Repression of YEATS2 induces cellular senescence in hepatocellular carcinoma and inhibits tumor growth.

Hepatocellular carcinoma (HCC) stands as the third leading cause of cancer-related fatalities globally. In this study, we observed a significant increase in the expression level of the YEATS2 gene in HCC patients, and it is negatively correlated with the patients' survival rate. While we have previously identified the association between YEATS2 and the survival of pancreatic cancer cells, the regulatory mechanisms and significance in HCC are still to be fully elucidated. Our study shows that knockdown (KD) of YEATS2 expression leads to DNA damage, which in turn results in an upregulation of γ-H2A.X expression and activation of the canonical senescence-related pathway p53/p21Cip1. Moreover, our transcriptomic analysis reveals that YEATS2 KD cells can enhance the expression of p21Cip1 via the c-Myc/miR-93-5p pathway, consequently fostering the senescence of HCC cells. The initiation of cellular senescence through dual-channel activation suggests that YEATS2 plays a pivotal regulatory role in the process of cell proliferation. Ultimately, our in vivo research utilizing a nude mouse tumor model revealed a notable decrease in both tumor volume and weight after the suppression of YEATS2 expression. This phenomenon is likely attributable to the attenuation of proliferative cell activity, coupled with a concurrent augmentation in the population of natural killer (NK) cells. In summary, our research results have supplemented the understanding of the regulatory mechanisms of HCC cell proliferation and indicated that targeting YEATS2 may potentially inhibit liver tumor growth.

Aging induces region-specific dysregulation of hormone synthesis in the primate adrenal gland.

Adrenal glands, vital for steroid secretion and the regulation of metabolism, stress responses and immune activation, experience age-related decline, impacting systemic health. However, the regulatory mechanisms underlying adrenal aging remain largely uninvestigated. Here we established a single-nucleus transcriptomic atlas of both young and aged primate suprarenal glands, identifying lipid metabolism and steroidogenic pathways as core processes impacted by aging. We found dysregulation in centripetal adrenocortical differentiation in aged adrenal tissues and cells in the zona reticularis region, responsible for producing dehydroepiandrosterone sulfate (DHEA-S), were highly susceptible to aging, reflected by senescence, exhaustion and disturbed hormone production. Remarkably, LDLR was downregulated in all cell types of the outer cortex, and its targeted inactivation in human adrenal cells compromised cholesterol uptake and secretion of dehydroepiandrosterone sulfate, as observed in aged primate adrenal glands. Our study provides crucial insights into endocrine physiology, holding therapeutic promise for addressing aging-related adrenal insufficiency and delaying systemic aging.

DNA methylation clocks for estimating biological age in Chinese cohorts.

Epigenetic clocks are accurate predictors of human chronological age based on the analysis of DNA methylation at specific CpG sites. However, available DNA methylation (DNAm) age predictors are based on datasets with limited ethnic representation. Moreover, a systematic comparison between DNAm data and other omics datasets has not yet been performed. To address these knowledge gaps, we generated and analyzed DNA methylation datasets from two independent Chinese cohorts, revealing age-related DNAm changes. Additionally, a DNA methylation (DNAm) aging clock (iCAS-DNAmAge) and a group of DNAm-based multi-modal clocks for Chinese individuals were developed, with most of them demonstrating strong predictive capabilities for chronological age. The clocks were further employed to predict factors influencing aging rates. The DNAm aging clock, derived from multi-modal aging features (compositeAge-DNAmAge), exhibited a close association with multi-omics changes, lifestyles, and disease status, underscoring its robust potential for precise biological age assessment. Our findings offer novel insights into the regulatory mechanism of age-related DNAm changes and extend the application of the DNAm clock for measuring biological age and aging pace, providing basis for evaluating aging intervention strategies.

MicroRNA-377-3p exacerbates chronic obstructive pulmonary disease through suppressing ZFP36L1 expression and inducing lung fibroblast senescence.

