were established. The molecular mechanisms were investigated by RIP, RNA pull-down and ChIP assays. The methylation level was detected by MSP. Herein, we found that CALML3-AS1 was upregulated, while butyrophilin-like 9 (BTNL9) was downregulated in NSCLC. Functionally, CALML3-AS1 depletion repressed NSCLC cell malignant phenotypes, in vivo tumor growth, and liver metastasis. Mechanistically, AlkB homolog 5 (ALKBH5) enhanced CALML3-AS1 stability via N6-methyladenosine (m6A) demethylation, whereas m6A reader YTH domain-containing 2 (YTHDC2) destabilized CALML3-AS1. Moreover, CALML3-AS1 inhibited BTNL9 transcription and expression through the recruitment of Zeste homolog 2 (EZH2). Rescue experiments demonstrated that BTNL9 downregulation counteracted sh-CALML3-AS1-mediated antitumor effects on NSCLC. Taken together, CALML3-AS1 modulated by ALKBH5 and YTHDC2 in an m6A modification dependent manner drives NSCLC progression via epigenetically repressing BTNL9.

ATF5 Attenuates the Secretion of Pro-Inflammatory Cytokines in Activated Microglia.

The highly dynamic changes in microglia necessary to achieve a rapid neuroinflammatory response require a supply of energy from mitochondrial respiration, which leads to the accumulation of unfolded mitochondrial proteins. We previously reported that microglial activation is correlated with the mitochondrial unfolded protein response (UPRmt) in a kaolin-induced hydrocephalus model, but we still do not know the extent to which these changes in microglia are involved in cytokine release. Here, we investigated the activation of BV-2 cells and found that treatment with lipopolysaccharide (LPS) for 48 h increased the secretion of pro-inflammatory cytokines. This increase was accompanied by a concurrent decrease in oxygen consumption rate (OCR) and mitochondrial membrane potential (MMP), in association with the up-regulation of the UPRmt. Inhibition of the UPRmt by knockdown of ATF5, a key upstream regulator of the UPRmt, using small-interfering RNA against ATF5 (siATF5) not only increased production of the pro-inflammatory cytokines, interleukin-6 (IL-6), IL-1ß and tumor necrosis factor-α (TNF-α), but also decreased MMP. Our results suggest that ATF5-dependent induction of the UPRmt in microglia acts as a protective mechanism during neuroinflammation and may be a potential therapeutic target for reducing neuroinflammation.

LINC00963 promotes the malignancy and metastasis of lung adenocarcinoma by stabilizing Zeb1 and exosomes-induced M2 macrophage polarization.

BACKGROUND: Long intergenic non-coding RNA 00963 (LINC00963) is an oncogenic lncRNA in human cancers. However, little is known on how it impacts the pathogenesis of lung adenocarcinoma (LUAD). METHODS: Biological effects on proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT) were examined by CCK-8, colony formation, EdU incorporation, transwell, and immunofluorescence assays, respectively. Macrophage polarization was evaluated by flow cytometry. Ubiquitination of Zeb1 was examined by co-immunoprecipitation. The location of LINC00963 in LUAD tissues and cell lines was tested by FISH assay. The LINC00963/HNRNPA2B1/Siah1 mRNA complex interaction was verified using RNA pull-down and immunoprecipitation assays. The exact roles of LINC00963 were further validated in metastasis and xenograft models. RESULTS: Higher LINC00963 expression in LUAD patients positively correlated with shorter overall survival, higher stages, and metastasis. LINC00963 mainly localized in the cytoplasm and aggravated malignant phenotypes of LUAD cells in vitro and metastasis in vivo. Mechanistically, LINC00963 directly interacted HNRNPA2B1 protein to trigger the degradation of Siah1 mRNA, which inhibited the ubiquitination and degradation of Zeb1. Moreover, exosomal LINC00963 derived from LUAD cells induced M2 macrophage polarization and promoted LUAD growth and metastasis. CONCLUSION: By stabilizing Zeb1 in cancer cells and delivering exosomes to induce M2 macrophage polarization, LINC00963 promoted the malignancy and metastasis of LUAD. Targeting LINC00963 may become a valuable therapeutic target for LUAD.

A transcriptomic analysis of cerebral microvessels reveals the involvement of Notch1 signaling in endothelial mitochondrial-dysfunction-dependent BBB disruption.

