第11卷，第2期

Fibroblast growth factor 21 (FGF21) is a hormone that can balance nutrient fluctuations, control metabolic processes, and maintain energy homeostasis. Endogenous FGF21 is produced by a variety of cell types, including hepatocytes, adipocytes, bone cells, myocardial cells, and pancreas cells, and act on various effector tissues such as brain, adipose tissue, liver, heart, and skeletal muscle. The target that FGF21 interacts with is the cell-surface receptor composed of FGF receptors in complex with the single-pass transmembrane protein β-Klotho. These receptors are abundantly expressed, both in peripheral tissues and some regions of the CNS, such as the hypothalamus and amygdala and the locus coeruleus (LC). Physiologically, FGF21 can be induced by various metabolic stresses, including hunger, proteindeficiency, monosaccharides, andalcohol, by acting on β-Klotho receptor in corresponding tissues, ultimately exerting regulatory effects.

1640

101

Tumor-protein 53 (p53) is a transcription factor (TF) encoded by TP53 (Trp53 in mice) and is a master regulator of tumor-suppressive programs. Importantly, TP53 is the most frequently mutated gene in human cancer, and its loss of function contributes to tumor development, exemplified by variable p53 mutations detected in 50%-75% of pancreatic cancers. From these perceptions, a number of targets transcribed by p53 that confer tumor suppression have been identified. Nevertheless, our understanding of how p53 prevents normal cells from initiating tumorigenesis is still incomplete. To complement this limitation, herein, we established a new approach to identify underlying effectors transcribed by p53 in normal physiology.

1525

Recently, Peter et al reported a novel form of cell death (cuproptosis) that was different from other known death mechanisms. They found that accumulation of copper ions could induce destabilization of FeeS cluster proteins and aggregation of mitochondrial lipoylated proteins, ultimately resulting in cuproptosis. Of note, ten genes were identified as cuproptosis-related genes (CRGs) in copper ion-induced cell death, including PDHB, PDHA1, DLAT, DLD, LIPT1, LIAS, FDX1, CDKN2A, GLS, and MTF1. Since dysregulation of copper metabolism is involved in many cancers, cuproptosis and CRGs may play vital roles in cancer development and treatment. Herein, our pancancer analysis revealed the coordinated upregulation of 9 of 10 CRGs in human liver hepatocellular carcinoma (LIHC) across 33 solid tumors, suggesting the distinct role of CRGs in LIHC. We then explored the genetic landscape, biological function, and clinical significance of CRGs in LIHC, and validated our bioinformatic findings by in vitro experiments and clinical cohorts. The detailed methods were described in the supplementary material.

1476

Primary liver cancer, which is mainly composed of hepatocellular carcinoma (HCC), is the sixth most common type of cancer worldwide and the third most common cause of cancer mortality. The total number of mutations present in tumor specimens is called tumor mutation burden (TMB) anditisanemergingbiomarkerofimmunotherapy response. TMB can predict clinical responses to immunotherapy such as ICI (immune checkpoint inhibitor) treatments and higher TMB is related to better survival. TP53, a gene encoding a tumor suppressor protein that triggers apoptosis and cell cycle arrest, is one of the most prevalent mutations in 25% e30% of HCC patients. Research shows that TP53 mutations in HCC patients are associated with advanced tumor grade and poor prognosis. To identify the TP53 mutation-related genes which can predict HCC patients' prognosis and explore the immune cell infiltration, we constructed a risk model based on six TP53 mutation-related genes which can accurately predict patients' prognosis. Besides, six immune cells with a similar expression pattern were identified in The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) databases.

1409

Germline activating variants in WWP1, which encodes an E3 ubiquitin ligase that antagonizes PTEN tumor suppressive function, have been proposed as an alternative mechanism of PTEN inactivation in PTEN-hamartoma-tumor syndrome (PHTS) -like patients with wildtype PTEN. More specifically, heterozygous, potentially activating WWP1 variants were first identified by Lee et al in patients affected with gastrointestinal oligopolyposis, including adenomatous, hyperplastic/serrated, and hamartomatous polyps, and occasionally with colorectal cancer. Subsequently, based on the PHTS phenotypic features, WWP1 mutational screening was performed in patients with thyroid nodules, or normocephalic autism spectrum disorder (ASD), where germline WWP1 variants were also identified.

1257

Triple-negative breast cancer (TNBC) has aggressive characteristics as exemplified by the high risk of recurrence, metastasis, andpatientmortality. Cytotoxicchemotherapy may reduce tumor burden initially, but leave behind chemo-resistant breast cancer stem cells (BCSCs) that lead to tumor recurrence and metastasis. Chemotherapy induces BCSC enrichment through the activation of glutathione biosynthesis pathways, which increases intracellular glutathione levels and specifies the BCSC phenotype through complicated downstream signaling pathways. Glutathione S-transferases (GSTs), a superfamily (which contains seven cytosolic classes: Alpha, Kappa, Mu, Omega, Pi, Theta, and Zeta, and one microsomal class MGSTs) of phase II enzymes that catalyze the conjugation of glutathione with electrophilic compounds, play a critical role in detoxification and chemotherapy resistance of cancer cells. However, the role of GSTs in the regulation of BCSCs is largely elusive. In the present study, we first investigated the expression of all GST family members in response to chemotherapeutic drug carboplatin treatment at the dose of IC₅₀ in multiple TNBC cell lines, and found four members of the GST Mu family (GSTM1, GSTM2, GSTM3, and GSTM4) were markedly repressed in all four cell lines. GSTM1/2/3/4 mRNA and protein were also repressed by other FDA-approved chemotherapy drugs paclitaxel and gemcitabine at the dose of IC₅₀ in TNBC cell lines. Carboplatin also repressed GSTM1/2/3/4 expression in vivo, as shown in SCID (severe combined immunodeficiency) mice transplanted with MDA-MB-231 cells and in MMTV-PyMT transgenic mice (a genetically engineered autochthonous breast cancer model) that were treated by carboplatin.

1216

The biogenesis and functions of extrachromosomal circular DNA (eccDNA) have been studied for several decades. However, the heterogeneity of eccDNA is largely ignored. In this study, we purified and sequenced eccDNA and RNA using a method that simultaneously extracts DNA and RNA from cultured cells treated with iron nanoparticles. We discovered that the characteristics of eccDNA are altered by iron stress, particularly on genic and CpG islands regions, suggesting a biogenesis mechanism induced by transcription. However, we found that only a few eccDNA-annotated genes were shared by differentially expressed genes identified by RNA-seq, indicating that eccDNA has a complex background that may be the result accumulated from previous states. In addition, we observed little variation in the distribution pattern of eccDNA in ferroptosis cells with fewer differentially expressed genes, indicating that eccDNA is generated randomly in cells when they are unable to regulate many more genes to resist environmental stress. Collectively, our study characterized eccDNA induced by iron stress, uncovered their intricate background, and proposed the alterations in the distribution pattern of eccDNA as a potential biomarker.