Chronic obstructive pulmonary disease (COPD) is a leading aging related cause of global mortality. Small airway narrowing is recognized as an early and significant factor for COPD development. Senescent fibroblasts were observed to accumulate in lung of COPD patients and promote COPD progression through aberrant extracellular matrix (ECM) deposition and senescence-associated secretory phenotype (SASP). On the basis of our previous study, we further investigated the the causes for the increased levels of miR-377-3p in the blood of COPD patients, as well as its regulatory function in the pathological progression of COPD. We found that the majority of up-regulated miR-377-3p was localized in lung fibroblasts. Inhibition of miR-377-3p improved chronic smoking-induced COPD in mice. Mechanistically, miR-377-3p promoted senescence of lung fibroblasts, while knockdown of miR-377-3p attenuated bleomycin-induced senescence in lung fibroblasts. We also identified ZFP36L1 as a direct target for miR-377-3p that likely mediated its pro senescence activity in lung fibroblasts. Our data reveal that miR-377-3p is crucial for COPD pathogenesis, and may serve as a potential target for COPD therapy.

A critical signal for phenotype transition driven by negative feedback loops.

The biological rhythms governed by negative feedback loops have undergone extensive investigation. However, developing reliable and versatile warning signals to predict periodic fluctuations in physiological processes and behaviors associated with these rhythms remains a challenge. Here, we monitored the heart rate and tracked ovulation dates of 91 fertile women. The finding strongly links the velocity (derivative) of heart rate with ovulation in menstrual cycles, providing a predictive warning signal. Similarly, an analysis of calcium signaling in the suprachiasmatic nucleus (SCN) of mice reveals that the maximum velocity of rising calcium signal aligns with locomotor activity offsets. To demonstrate the generality of derivative-transitions link, numerical simulations using a negative feedback loop model were conducted. Statistical analysis indicated that over 90% of the oscillations exhibited a correlation between maximum velocity and transition points. Consequently, the maximum velocity derived from oscillatory curves holds significant potential as an early warning signal for critical transitions.

HALL: a comprehensive database for human aging and longevity studies.

Diverse individuals age at different rates and display variable susceptibilities to tissue aging, functional decline and aging-related diseases. Centenarians, exemplifying extreme longevity, serve as models for healthy aging. The field of human aging and longevity research is rapidly advancing, garnering significant attention and accumulating substantial data in recent years. Omics technologies, encompassing phenomics, genomics, transcriptomics, proteomics, metabolomics and microbiomics, have provided multidimensional insights and revolutionized cohort-based investigations into human aging and longevity. Accumulated data, covering diverse cells, tissues and cohorts across the lifespan necessitates the establishment of an open and integrated database. Addressing this, we established the Human Aging and Longevity Landscape (HALL), a comprehensive multi-omics repository encompassing a diverse spectrum of human cohorts, spanning from young adults to centenarians. The core objective of HALL is to foster healthy aging by offering an extensive repository of information on biomarkers that gauge the trajectory of human aging. Moreover, the database facilitates the development of diagnostic tools for aging-related conditions and empowers targeted interventions to enhance longevity. HALL is publicly available at https://ngdc.cncb.ac.cn/hall/index.

Multitissue Circadian Proteome Atlas of WT and Per1-/-/Per2-/- Mice.

The molecular basis of circadian rhythm, driven by core clock genes such as Per1/2, has been investigated on the transcriptome level, but not comprehensively on the proteome level. Here we quantified over 11,000 proteins expressed in eight types of tissues over 46 h with an interval of 2 h, using WT and Per1/Per2 double knockout mouse models. The multitissue circadian proteome landscape of WT mice shows tissue-specific patterns and reflects circadian anticipatory phenomena, which are less obvious on the transcript level. In most peripheral tissues of double knockout mice, reduced protein cyclers are identified when compared with those in WT mice. In addition, PER1/2 contributes to controlling the anticipation of the circadian rhythm, modulating tissue-specific cyclers as well as key pathways including nucleotide excision repair. Severe intertissue temporal dissonance of circadian proteome has been observed in the absence of Per1 and Per2. The γ-aminobutyric acid might modulate some of these temporally correlated cyclers in WT mice. Our study deepens our understanding of rhythmic proteins across multiple tissues and provides valuable insights into chronochemotherapy. The data are accessible at https://prot-rhythm.prottalks.com/.