BACKGROUND: Endothelial cells (ECs) in cerebral vessels are considered the primary targets in acute hemorrhagic brain injuries. EC dysfunction can aggravate neuronal injuries by causing secondary inflammatory responses and blood-brain barrier (BBB) disruption. Previous studies have reported that enhancement of mitochondrial function within ECs may reduce BBB disruption and decrease the severity of acute brain injuries. However, the molecular signaling pathways through which enhanced EC mitochondrial function is enhanced to exert this BBB protective effect have not been fully elucidated. METHODS: To identify signaling pathways involved in linking EC-specific mitochondrial dysfunction and BBB disruption, we first performed RNA sequencing using isolated cerebral vessels from TEKCRIF1 KO mice, a mouse strain that displays EC-specific mitochondrial dysfunction. After identification, we assessed the significance of candidate signaling pathways using an intracerebral hemorrhage (ICH) mouse model. BBB integrity was assessed using an IgG leakage assay, and symptomatic changes were evaluated using behavioral assays. RESULTS: Transcriptome analyses of the TEKCRIF1 KO mouse revealed significant changes in Notch1 signaling, a pathway intimately involved in BBB maintenance. We also observed a decrease in Notch1 signaling and expression of the mitochondrial oxidative phosphorylation (OxPhos) complex in the ICH mouse model, which also exhibits BBB disruption. To further assess the function of Notch1 signaling in relation to BBB disruption, we injected ICH model mice with adropin, a protein that interacts with the Notch1 ligand NB-3 and activates Notch1 signaling. We found that adropin prevented BBB disruption and reduced the extent (area) of the injury compared with that in vehicle controls, in association with alteration of mitochondrial function. CONCLUSION: These results suggest that the Notch1 signaling pathway acts as an upstream regulator of DEGs and can be a target to regulate the changes involved with endothelial mitochondrial dysfunction-dependent BBB disruption. Thus, treatment methods that activate Notch1 may be beneficial in acute brain injuries by protecting BBB integrity.

m6A methyltransferase METTL3-induced lncRNA SNHG17 promotes lung adenocarcinoma gefitinib resistance by epigenetically repressing LATS2 expression.

Gefitinib has been widely applied for the treatment of lung adenocarcinoma (LUAD). However, the long-term application of gefitinib usually leads to acquired drug resistance in tumour patients, resulting in clinical treatment failure. Small nucleolar host gene 17 (SNHG17) has been shown to play a regulatory role in LUAD progression. Nevertheless, the role of SNHG17 in LUAD gefitinib resistance remains elusive. The expression pattern of SNHG17 was examined in tissues and cell lines of gefitinib-sensitive and gefitinib-resistant LUAD, respectively. Gain- and loss-of-function experiments were employed to assess the biological functions of SNHG17 in cell proliferation and apoptosis, as well as aggressive phenotypes of LUAD cells. MeRIP-qPCR and colorimetric quantificational analysis were performed to detect m6A modifications and contents. Fluorescence in situ hybridisation (FISH) and subcellular fractionation analysis were used to reveal the distribution of SNHG17. RIP and ChIP assays were performed to further validate the SNHG17/EZH2/LATS2 regulatory axis. A xenograft tumour growth assay was conducted to evaluate the role of SNHG17 in LUAD gefitinib resistance in vivo. SNHG17 was upregulated in gefitinib-resistant LUAD tissues and cell lines. Functional assays showed that SNHG17 aggravated the malignant phenotypes of gefitinib-resistant LUAD cells. In addition, METTL3-mediated N6-methyladenosine modification could induce the upregulation of SNHG17by stabilising its RNA transcript. Mechanistically, SNHG17 epigenetically repressed the expression of LATS2 by recruiting EZH2 to the promoter region of LATS2. The regulatory role of the SNHG17/EZH2/LATS2 axis in LUAD gefitinib resistance was further supported in vivo. Collectively, our findings suggested that SNHG17 induced by METTL3 could promote LUAD gefitinib resistance by epigenetically repressing LATS2 expression.

Reactive microglia and mitochondrial unfolded protein response following ventriculomegaly and behavior defects in kaolin-induced hydrocephalus.

Ventriculomegaly induced by the abnormal accumulation of cerebrospinal fluid (CSF) leads to hydrocephalus, which is accompanied by neuroinflammation and mitochondrial oxidative stress. The mitochondrial stress activates mitochondrial unfolded protein response (UPRmt), which is essential for mitochondrial protein homeostasis. However, the association of inflammatory response and UPRmt in the pathogenesis of hydrocephalus is still unclear. To assess their relevance in the pathogenesis of hydrocephalus, we established a kaolin-induced hydrocephalus model in 8-week-old male C57BL/6J mice and evaluated it over time. We found that kaolin-injected mice showed prominent ventricular dilation, motor behavior defects at the 3-day, followed by the activation of microglia and UPRmt in the motor cortex at the 5-day. In addition, PARP-1/NF-κB signaling and apoptotic cell death appeared at the 5-day. Taken together, our findings demonstrate that activation of microglia and UPRmt occurs after hydrocephalic ventricular expansion and behavioral abnormalities which could be lead to apoptotic neuronal cell death, providing a new perspective on the pathogenic mechanism of hydrocephalus. [BMB Reports 2022; 55(4): 181-186].

Auraptene Enhances Junction Assembly in Cerebrovascular Endothelial Cells by Promoting Resilience to Mitochondrial Stress through Activation of Antioxidant Enzymes and mtUPR.