1611

SARS-CoV-2 has been demonstrated to be highly susceptible to neuron-and glial-like cells, but there is no consensus regarding how the virus affects neurons following neuroinvasion. It is therefore unknown if and how SARS-CoV-2 maintains the homeostasis of neuronal cells. Herein, we revealed that the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein acted as an extracellular remodeling component to undermine epigenetic regulation, perturb nascent RNA (nsRNA) transcription, promote mitochondrial biogenesis, and increase cellular viability in SH-SY5Y cells. Through boosting the expression of KDM6B, RBD proteins decreased H3K27me3. Down-regulation of KDM6B was able to decrease molecular and cellular abnormalities previously mediated by all RBD mutants (K417N/L452R/T478K: KLT;L452R/E484Q: LE;N501Y), including NRF2 expression levels, ROS scavenging capacity, mitochondrial activity, and cell proliferation capacity. Further, we also investigated whether RBD proteins affected mitochondrial biogenesis epigenetically. After treatment with the LE mutant, ChIP-qPCR revealed that H3K27me3 was repressed in the NRF2 promoter regions, resulting in NRF2 activating. This study, therefore, uncovered a novel epigenetic mechanism by which SARS-CoV-2 RBD proteins regulate mitochondrial biogenesis via an extracellular signal transduction channel and remodel neuronal cell proliferation.

1294

Juvenile idiopathic arthritis (JIA) is one of the most common chronic inflammatory rheumatic diseases in children, with onset before age 16 and lasting for more than 6 weeks. JIA is a highly heterogeneous condition with various consequences for health and quality of life. For some JIA patients, early detection and intervention remain challenging. As a result, further investigation of the complex and unknown mechanisms underlying JIA is required. Advances in technology now allow us to describe the biological heterogeneity and function of individual cell populations in JIA. Through this review, we hope to provide novel ideas and potential targets for the diagnosis and treatment of JIA by summarizing the current findings of single-cell RNA sequencing studies and understanding how the major cell subsets drive JIA pathogenesis.

1886

The tumor heterogeneity results in different clinical characteristics of tumor patients, so precise therapy is a popular treatment strategy clinically. Microsatellite instability (MSI) is found in 10% e15% of colorectal cancer (CRC) patients and exhibits different clinical outcomes than microsatellite stable (MSS) patients. Surprisingly, MSI patients have a better prognosis than patients with stable microsatellites. MSI patients exhibit clinical features that are not responsive to chemotherapy, however, MSI patients showed excellent responses to immunotherapy. Although the subtype classification of MSI is beneficial to the clinical treatment of CRC patients, the mechanism of MSI remains unclear.

1279

Osteoblasts are essential in the maintenance of human bone homeostasis. The abnormal formation and impaired differentiation ability of osteoblasts are pivotal factors leading to bone-related diseases, suggesting that an indepth study of osteoblasts can provide novel treatment strategies for these diseases. Human primary osteoblasts arep recious experimental models in bone-related research. However, the isolation and culture of human primary osteoblasts still have some difficulties and limitations. Here, we refined a method to isolate and culture human primary osteoblasts by taking cancellous bone through a bone tissue sampler and pre-digesting it with pancreatin, which makes the process easier, cheaper, and more efficient. Furthermore, we compared the effects of the subtle changes in isolation and culture methods on the phenotype of human primary osteoblasts and examined the differences in morphology, proliferation, and differentiation between micro-explants cultured osteoblasts (MEC-OBs) and explants cultured osteoblasts (EC-OBs). Our results showed that although both MEC-OBs and EC-OBs have prominent osteoblastic phenotypes, they still have their own characteristics. Collectively, this improved method could reduce the difficulty and provide benefits for the research and application of human primary osteoblasts, such as human bone-related organoid culture and medical treatment of bone-related diseases.

1271

PR/SET domain 1 (PRDM1) gene is located on chromosome 6q21, encoding the B lymphocyte-induced maturation protein 1 (BLIMP1). It is reported that loss of PRDM1 function is exacerbated in activated B-cell-like (ABC) -diffuse large B cell lymphoma (DLBCL) and associated with inferior survival. However, it remains unclear what leads to PRDM1 inactivation and the drug resistance mechanism caused by abnormal inactivation of PRDM1. We investigated the contribution of PRDM1 gene as a prognosis and potential therapeutic target for ABC-DLBCL patients and further clarified the possible mechanism of PRDM1 abnormal inactivation. We first proposed that TP53 could regulate PRDM1 by histone ubiquitination modification at the post-transcriptional level. Moreover, the therapeutic effect of bortezomib was dependent on PRDM1 and TP53, and a synergistic effect of lenalidomide and bortezomib was observed in PRDM1-mutated ABC DLBCL cell lines, which provided a theoretical reference for overcoming drug resistance in PRDM1-mutated ABC-DLBCL patients.

1284

The high mortality of patients with lung squamous cell carcinoma (LUSC) results from metastasis rather than primary tumors, whereas the molecular pathogenesis of cancer metastasis remains poorly understood. PLAU encodes urokinase-type plasminogen activator (uPA) which is closely related to tumor diagnosis, treatment target, and prognosis of patients. We previously predicted that PLAU is a key gene negatively associated with overall survival by integrated multidimensional analyses of The Cancer Genome Atlas (TCGA) datasets from 504 samples of LUSC tumor tissues and 46 samples of adjacent non-tumorous lung tissues. However, whether the role of PLAU contributes to the metastasis of LUSC remains unclear. Here, we analyzed the association between uPA levels and LUSC and found that uPA levels were associated with pathological variations in LUSC patients. Furthermore, we found that overexpression or interference of PLAU resulted in significant changes in the migration abilities and metastasis-associated gene expression in lung and LUSC cells, respectively. These findings uncover a key role for PLAU in LUSC metastasis and provide a potential novel early diagnosis and therapeutic target for metastatic LUSC intervention. We demonstrated that PLAU (uPA) is vital to LUSC metastasis; thus, it could be a promising biomarker for this disease, which contributes to the prevention of LUSC metastasis by encouraging the development of diagnostic and therapeutic strategies that target the identified PLAU (uPA) -associated genes and pathways.

1380

Intervertebral disc degeneration (IDD) is the main cause of lower back pain. Lower back pain places a huge burden on society, and all current treatments for IDD cannot restore the original function of the intervertebral disc. Proposing new treatments for IDD requires clarifying the mechanisms of IDD. Physiological hypoxia is an important feature of the nucleus pulposus because of its special anatomical structure. Using RNA sequencing (RNA-seq), we obtained the whole transcriptome of nucleus pulposus cells (NPCs) under hypoxia and hypoxia-inducible factor (HIF1A) deletion. Results demonstrated possible effects of oxygen concentration and HIF1A on NPCs. In addition, our results showed that hypoxia can affect lipid metabolism in NPCs.

1219

Absolute erythrocytosis, due to an increased production of red blood cells, becomes manifest with hemoglobin (Hb) levels above 165 or 160 g/L or with a hematocrit (HCT) above 51% and 48% in males and females, respectively. A minority of patients have polycythemia vera (PV); acquired secondary erythrocytosis frequently occurs due to appropriate or inappropriate erythropoietin (EPO) excess, while congenital secondary erythrocytosis may derive from genetic defects causing tissue hypoxia. Hereditary erythrocytosis occurs in patients with genetic mutations of the oxygen-sensing pathway (VHL, PHD2, HIF-1-alpha) or the erythropoietin receptor (EPOR) genes. Recently, HFE gene variants have been described in sporadic erythrocytoses. In a high proportion (about 70%) of patients with erythrocytosis, a specific etiology remains elusive despite extensive testing, and the diagnosis of idiopathic erythrocytosis (IE) is ruled out.