TAF1B depletion leads to apoptotic cell death by inducing nucleolar stress and activating p53-miR-101 circuit in hepatocellular carcinoma.

Background: TAF1B (TATA Box Binding Protein (TBP)-Associated Factor) is an RNA polymerase regulating rDNA activity, stress response, and cell cycle. However, the function of TAF1B in the progression of hepatocellular carcinoma (HCC) is unknown. Objective: In this study, we intended to characterize the crucial role and molecular mechanisms of TAF1B in modulating nucleolar stress in HCC. Methods: We analyzed the differential expression and prognostic value of TAF1B in hepatocellular carcinoma based on The Cancer Genome Atlas (TCGA) database, tumor and paraneoplastic tissue samples from clinical hepatocellular carcinoma patients, and typical hepatocellular carcinoma. We detected cell proliferation and apoptosis by lentiviral knockdown of TAF1B expression levels in HepG2 and SMMC-7721 cells using clone formation, apoptosis, and Western blotting (WB) detection of apoptosis marker proteins. Simultaneously, we investigated the influence of TAF1B knockdown on the function of the pre-initiation complex (PIC) by WB, and co-immunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) assays verified the interaction between the complexes and the effect on rDNA activity. Immunofluorescence assays measured the expression of marker proteins of nucleolus stress, fluorescence in situ hybridization (FISH) assays checked the rDNA activity, and qRT-PCR assays tested the pre-rRNA levels. Regarding molecular mechanisms, we investigated the role of p53 and miR-101 in modulating nucleolar stress and apoptosis. Finally, the impact of TAF1B knockdown on tumor growth, apoptosis, and p53 expression was observed in xenograft tumors. Result: We identified that TAF1B was highly expressed in hepatocellular carcinoma and associated with poor prognosis in HCC patients. TAF1B depletion modulated nucleolar stress and apoptosis in hepatocellular carcinoma cells through positive and negative feedback from p53-miR-101. RNA polymerase I transcription repression triggered post-transcriptional activation of miR-101 in a p53-dependent manner. In turn, miR-101 negatively feeds back through direct inhibition of the p53-mediated PARP pathway. Conclusion: These findings broaden our comprehension of the function of TAF1B-mediated nucleolar stress in hepatocellular carcinoma and may offer new biomarkers for exploring prospective therapeutic targets in HCC.

Osteoporotic bone loss from excess iron accumulation is driven by NOX4-triggered ferroptosis in osteoblasts.

Excess iron accumulation is a risk factor for osteopenia and osteoporosis, and ferroptosis is becoming well understood as iron-dependent form of cell death resulting from lipid peroxide accumulation. However, any pathological impacts of ferroptosis on osteoporosis remain unknown. Here, we show that ferroptosis is involved in excess-iron-induced bone loss and demonstrate that osteoporotic mice and humans have elevated skeletal accumulation of the NADPH oxidase 4 (NOX4) enzyme. Mechanistically, we found that the NOX4 locus contains iron-response element-like (IRE-like) sequences that are normally bound (and repressed) by the iron regulatory protein 1 (IRP1) protein. Binding with iron induces dissociation of IRP1 from the IRE-like sequences and thereby activates NOX4 transcription. Elevated NOX4 increases lipid peroxide accumulation and causes obvious dysregulation of mitochondrial morphology and function in osteoblasts. Excitingly, the osteoporotic bone loss which we initially observed in an excessive-iron accumulating mouse line (Hepc1-/-) was blocked upon treatment with the ferroptosis-inhibitor ferrostatin-1 (Ferr-1) and with the iron chelator deferoxamine (DFO), suggesting a potential therapeutic strategy for preventing osteoporotic bone loss based on disruption of ferroptosis.

Disturbance of suprachiasmatic nucleus function improves cardiac repair after myocardial infarction by IGF2-mediated macrophage transition.