Junctional proteins in cerebrovascular endothelial cells are essential for maintaining the barrier function of the blood-brain barrier (BBB), thus protecting the brain from the infiltration of pathogens. The present study showed that the potential therapeutic natural compound auraptene (AUR) enhances junction assembly in cerebrovascular endothelial cells by inducing antioxidant enzymes and the mitochondrial unfolded protein response (mtUPR). Treatment of mouse cerebrovascular endothelial cells with AUR enhanced the expression of junctional proteins, such as occludin, zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin), by increasing the levels of mRNA encoding antioxidant enzymes. AUR treatment also resulted in the depolarization of mitochondrial membrane potential and activation of mtUPR. The ability of AUR to protect against ischemic conditions was further assessed using cells deprived of oxygen and glucose. Pretreatment of these cells with AUR protected against damage to junctional proteins, including occludin, claudin-5, ZO-1 and VE-cadherin, accompanied by a stress resilience response regulated by levels of ATF5, LONP1 and HSP60 mRNAs. Collectively, these results indicate that AUR promotes resilience against oxidative stress and improves junction assembly, suggesting that AUR may help maintain intact barriers in cerebrovascular endothelial cells.

Angiopoietin-Like Growth Factor Involved in Leptin Signaling in the Hypothalamus.

The hypothalamic regulation of appetite governs whole-body energy balance. Satiety is regulated by endocrine factors including leptin, and impaired leptin signaling is associated with obesity. Despite the anorectic effect of leptin through the regulation of the hypothalamic feeding circuit, a distinct downstream mediator of leptin signaling in neuron remains unclear. Angiopoietin-like growth factor (AGF) is a peripheral activator of energy expenditure and antagonizes obesity. However, the regulation of AGF expression in brain and localization to mediate anorectic signaling is unknown. Here, we demonstrated that AGF is expressed in proopiomelanocortin (POMC)-expressing neurons located in the arcuate nucleus (ARC) of the hypothalamus. Unlike other brain regions, hypothalamic AGF expression is stimulated by leptin-induced signal transducers and activators of transcription 3 (STAT3) phosphorylation. In addition, leptin treatment to hypothalamic N1 cells significantly enhanced the promoter activity of AGF. This induction was abolished by the pretreatment of ruxolitinib, a leptin signaling inhibitor. These results indicate that hypothalamic AGF expression is induced by leptin and colocalized to POMC neurons.

Schisandra Extract and Ascorbic Acid Synergistically Enhance Cognition in Mice Through Modulation of Mitochondrial Respiration.

Cognitive decline is observed in aging and neurodegenerative diseases, including Alzheimer's disease (AD) and dementia. Intracellular energy produced via mitochondrial respiration is used in the regulation of synaptic plasticity and structure, including dendritic spine length and density, as well as for the release of neurotrophic factors involved in learning and memory. To date, a few synthetic agents for improving mitochondrial function have been developed for overcoming cognitive impairment. However, no natural compounds that modulate synaptic plasticity by directly targeting mitochondria have been developed. Here, we demonstrate that a mixture of Schisandra chinensis extract (SCE) and ascorbic acid (AA) improved cognitive function and induced synaptic plasticity-regulating proteins by enhancing mitochondrial respiration. Treatment of embryonic mouse hippocampal mHippoE-14 cells with a 4:1 mixture of SCE and AA increased basal oxygen consumption rate. We found that mice injected with the SCE-AA mixture showed enhanced learning and memory and recognition ability. We further observed that injection of the SCE-AA mixture in mice significantly increased expression of postsynaptic density protein 95 (PSD95), an increase that was correlated with enhanced brain-derived neurotrophic factor (BDNF) expression. These results demonstrate that a mixture of SCE and AA improves mitochondrial function and memory, suggesting that this natural compound mixture could be used to alleviate AD and aging-associated memory decline.

Endothelial-specific Crif1 deletion induces BBB maturation and disruption via the alteration of actin dynamics by impaired mitochondrial respiration.

Cerebral endothelial cells (ECs) require junctional proteins to maintain blood-brain barrier (BBB) integrity, restricting toxic substances and controlling peripheral immune cells with a higher concentration of mitochondria than ECs of peripheral capillaries. The mechanism underlying BBB disruption by defective mitochondrial oxidative phosphorylation (OxPhos) is unclear in a mitochondria-related gene-targeted animal model. To assess the role of EC mitochondrial OxPhos function in the maintenance of the BBB, we developed an EC-specific CR6-interactin factor1 (Crif1) deletion mouse. We clearly observed defects in motor behavior, uncompacted myelin and leukocyte infiltration caused by BBB maturation and disruption in this mice. Furthermore, we investigated the alteration in the actin cytoskeleton, which interacts with junctional proteins to support BBB integrity. Loss of Crif1 led to reorganization of the actin cytoskeleton and a decrease in tight junction-associated protein expression through an ATP production defect in vitro and in vivo. Based on these results, we suggest that mitochondrial OxPhos is important for the maturation and maintenance of BBB integrity by supplying ATP to cerebral ECs.