1277

Gastric cancer (GC) is the third most common cause of cancer death globally and a large portion of patients are diagnosed at advanced stages with cancer invasion and metastasis. However, the mechanisms underlying the invasion and metastasis of GC remain to be delineated. ZYX plays critical roles in cell mobility via cytoskeleton regulation in various cell types. In this study, we further reported that ZYX promoted migration, invasion, and metastasis of GC cells. Mechanistically, ZYX promoted WNK1 activation and SNAI1 up-regulation, inducing epithelial-mesenchymal transition (EMT) to enhance the mobility of GC cells. Inhibition of WNK1 impaired the mobility of GC cells. Therefore, ZYX/WNK1 could be potential therapeutic targets for GC treatment.

1332

Immunogenic death is a form of programmed cell death that is common in the development and progression of cancer. However, its impact on tumor progression differs depending on the location of the tumor. Meanwhile, it also plays an important role in antitumor immunity because the process of immunogenic death can release substances that activate immunity. Therefore, in this study, the key mechanism of mediating immunogenic death in hepatocellular carcinoma (HCC) was found from the excavation of the immunogenic death characteristics of pan-cancer and the in-depth investigation of its effects on different tumor types

1277

Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by extensive tumor heterogeneity, with limited treatment options and a poor prognosis. We used cell type annotation and trajectory analysis to identify two distinct differentiation states of epithelial cells, while tumor cells predominantly resided on one of these branches. Inferring the malignant state of epithelial cells based on copy number variation, 280 tumor cells were eventually identified from epithelial cells. Differential genes between tumor cells and non-tumor cells were mainly enriched in immune-related functions and pathways. To explore the immune roles of differential genes, we finally identified 106 key lncRNAs and mRNAs associated with immune processes by integrating single-cell and bulk RNA-seq data from TNBC. Moreover, based on the LASSO-COX prognostic model, we systematically identified five lncRNAs and six mRNAs that were significantly associated with TNBC survival. The immune-related 11 lncRNA-mRNA signature can be used as a prognostic signature to independently divide the overall survival time of patients. Our immune-related prognostic signature has prognostic significance for 15 cancers and exhibited excellent predictive performance compared to the 20 prognostic models, indicating that it may have clinical value in improving the overall survival of patients.

1272

Neutrophils constitute a significant portion of the immune cells present within the tumor microenvironment. Evidence generated by our group has confirmed neutrophils to be an adverse independent prognostic factor affecting the disease-free survival of gastric carcinoma (GC) patients. Neutrophil extracellular traps (NETs), protein-covered DNA webs that interact with tumor cells in the tumor microenvironment, were detected in GC tissues and found to clinically associated with disease progression in our previous study. NETosis could be mediated by a series of agonists, such as interleukin 8 (IL-8). However, the mechanism of IL-8 in the progression of GC occurs via NETs remains unclear. In this study, we identified that IL-8-mediated NETosis can promote the proliferation, migration, and invasion of GC cells in vitro, which can be abrogated by NET degradation through deoxyribonuclease I (DNase I) or IL-8 suppression through neutralizing antibodies. Disruption of NETs by DNase I or blockade of the IL-8-CXC chemokine receptor 2 (CXCR2) axis could result in growth retardation in mouse models. We also confirmed that NETs may influence the biological behavior of GC cells by up-regulating the expression of transcription factor IIB-related factor 1 (BRF1) and cyclin p21/p27.

1283

Colorectal carcinoma (CRC), the third most commonly diagnosed cancer, accounts for 9.7% of all newly diagnosed cancer cases and 9.4% of cancer-related deaths globally. Recent studies have demonstrated that post-transcriptional RNA modifications, such as N⁶-methyladenosine, N -methylcytosine, and N⁷-methylguanosine, play critical roles in the regulation of mRNA stability and translation, primary microRNA processing, and lncRNA-protein complex that contributes to the progression of human cancer. Here, we found that the expression of the member of methyltransferase-like (METTL) family-METTL1, the m⁷G "writers", was remarkably up-regulated in colorectal cancer tissue and positively correlated with poor prognosis. METTL1 knockdown suppressed colorectal cancer cell growth and G1/S phase transition. Further functional experiments indicate that METTL1 could directly interact with checkpoint kinase 2 (CHEK2) and suppress its protein expression, which was abrogated by BML-277, the CHEK2 inhibitor. Our data uncover that METTL1 plays an important supportive role in colorectal cancer proliferation and progression, providing a potential therapeutic target for colorectal cancer.

1324

Non-small cell lung cancer (NSCLC) is a malignant tumor that poses a serious threat to human health. Adenocarcinoma (LUAD) and squamous cell carcinoma (LUSC) are two common types of NSCLC. They originate from glandular and squamous cells of the bronchial epithelium, respectively. Identifying gene changes during tumorigenesis is conducive to the selection of suitable treatment methods for patients. CD98 is a heterodimeric transmembrane glycoprotein, which is composed of a heavy chain and a light chain. CD98 heavy chain (CD98hc), also known as 4F2hc or SLC3A2, is an 85 kDa type II transmembrane glycoprotein, which consists of a cytoplasmic region (NH2 terminal), a single chain transmembrane region, and a huge extracellular region (COOH terminal). CD98hc can combine with the CD98 light chain, LAT1 (SLC7A5), to form CD98 protein through disulfide bonds. SLC7A5 is a 12 times transmembrane helix bundle protein composed of 501-535 amino acid residues. The overexpression of CD98 is closely related to the occurrence and development of NSCLC. Therefore, we screened the expression profiles of SLC3A2 and SLC7A5 in smoking and non-smoking patients with LUSC and LUAD. In addition, we attempted to determine the mechanisms underlying nicotine-induced SLC7A5 expression in LUSC and LUAD cells.

1350

Pregnancy is a unique physiological state in which several changes occur. One of the most important aspects is the maternal immune system, which sets to tolerate the presence of the developing fetus (a semi-allogeneic organism), while at the same time providing protection against pathogens. Pregnancy has long been known to affect the risk of certain cancers including breast cancer in the short term, gestational trophoblastic disease (GTD), cervical cancer, and melanoma. The reasons for the increased risk of these cancers during pregnancy are not fully understood, but several hypotheses have been proposed as the hormonal and immune system changes. Non-invasive prenatal testing (NIPT) offers a safer and more accurate alternative to amniocentesis and chorionic villus sampling (CVS) for screening against chromosomal abnormality associated with severe malformations and neurological alterations[American College of Obstetricians and Gynecologists, and the International Society for Prenatal Diagnosis]. However, this is a complex and delicate issue, and structured recommendations, in this context, have proven to be inefficient since the decision to undergo prenatal testing is frequently a personal one.

1262

Long-read sequencing with nanopore technology has been widely utilized for the identification of the full-length sequence of RNA molecules. It is especially suitable for the identification of circular RNAs (circRNAs), which are regulatory molecules that are regarded as promising non-invasive biomarkers for various human malignancies, including breast cancer. The impact of circRNAs derived from the protein arginine methyltransferase 1 (PRMT1) gene has not been clarified yet, even though the effect of PRMT1-mediated methylation on breast cancer cell characteristics has been well established. We have previously shown that several circRNAs are produced by PRMT1, and thus we aimed at identifying novel PRMT1 circRNAs in 12 breast cell lines, following a PCR-based, targeted nanopore sequencing work flow (described in detail in the supplementary materials and methods). This pipeline leads to the identification of the full circRNA sequence, including the backesplice junction, as a single read. Contrary to circRNA profiling studies, our targeted approach allows for the amplification and sequencing of circRNAs originating from the PRMT1 gene with higher specificity. Meanwhile, it enables the identification of rare circular transcripts that may be missed by a direct circRNA sequencing approach. In our study, a wide variety of novel PRMT1 circRNAs were identified. Their intricacy in structure and distinct expression profiles among breast cancer cells raises new questions about the functions of circRNAs and the regulatory role of PRMT1 in breast cancer.