Suprachiasmatic nucleus (SCN) in mammals functions as the master circadian pacemaker that coordinates temporal organization of physiological processes with the environmental light/dark cycles. But the causative links between SCN and cardiovascular diseases, specifically the reparative responses after myocardial infarction (MI), remain largely unknown. In this study we disrupted mouse SCN function to investigate the role of SCN in cardiac dysfunction post-MI. Bilateral ablation of the SCN (SCNx) was generated in mice by electrical lesion; myocardial infarction was induced via ligation of the mid-left anterior descending artery (LAD); cardiac function was assessed using echocardiography. We showed that SCN ablation significantly alleviated MI-induced cardiac dysfunction and cardiac fibrosis, and promoted angiogenesis. RNA sequencing revealed differentially expressed genes in the heart of SCNx mice from D0 to D3 post-MI, which were functionally associated with the inflammatory response and cytokine-cytokine receptor interaction. Notably, the expression levels of insulin-like growth factor 2 (Igf2) in the heart and serum IGF2 concentration were significantly elevated in SCNx mice on D3 post-MI. Stimulation of murine peritoneal macrophages in vitro with serum isolated from SCNx mice on D3 post-MI accelerated the transition of anti-inflammatory macrophages, while antibody-mediated neutralization of IGF2 receptor blocked the macrophage transition toward the anti-inflammatory phenotype in vitro as well as the corresponding cardioprotective effects observed in SCNx mice post-MI. In addition, disruption of mouse SCN function by exposure to a desynchronizing condition (constant light) caused similar protective effects accompanied by elevated IGF2 expression on D3 post-MI. Finally, mice deficient in the circadian core clock genes (Ckm-cre; Bmal1f/f mice or Per1/2 double knockout) did not lead to increased serum IGF2 concentration and showed no protective roles in post-MI, suggesting that the cardioprotective effect observed in this study was mediated particularly by the SCN itself, but not by self-sustained molecular clock. Together, we demonstrate that inhibition of SCN function promotes Igf2 expression, which leads to macrophage transition and improves cardiac repair post-MI.

Nuclear receptor modulators inhibit osteosarcoma cell proliferation and tumour growth by regulating the mTOR signaling pathway.

Osteosarcoma is the most common primary malignant bone tumour in children and adolescents. Chemoresistance leads to poor responses to conventional therapy in patients with osteosarcoma. The discovery of novel effective therapeutic targets and drugs is still the main focus of osteosarcoma research. Nuclear receptors (NRs) have shown substantial promise as novel therapeutic targets for various cancers. In the present study, we performed a drug screen using 29 chemicals that specifically target 17 NRs in several different human osteosarcoma and osteoblast cell lines. The retinoic acid receptor beta (RARb) antagonist LE135, peroxisome proliferator activated receptor gamma (PPARg) antagonist T0070907, liver X receptor (LXR) agonist T0901317 and Rev-Erba agonist SR9011 significantly inhibited the proliferation of malignant osteosarcoma cells (U2OS, HOS-MNNG and Saos-2 cells) but did not inhibit the growth of normal osteoblasts. The effects of these NR modulators on osteosarcoma cells occurred in a dose-dependent manner and were not observed in NR-knockout osteosarcoma cells. These NR modulators also significantly inhibited osteosarcoma growth in vivo and enhanced the antitumour effect of doxorubicin (DOX). Transcriptomic and immunoblotting results showed that these NR modulators may inhibit the growth of osteosarcoma cells by regulating the PI3K/AKT/mTOR and ERK/mTOR pathways. DDIT4, which blocks mTOR activation, was identified as one of the common downstream target genes of these NRs. DDIT4 knockout significantly attenuated the inhibitory effects of these NR modulators on osteosarcoma cell growth. Together, our results revealed that modulators of RARb, PPARg, LXRs and Rev-Erba inhibit osteosarcoma growth both in vitro and in vivo through the mTOR signaling pathway, suggesting that treatment with these NR modulators is a novel potential therapeutic strategy.

Long-term SCN calcium signal recording in freely moving mice.