1336

Hepatocellular carcinoma (HCC) is characterized by both inter-and intra-tumor heterogeneity and has distinct clinical outcomes. A promising clinical tool to perform patient stratification, prognosis evaluation, and treatment recommendations is indispensable. Here, we enrolled a total of 1595tumorpatientsfrom13independentcohorts, including seven cohorts with survival data, four cohorts with immunotherapy information, and two cohorts with transcatheter arterial chemoembolization (TACE) and Sorafenib information, respectively (Table S1). Using 96 algorithms combinations derived from 10 popular machinelearning approaches, a novel framework was constructed and described in Figure S1. Firstly, a total of 26 stable consensus prognostic genes were screened in seven cohorts harboring complete survival information via univariate Cox regression analysis. Then, these 26 genes were further subjected to our integrative machine learning-based framework to establish a consensus prognostic signature (CPIS). For a prognostic signature, superior generalization capability is a target leading research to be pursued, which means the signature still retains robust performance across different validation cohorts. Hence, the C-index was measured in the other six validation cohorts and the signature with the highest average C-index (0.682) was regarded as the optimal one, which was derived by the classical machine learning algorithm Ridge.

1319

With the development of sequencing technology, transfer RNA (tRNA) -derived small RNAs (tsRNAs) have received extensive attention as a new type of small noncoding RNAs. Based on the differences in the cleavage sites of nucleases on tRNAs, tsRNAs can be divided into two categories, tRNA halves (tiRNAs) and tRNA-derived fragments (tRFs), each with specific subcellular localizations. Additionally, the biogenesis of tsRNAs is tissue-specific and can be regulated by tRNA modifications. In this review, we first elaborated on the classification and biogenesis of tsRNAs. After summarizing the latest mechanisms of tsRNAs, including transcriptional gene silencing, post-transcriptional gene silencing, nascent RNA silencing, translation regulation, rRNA regulation, and reverse transcription regulation, we explored the representative biological functions of tsRNAs in tumors. Furthermore, this review summarized the clinical value of tsRNAs in cancers, thus providing theoretical support for their potential as novel biomarkers and therapeutic targets.

1444

An accumulation of previous work has established organoids as good preclinical models of human tumors, facilitating translation from basic research to clinical practice. They are changing the paradigm of preclinical cancer research because they can recapitulate the heterogeneity and pathophysiology of human cancers and more closely approximate the complex tissue environment and structure found in clinical tumors than in vitro cell lines and animal models. However, the potential applications of cancer organoids remain to be comprehensively summarized. In the review, we firstly describe what is currently known about cancer organoid culture and then discuss in depth the basic mechanisms, including tumorigenesis and tumor metastasis, and describe recent advances in patient-derived tumor organoids (PDOs) for drug screening and immunological studies. Finally, the present challenges faced by organoid technology in clinical practice and its prospects are discussed. This review highlights that organoids may offer a novel therapeutic strategy for cancer research.

1363

1367

Lysine-specific demethylase 4 A (KDM4A, also named JMJD2A, KIA0677, or JHDM3A) is a demethylase that can remove methyl groups from histones H3K9me2/3, H3K36me2/3, and H1.4K26me2/me3. Accumulating evidence suggests that KDM4A is not only involved in body homeostasis (such as cell proliferation, migration and differentiation, and tissue development) but also associated with multiple human diseases, especially cancers. Recently, an increasing number of studies have shown that pharmacological inhibition of KDM4A significantly attenuates tumor progression in vitro and in vivo in a range of solid tumors and acute myeloid leukemia. Although there are several reviews on the roles of the KDM4 subfamily in cancer development and therapy, all of them only briefly introduce the roles of KDM4A in cancer without systematically summarizing the specific mechanisms of KDM4A in various physiological and pathological processes, especially in tumorigenesis, which greatly limits advances in the understanding of the roles of KDM4A in a variety of cancers, discovering targeted selective KDM4A inhibitors, and exploring the adaptive profiles of KDM4A antagonists. Herein, we present the structure and functions of KDM4A, simply outline the functions of KDM4A in homeostasis and non-cancer diseases, summarize the role of KDM4A and its distinct target genes in the development of a variety of cancers, systematically classify KDM4A inhibitors, summarize the difficulties encountered in the research of KDM4A and the discovery of related drugs, and provide the corresponding solutions, which would contribute to understanding the recent research trends on KDM4A and advancing the progression of KDM4A as a drug target in cancer therapy.

1399

Osteosarcoma is the most common malignant bone tumor affecting children and adolescents. Currently, the most common treatment is surgery combined with neoadjuvant chemotherapy. Although the survival rate of patients with osteosarcoma has improved in recent years, it remains poor when the tumor (s) progress and distant metastases develop. Therefore, better animal models that more accurately replicate the natural progression of the disease are needed to develop improved prognostic and diagnostic markers, as well as targeted therapies for both primary and metastatic osteosarcoma. The present review described animal models currently being used in research investigating osteosarcoma, and their characteristics, advantages, and disadvantages. These models may help elucidate the pathogenic mechanism (s) of osteosarcoma and provide evidence to support and develop clinical treatment strategies.

1419

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is an acquired immune system of many bacteria and archaea, comprising CRISPR loci, Cas genes, and its associated proteins. This system can recognize exogenous DNA and utilize the Cas9 protein's nuclease activity to break DNA doublestrand and to achieve base insertion or deletion by subsequent DNA repair. In recent years, multiple laboratory and clinical studies have revealed the therapeutic role of the CRISPR/Cas9 system in neurological diseases. This article reviews the CRISPR/Cas9-mediated gene editing technology and its potential for clinical application against neurological diseases.

1973

Neuregulin 4 (Nrg4), an epidermal growth factor (EGF) family member, can bind to and activate the ErbB4 receptor tyrosine kinase. Nrg4 has five different isoforms by alternative splicing and performs a wide variety of functions. Nrg4 is involved in a spectrum of physiological processes including neurobiogenesis, lipid metabolism, glucose metabolism, thermogenesis, and angiogenesis. In pathological processes, Nrg4 inhibits inflammatory factor levels and suppresses apoptosis in inflammatory diseases. In addition, Nrg4 could ameliorate obesity, insulin resistance, and cardiovascular diseases. Furthermore, Nrg4 improves non-alcoholic fatty liver disease (NAFLD) by promoting autophagy, improving lipid metabolism, and inhibiting cell death of hepatocytes. Besides, Nrg4 is closely related to the development of cancer, hyperthyroidism, and some other diseases. Therefore, elucidation of the functional role and mechanisms of Nrg4 will provide a clearer view of the therapeutic potential and possible risks of Nrg4.

1384

B-cell CLL/lymphoma 9 (BCL9) is considered a key developmental regulator and a well-established oncogenic driver in multiple cancer types, mainly through potentiating the Wnt/β-catenin signaling. However, increasing evidences indicate that BCL9 also plays multiple Wnt-independent roles. Herein, we summarized the updates of the canonical and non-canonical functions of BCL9 in cellular, physiological, or pathological processes. Moreover, we also concluded that the targeted inhibitors disrupt the interaction of β-catenin with BCL9 reported recently.

1418

N6-methyladenosine (m⁶A) is a dynamic and reversible epigenetic regulation. As the most prevalent internal post-transcriptional modification in eukaryotic RNA, it participates in the regulation of gene expression through various mechanisms, such as mRNA splicing, nuclear export, localization, translation efficiency, mRNA stability, and structural transformation. The involvement of m6A in the regulation of gene expression depends on the specific recognition of m6A-modified RNA by reader proteins. In the pathogenesis and treatment of liver disease, studies have found that the expression levels of key genes that promote or inhibit the development of liver disease are regulated by m⁶A modification, in which abnormal expression of reader proteins determines the fate of these gene transcripts. In this review, we introduce m⁶A readers, summarize the recognition and regulatory mechanisms of m⁶A readers on mRNA, and focus on the biological functions and mechanisms of m⁶A readers in liver cancer, viral hepatitis, non-alcoholic fatty liver disease (NAFLD), hepatic fibrosis (HF), acute liver injury (ALI), and other liver diseases. This information is expected to be of high value to researchers deciphering the links between m⁶A readers and human liver diseases.

1330

Hepatocellular carcinoma (HCC) is a liver cancer, highly heterogeneous both at the histopathological and molecular levels. It arises from hepatocytes as the result of the accumulation of numerous genomic alterations in various signaling pathways, including canonical WNT/β-catenin, AKT/mTOR, MAPK pathways as well as signaling associated with telomere maintenance, p53/cell cycle regulation, epigenetic modifiers, and oxidative stress. The role of WNT/β-catenin signaling in liver homeostasis and regeneration is well established, whereas in development and progression of HCC is extensively studied. Herein, we review recent advances in our understanding of how WNT/β-catenin signaling facilitates the HCC development, acquisition of stemness features, metastasis, and resistance to treatment. We outline genetic and epigenetic alterations that lead to activated WNT/β-catenin signaling in HCC. We discuss the pivotal roles of CTNNB1 mutations, aberrantly expressed non-coding RNAs and complexity of crosstalk between WNT/β-catenin signaling and other signaling pathways as challenging or advantageous aspects of therapy development and molecular stratification of HCC patients for treatment.

1507

In the mammalian heart, cardiomyocytes are forced to withdraw from the cell cycle shortly after birth, limiting the ability of the heart to regenerate and repair. The development of multimodal regulation of cardiac proliferation has verified that pre-existing cardiomyocyte proliferation is an essential driver of cardiac renewal. With the continuous development of genetic lineage tracking technology, it has been revealed that cell cycle activity produces polyploid cardiomyocytes during the embryonic, juvenile, and adult stages of cardiogenesis, but newly formed mononucleated diploid cardiomyocytes also elevated sporadically during myocardial infarction. It implied that adult cardiomyocytes have a weak regenerative capacity under the condition of ischemia injury, which offers hope for the clinical treatment of myocardial infarction. However, the regeneration frequency and source of cardiomyocytes are still low, and the mechanism of regulating cardiomyocyte proliferation remains further explained. It is noteworthy to explore what force triggers endogenous cardiomyocyte proliferation and heart regeneration. Here, we focused on summarizing the recent research progress of emerging endogenous key modulators and crosstalk with other signaling pathways and furnished valuable insights into the internal mechanism of heart regeneration. In addition, myocardial transcription factors, non-coding RNAs, cyclins, and cell cycle-dependent kinases are involved in the multimodal regulation of pre-existing cardiomyocyte proliferation. Ultimately, awakening the myocardial proliferation endogenous modulator and regeneration pathways may be the final battlefield for the regenerative therapy of cardiovascular diseases.

1337

Breast cancer is a molecularly heterogeneous disease and the most common female malignancy. In recent years, therapy approaches have evolved to accommodate molecular diversity, with a focus on more biologically based therapies to minimize negative consequences. To regulate cell fate in human breast cells, the Hippo signaling pathway has been associated with the alpha subtype of estrogen receptors. This pathway regulates tissue size, regeneration, and healing, as well as the survival of tissue-specific stem cells, proliferation, and apoptosis in a variety of organs, allowing for cell differentiation. Hippo signaling is mediated by the kinases MST1, MST2, LATS1, and LATS2, as well as the adaptor proteins SAV1 and MOB. These kinases phosphorylate the downstream effectors of the Hippo pathway, yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ), suppressing the expression of their downstream target genes. The Hippo signaling pathway kinase cascade plays a significant role in all cancers. Understanding the principles of this kinase cascade would prevent the occurrence of breast cancer. In recent years, small noncoding RNAs, or microRNAs, have been implicated in the development of several malignancies, including breast cancer. The interconnections between miRNAs and Hippo signaling pathway core proteins in the breast, on the other hand, remain poorly understood. In this review, we focused on highlighting the Hippo signaling system, its key parts, its importance in breast cancer, and its regulation by miRNAs and other related pathways.

1288

The physicochemical characteristics of RNA admit non-coding RNAs to perform a different range of biological acts through various mechanisms and are involved in regulating a diversity of fundamental processes. Notably, some reports of pathological conditions have proved abnormal expression of many non-coding RNAs guides the ailment. Vault RNAs are a class of non-coding RNAs containing stem regions or loops with well-conserved sequence patterns that play a fundamental role in the function of vault particles through RNA-ligand, RNA eRNA, or RNA-protein interactions. Taken together, vault RNAs have been proposed to be involved in a variety of functions such as cell proliferation, nucleocytoplasmic transport, intracellular detoxification processes, multidrug resistance, apoptosis, and autophagy, and serve as microRNA precursors and signaling pathways. Despite decades of investigations devoted, the biological function of the vault particle or the vault RNAs is not yet completely cleared. In this review, the current scientific assertions of the vital vault RNAs functions were discussed.

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R-spondins are secretory proteins localized in the endoplasmic reticulum and Golgi bodies and are processed through the secretory pathway. Among the R-spondin family, RSPO2 has emanated as a novel regulator of Wnt signaling, which has now been acknowledged in numerous in vitro and in vivo studies. Cancer is an abnormal growth of cells that proliferates and spreads uncontrollably due to the accumulation of genetic and epigenetic factors that constitutively activate Wnt signaling in various types of cancer. Colorectal cancer (CRC) begins when cells in the colon and rectum follow an indefinite pattern of division due to aberrant Wnt activation as one of the key hallmarks. Decades-long progress in research on R-spondins has demonstrated their oncogenic function in distinct cancer types, particularly CRC. As a critical regulator of the Wnt pathway, it modulates several phenotypes of cells, such as cell proliferation, invasion, migration, and cancer stem cell properties. Recently, RSPO mutations, gene rearrangements, fusions, copy number alterations, and altered gene expression have also been identified in a variety of cancers, including CRC. In this review, we addressed the recent updates regarding the recurrently altered R-spondins with special emphasis on the RSPO2 gene and its involvement in potentiating Wnt signaling in CRC. In addition to the compelling physiological and biological roles in cellular fate and regulation, we propose that RSPO2 would be valuable as a potential biomarker for prognostic, diagnostic, and therapeutic use in CRC.