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker of the mammalian biological clock. Here, we provide a detailed protocol for long-term recording of calcium signals in SCN neurons of freely moving mice through a multichannel optical fiber recording system. This system can simultaneously collect calcium signals from up to seven animals. The calcium signals can be visualized by the appropriate software and code. This protocol can be used to explore the long-term response of SCN to external environmental stimulation. For complete details on the use and execution of this protocol, please refer to Zhai et al. (2022).

Time-restricted feeding entrains long-term behavioral changes through the IGF2-KCC2 pathway.

The suprachiasmatic nucleus (SCN) integrates light and systemic signals from peripheral tissues to coordinate physiology and behavior daily rhythms. However, the contribution that nutrients and feeding patterns provide to the SCN network regulation remains controversial. Here, we found that time-restricted feeding (TRF) in ZT0-4 (Zeitgeber Time) generates a robust and long-term shift in locomotor behavior and increased wakefulness. Intracellular Ca2+ signals in SCN GABAergic neurons of freely moving mice showed significant activation after ZT0-4 TRF treatment. Furthermore, RNA-seq profiling of SCN showed that TRF during ZT0-4 increased Insulin-like Growth Factor 2 (Igf2) expression and dysregulated ion transporters, including the downregulation of Kcc2. SCN neuron-specific loss of function of Kcc2 amplified ZT0-4 TRF induced aftereffect. Moreover, overexpression of IGF2 in SCN GABAergic neurons extended the locomotion range, mirroring the TRF aftereffect. In summary, our study showed that the IGF2-KCC2 pathway plays an important role for TRF induced behavior changes.

Impaired function of the suprachiasmatic nucleus rescues the loss of body temperature homeostasis caused by time-restricted feeding.

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker that drives body temperature rhythm. Time-restricted feeding (TRF) has potential as a preventative or therapeutic approach against many diseases. The potential side effects of TRF remain unknown. Here we show that a 4-hour TRF stimulus in mice can severely impair body temperature homeostasis and can result in lethality. Nearly half of the mice died at 21 °C, and all mice died at 18 °C during 4-hour TRF. Moreover, this effect was modulated by the circadian clock and was associated with severe hypothermia due to loss of body temperature homeostasis, which is different from "torpor", an adaptive response under food deprivation. Disrupting the circadian clock by the SCN lesions or a non-invasive method (constant light) which disrupts circadian clock rescued lethality during TRF. Analysis of circadian gene expression in the dorsomedial hypothalamus (DMH) demonstrated that TRF reprograms rhythmic transcriptome in DMH and suppresses expression of genes, such as Ccr5 and Calcrl, which are involved in thermoregulation. We demonstrate a side effect of 4-hour TRF on the homeostasis of body temperature and a rescue function by impairing the SCN function. Altogether, our results suggested that constructing a circadian arrhythmicity may have a beneficial effect on the host response to an acute stress.

High-throughput discovery of genetic determinants of circadian misalignment.

Circadian systems provide a fitness advantage to organisms by allowing them to adapt to daily changes of environmental cues, such as light/dark cycles. The molecular mechanism underlying the circadian clock has been well characterized. However, how internal circadian clocks are entrained with regular daily light/dark cycles remains unclear. By collecting and analyzing indirect calorimetry (IC) data from more than 2000 wild-type mice available from the International Mouse Phenotyping Consortium (IMPC), we show that the onset time and peak phase of activity and food intake rhythms are reliable parameters for screening defects of circadian misalignment. We developed a machine learning algorithm to quantify these two parameters in our misalignment screen (SyncScreener) with existing datasets and used it to screen 750 mutant mouse lines from five IMPC phenotyping centres. Mutants of five genes (Slc7a11, Rhbdl1, Spop, Ctc1 and Oxtr) were found to be associated with altered patterns of activity or food intake. By further studying the Slc7a11tm1a/tm1a mice, we confirmed its advanced activity phase phenotype in response to a simulated jetlag and skeleton photoperiod stimuli. Disruption of Slc7a11 affected the intercellular communication in the suprachiasmatic nucleus, suggesting a defect in synchronization of clock neurons. Our study has established a systematic phenotype analysis approach that can be used to uncover the mechanism of circadian entrainment in mice.