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Immune checkpoint inhibitors (ICIs) are monoclonal antibody antagonists, which can block cytotoxic T lymphocyte antigen-4 (CTLA-4), programmed death-1/ligand-1 (PD-1/PD-L1) pathways, and other molecules exploited by tumor cells to evade T cell-mediated immune response. ICIs have transformed the treatment landscape for various cancers due to their amazing efficacy. Many anti-tumor therapies, including targeted therapy, radiotherapy, and chemotherapy, combine ICIs to make the treatment more effective. However, the off-target immune activation caused by ICIs may lead to a broad spectrum of immune-related adverse events (irAEs) affecting multiple organ systems. Among irAEs, cardiotoxicity induced by ICIs, uncommon but fatal, has greatly offset survival benefits from ICIs, which is heartbreaking for both patients and clinicians. Consequently, such cardiotoxicity requires special vigilance, and it has become a common challenge both for patients and clinicians. This article reviewed the clinical manifestations and influence of cardiotoxicity from the view of patients and clinicians, elaborated on the underlying mechanisms in conjunction with animal studies, and then attempted to propose management strategies from a cardio-immuno-oncology multidisciplinary perspective.

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NLRP3 inflammasome, an intracellular multiprotein complex, can be activated by a range of pathogenic microbes or endogenous hazardous chemicals. Its activation results in the release of cytokines such as IL-1b and IL-18, as well as Gasdermin D which eventually causes pyroptosis. The activation of NLRP3 inflammasome is under strict control and regulation by numerous pathways and mechanisms. Its excessive activation can lead to a persistent inflammatory response, which is linked to the onset and progression of severe illnesses. Recent studies have revealed that the subcellular localization of NLRP3 changes significantly during the activation process. In this review, we review the current understanding of the molecular mechanism of NLRP3 inflammasome activation, focusing on the subcellular localization of NLRP3 and the associated regulatory mechanisms. We aim to provide a comprehensive understanding of the dynamic transportation, activation, and degradation processes of NLRP3.

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Leukemia is a malignancy in the blood that develops from the lymphatic system and bone marrow. Although various treatment options have been used for different types of leukemia, understanding the molecular pathways involved in the development and progression of leukemia is necessary. Recent studies showed that leukemia stem cells (LSCs) play essential roles in the pathogenesis of leukemia by targeting several signaling pathways, including Notch, Wnt, Hedgehog, and STAT3. LSCs are highly proliferative cells that stimulate tumor initiation, migration, EMT, and drug resistance. This review summarizes cellular pathways that stimulate and prevent LSCs' self-renewal, metastasis, and tumorigenesis.

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Gastric cancer (GC) is one of the most common and deadly cancers worldwide. Early detection offers the best chance for curative treatment and reducing its mortality. However, the optimal population-based early screening for GC remains unmet. Aberrant DNA methylation occurs in the early stage of GC, exhibiting cancer-specific genetic and epigenetic changes, and can be detected in the media such as blood, gastric juice, and feces, constituting a valuable biomarker for cancer early detection. Furthermore, DNA methylation is a stable epigenetic alteration, and many innovative methods have been developed to quantify it rapidly and accurately. Nonetheless, large-scale clinical validation of DNA methylation serving as tumor biomarkers is still lacking, precluding their implementation in clinical practice. In conclusion, after a critical analysis of the recent existing literature, we summarized the evolving roles of DNA methylation during GC occurrence, expounded the newly discovered noninvasive DNA methylation biomarkers for early detection of GC, and discussed its challenges and prospects in clinical applications.

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Microrchidia CW-type zinc finger 2 (MORC2) is a member of the MORC superfamily of nuclear proteins. Growing evidence has shown that MORC2 not only participates in gene transcription and chromatin remodeling but also plays a key in human disease and tumor development by regulating the expression of downstream oncogenes or tumor suppressors. The present review provides an updated overview of MORC2 in the aspect of cancer hallmark and therapeutic resistance and summarizes its upstream regulators and downstream target genes. This systematic review may provide a favorable theoretical basis for emerging players of MORC2 in tumor development and new insight into the potential clinical application of basic science discoveries in the future.

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Glioblastoma (GBM) is the most common intrinsic and aggressive primary brain tumor in adults, with a median survival of approximately 15 months. GBM heterogeneity is considered responsible for the treatment resistance and unfavorable prognosis. Proneuralmesenchymal transition (PMT) represents GBM malignant progression and recurrence, which might be a breakthrough to understand GBM heterogeneity and overcome treatment resistance. PMT is a complicated process influenced by crosstalk between GBM and tumor microenvironment, depending on intricate ligand-receptor interactions. In this review, we summarize the autocrine and paracrine pathways in the GBM microenvironment and related ligand-receptor interactions inducing PMT. We also discuss the current therapies targeting the PMT-related autocrine and paracrine pathways. Together, this review offers a comprehensive understanding of the failure of GBM-targeted therapy and ideas for future tendencies of GBM treatment.

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m⁶A methylation is the most frequent modification of mRNA in eukaryotes and plays a crucial role in cancer progression by regulating biological functions. Insulin-like growth factor 2 mRNA-binding proteins (IGF2BP) are newly identified m⁶A'readers'. They belong to a family of RNA-binding proteins, which bind to the m⁶A sites on different RNA sequences and stabilize them to promote cancer progression. In this review, we summarize the mechanisms by which different upstream factors regulate IGF2BP in cancer. The current literature analyzed here reveals that the IGF2BP family proteins promote cancer cell proliferation, survival, and chemoresistance, inhibit apoptosis, and are also associated with cancer glycolysis, angiogenesis, and the immune response in the tumor microenvironment. Therefore, with the discovery of their role as'readers' of m⁶A and the characteristic re-expression of IGF2BPs in cancers, it is important to elucidate their mechanism of action in the immunosuppressive tumor microenvironment. We also describe in detail the regulatory and interaction network of the IGF2BP family in downstream target RNAs and discuss their potential clinical applications as diagnostic and prognostic markers, as well as recent advances in IGF2BP biology and associated therapeutic value.

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Ubiquitously transcribed tetratricopeptide repeat on chromosome X (UTX), also known as lysine (K) -specific demethylase 6A (KDM6A), functions as a tumor suppressor gene or oncogene depending on the tumor type and context. However, its tumor-suppressive mechanisms remain largely unknown. Here, we investigated the clinical significance and biological effects of UTX expression in pancreatic ductal adenocarcinoma (PDA) and determined the potential mechanisms of its dysregulation. UTX expression and its association with clinicopathologic characteristics of PDA patients were analyzed using immunohistochemistry. UTX mRNA and protein expression and their regulation in PDA cell lines were measured using quantitative polymerase chain reaction and Western blot analyses. The biological functions of UTX in PDA cell growth, migration, and invasion were determined using gain-and loss-of-function assays with both in vitro and in vivo animal models. UTX expression was reduced in human PDA cell lines and specimens. Low UTX expression was associated with poor differentiation and prognosis in PDA. Forced UTX expression inhibited PDA proliferation, migration, and invasion in vitro and PDA growth and metastasis in vivo, whereas knockdown of UTX expression did the opposite. Mechanistically, UTX expression was trans-activated by GATA6 activation. GATA6-mediated PDA progression could be blocked, at least partially, by silencing UTX expression. In conclusion, loss of GATA6-mediated UTX expression was evident in human PDA and restored UTX expression suppressed PDA growth and metastasis. Thus, UTX is a tumor suppressor in PDA and may serve as a prognostic biomarker and therapeutic target.

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As a widely used plasticizer, di- (2-ethylhexyl) phthalate (DEHP) is known to induce significant testicular injury. However, the potential mechanism and effects of pubertal exposure to DEHP on testis development remain unclear.In vivo, postnatal day (PND) 21 male rats were gavaged with 0, 250, and 500 mg/kg DEHP for ten days. Damage to the seminiferous epithelium and disturbed spermatogenesis were observed after DEHP exposure. Meanwhile, oxidative stress-induced injury and pyroptosis were activated. Both endoplasmic reticulum (ER) stress and mitophagy were involved in this process. Monoethylhexyl phthalate (MEHP) was used as the biometabolite of DEHP in vitro. The GC-1 and GC-2 cell lines were exposed to 0, 100 mM, 200 mM, and 400 mM MEHP for 24 h. Reactive oxygen species (ROS) generation, oxidative stress damage, ER stress, mitophagy, and pyroptosis were significantly increased after MEHP exposure. The ultrastructure of the ER and mitochondria was destroyed. X-box binding protein 1 (XBP1) was observed to be activated and translocated into the nucleus. ROS generation was inhibited by acetylcysteine. The levels of antioxidative stress, ER stress, mitophagy, and pyroptosis were decreased as well. After the administration of the ER stress inhibitor 4-phenyl-butyric acid, both mitophagy and pyroptosis were inhibited. Toyocamycininduced XBP1 down-regulation decreased the levels of mitophagy and pyroptosis. The equilibrium between pyroptosis and mitophagy was disturbed by XBP1 accumulation. In summary, our findings confirmed that DEHP induced a ROS-mediated imbalance in pyroptosis and mitophagy in immature rat testes via XBP1. Moreover, XBP1 might be the key target in DEHP-related testis dysfunction.

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Osteosarcoma is the most common primary malignancy of bones and primarily occurs in adolescents and young adults. However, a second smaller peak of osteosarcoma incidence was reported in the elderly aged more than 60. Elderly patients with osteosarcoma exhibit different characteristics compared to young patients, which usually results in a poor prognosis. The mechanism underlying osteosarcoma development in elderly patients is intriguing and of significant value in clinical applications. Senescent cells can accelerate tumor progression by metabolic reprogramming. Recent research has shown that methylmalonic acid (MMA) was significantly up-regulated in the serum of older individuals and played a central role in the development of aggressive characteristics. We found that the significant accumulation of MMA in elderly patients imparted proliferative potential to osteosarcoma cells. The expression of MAFB was excessively up-regulated in osteosarcoma specimens and was further enhanced in response to MMA accumulation as the patient aged. Specifically, we first confirmed a novel molecular mechanism between cellular senescence and cancer, in which the MMA-driven transcriptional reprogramming of the MAFB-NOTCH3 axis accelerated osteosarcoma progression via the activation of PI3K-AKT pathways. Moreover, the down-regulation of the MAFB-NOTCH3 axis increased the sensitivity and effect of AKT inhibitors in osteosarcoma through significant inhibition of AKT phosphorylation. In conclusion, we confirmed that MAFB is a novel age-dependent biomarker for osteosarcoma, and targeting the MAFB-NOTCH3 axis in combination with AKT inhibition can serve as a novel therapeutic strategy for elderly patients with osteosarcoma in experimental and clinical trials.

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Mutation of the MAPK7 gene was related to human scoliosis. Mapk7 regulated the development of limb bones and skulls in mice. However, the role of MAPK7 in vertebral development is still unclear. In this study, we constructed Col2a1-cre; Mapk7^f/f transgenic mouse model to delete Mapk7 in cartilage, which displayed kyphosis and osteopenia. Mechanistically, Mapk7 loss decreased MEF2C expression and thus activated PTEN to oppose PI3K/AKT signaling in vertebral growth plate chondrocytes, which impaired chondrocyte hypertrophy and attenuated vertebral ossification. In vivo, systemic pharmacological activation of AKT rescued impaired chondrocyte hypertrophy and alleviated mouse vertebral defects caused by Mapk7 deficiency. Our study firstly clarified the mechanism by which MAPK7 was involved in vertebral development, which might contribute to understanding the pathology of spinal deformity and provide a basis for the treatment of developmental disorders of the spine.

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Protein post-translational modifications (PTMs) are at the heart status of cellular signaling events and broadly involved in tumor progression. CD147 is a tumor biomarker with various PTMs, promoting tumor metastasis and metabolism reprogramming. Nevertheless, the relationship between the PTMs of CD147 and apoptosis has not been reported. In our study, we produced a specific anti-CD147-K71 di-methylation (CD147-K71me2) antibody by immunizing with a di-methylated peptide and observed that the level of CD147-K71me2 in non-small cell lung cancer (NSCLC) tissues were lower than that in NSCLC adjacent tissues. SETDB1 was identified as the methyltransferase catalyzing CD147 to generate CD147-K71me2. RNA-seq showed that FOSB was the most significant differentially expressed gene (DEG) between wild-type CD147 (CD147-WT) and K71-mutant CD147 (CD147-K71R) groups. Subsequently, we found that CD147-K71me2 promoted the expression of FOSB by enhancing the phosphorylation of p38, leading to tumor cell apoptosis. In vivo experiments showed that CD147-K71me2 significantly inhibited tumor progression by promoting cell apoptosis. Taken together, our findings indicate the inhibitory role of CD147-K71me2 in tumor progression from the perspective of post-translational modification, which is distinct from the pro-cancer function of CD147 itself, broadening our perspective on tumor-associated antigen CD147.

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Chronic myeloid leukemia (CML) is a common adult leukemia. Both the acute phase of the disease and the adverse effects of anti-cancer treatments can lead to a poor prognosis. The N⁶-methyladenine (m⁶A) modification plays an important regulatory role in various physiological and pathological processes. KIAA1429 is a known m⁶A regulator, but the biological role of KIAA1429 in CML is unclear. In this study, we observed that the m⁶A levels and KIAA1429 expression were significantly up-regulated in patients with blast phase CML. Notably, KIAA1429 regulated the total level of RNA m⁶A modification in the CML cells and promoted the malignant biological behaviors of CML cells, including proliferation, migration, and imatinib resistance. Inhibiting KIAA1429 in CML cells reduced the stability of RAB27B mRNA through the m⁶A/YTHDF1 axis, consequently inhibiting CML proliferation and drug efflux, ultimately increasing the sensitivity of CML cells to imatinib. Moreover, the knockdown of RAB27B also inhibited the proliferation and drug resistance of CML cells and promoted their apoptosis. Rucaparib, a recently developed anti-cancer agent, suppressed the expression of KIAA1429 and CML cell proliferation and promoted cell apoptosis. Rucaparib also inhibited the tumorigenesis of CML cells in vivo. The combined use of rucaparib and imatinib enhanced the sensitivity of CML cells to imatinib. Our study provides evidence that elevated KIAA1429 expression in the blast phase of CML enhances the stability of RAB27B mRNA through the m⁶A/YTHDF1 axis to up-regulate RAB27B expression, thereby promoting CML progression. Rucaparib exerts inhibitory effects on KIAA1429 expression and thus reduces CML progression.

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No well-established biomarkers are available for the clinical diagnosis of major depressive disorder (MDD). Vitamin D-binding protein (VDBP) is altered in plasma and postmortem dorsolateral prefrontal cortex (DLPFC) tissues of MDD patients. Thereby, the role of VDBP as a potential biomarker of MDD diagnosis was further assessed. Total extracellular vesicles (EVs) and brain cell-derived EVs (BCDEVs) were isolated from the plasma of first-episode drug-naïve or drug-free MDD patients and well-matched healthy controls (HCs) in discovery (20 MDD patients and 20 HCs) and validation cohorts (88 MDD patients and 38 HCs). VDBP level in the cerebrospinal fluid (CSF) from chronic glucocorticoid-induced depressed rhesus macaques or prelimbic cortex from lipopolysaccharide (LPS) -induced depressed mice and wild control groups was measured to evaluate its relationship with VDBP in plasma microgliaderived extracellular vesicles (MDEVs). VDBP was significantly decreased in MDD plasma MDEVs compared to HCs, and negatively correlated with HAMD-24 score with the highest diagnostic accuracy among BCDEVs. VDBP in plasma MDEVs was decreased both in depressed rhesus macaques and mice. A positive correlation of VDBP in MDEVs with that in CSF was detected in depressed rhesus macaques. VDBP levels in prelimbic cortex microglia were negatively correlated with those in plasma MDEVs in depressed mice. The main results suggested that VDBP in plasma MDEVs might serve as a prospective candidate biomarker for MDD diagnosis.

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Identified as the pathogenic genes of Alzheimer's disease (AD), APP, PSEN1, and PSEN2 mainly lead to early-onset AD, whose course is more aggressive, and atypical symptoms are more common than sporadic AD. Here, a novel missense mutation, APP E674Q (also named "Shanghai APP"), was detected in a Chinese index patient with typical late-onset AD (LOAD) who developed memory decline in his mid-70s. The results from neuroimaging were consistent with AD, where widespread amyloid b deposition was demonstrated in¹⁸F-florbetapir Positron Emission Tomography (PET).APP E674Q is close to the β-secretase cleavage site and the well-studied Swedish APP mutation (KM670/671NL), which was predicted to be pathogenic in silico. Molecular dynamics simulation indicated that the E674Q mutation resulted in a rearrangement of the interaction mode between APP and BACE1 and that the E674Q mutation was more prone to cleavage by BACE1. The in vitro results suggested that the E674Q mutation was pathogenic by facilitating the BACE1-mediated processing of APP and the production of Ab. Furthermore, we applied an adeno-associated virus (AAV) -mediated transfer of the human E674Q mutant APP gene to the hippocampi of two-month-old C57Bl/6 J mice. AAV-E674Q-injected mice exhibited impaired learning behavior and increased pathological burden in the brain, implying that the E674Q mutation had a pathogenicity that bore a comparison with the classical Swedish mutation. Collectively, we report a strong amyloidogenic effect of the E674Q substitution in AD. To our knowledge, E674Q is the only pathogenic mutation within the amyloid processing sequence causing LOAD.

1331

The endoplasmic reticulum (ER) membrane protein complex (EMC) is responsible for monitoring the biogenesis and synthetic quality of membrane proteins with tail-anchored or multiple transmembrane domains. The EMC subunit EMC6 is one of the core members of EMC and forms an enclosed hydrophilic vestibule in cooperation with EMC3. Despite studies demonstrating that deletion of EMC3 led to rhodopsin mislocalization in rod photoreceptors of mice, the precise mechanism leading to the failure of rhodopsin trafficking remains unclear. Here, we generated the first rod photoreceptor-specific knockout of Emc6 (RKO) and cone photoreceptor-specific knockout of Emc6 (CKO) mouse models. Deficiency of Emc6 in rod photoreceptors led to progressive shortening of outer segments (OS), impaired visual function, mislocalization and reduced expression of rhodopsin, and increased gliosis in rod photoreceptors. In addition, CKO mice displayed the progressive death of cone photoreceptors and abnormal localization of cone opsin protein. Subsequently, proteomics analysis of the RKO mouse retina illustrated that several cilium-related proteins, particularly anoctamin-2 (ANO2) and transmembrane protein 67 (TMEM67), were significantly down-regulated prior to OS degeneration. Detrimental rod photoreceptor cilia and mislocalized membrane disc proteins were evident in RKO mice. Our data revealed that in addition to monitoring the synthesis of rhodopsin-dominated membrane disc proteins, EMC6 also impacted rod photoreceptors' ciliogenesis by regulating the synthesis of membrane proteins associated with cilia, contributing to the mislocalization of membrane disc proteins.

1320

Bladder cancer (BC) is one of the most common malignant tumors in the urinary system. Due to the poor prognosis and high mortality rate of the disease, it is urgent to develop new drugs with high efficacy and low toxicity to treat BC. Echinatin (Ecn) is a bioactive naturalflavonoid oflicorice that has attracted special attention for its promising anti-tumor potential. Herein, we explored the inhibitory effects of Echinatin on BC cells and probed the possible molecular mechanism. We found that Ecnin vitro inhibited the proliferation, migration, and invasion, arrested the cell cycle at the G2/M phase, and promoted apoptosis in BC cells. Besides, Ecn had no notable cytotoxicity towards human normal cells. We subsequently confirmed that Ecn restrained xenograft tumor growth and metastasis of BC cells in vivo. Mechanistically, Ecn activated the p38 signaling pathway but inactivated the Wnt/β-catenin signaling pathway, while over-expression of β-catenin and the p38 inhibitor both attenuated the inhibitory effects of Ecn on BC cells. Remarkably, Ecn combined with cisplatin (DDP) or gemcitabine (Gem) had synergistic inhibitory effects on BC cells. In summary, our results validate that Ecn inhibits the tumor growth of human BC cells via p38 and Wnt/β-catenin signaling pathways. More meaningfully, our results suggest a potential strategy to enhance DDP-or Gem-induced inhibitory effects on BC cells by combining with Ecn.

1369

Vitamin A (VA) plays an essential role in modulating both the gut microbiota and gut barrier function. Short-chain fatty acids (SCFAs), as metabolites of the gut microbiota, protect the physiological intestinal barrier; however, they are compromised when VA is deficient. Thus, there is an urgent need to understand how and which SCFAs modulate colonic epithelial barrier integrity in VA deficiency (VAD). Herein, compared with normal VA rats (VAN), at the beginning of pregnancy, we confirmed that the colonic desmosome junction was impaired in the VAD group, and the amounts of acetate, propionate, and butyrate declined because of the decreased abundance of SCFA-producing bacteria (Romboutsia, Collinsella, and Allobaculum). The differentially expressed genes correlated with the gut barrier and the histone deacetylase complex between the VAD and VAN groups were enriched by RNA sequencing. In the VAD group, the expression levels of colonic CEA cell adhesion molecule 1 (CEACAM1) were down-regulated, and the levels of histone deacetylase 1 (HDAC1) and HDAC3 were up-regulated. Intriguingly, the above changes in the VAD groups were rescued by VA supplementation in the early postnatal period. Further study indicated that in Caco-2 cells, butyrate treatment significantly repressed the enrichment of HDAC3 on the promoter of the CEACAM1 gene to induce its expression. Our findings support that butyrate intervention can alleviate the impairment of colonic barrier function caused by VAD, and timely postnatal VA intervention may reverse the damage caused by VAD on gut barrier integrity during pregnancy.

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