Alternative polyadenylation reprogramming of MORC2 induced by NUDT21 loss promotes KIRC carcinogenesis.

Alternative polyadenylation (APA), a posttranscriptional mechanism of gene expression via determination of 3'UTR length, has an emerging role in carcinogenesis. Although abundant APA reprogramming is found in kidney renal clear cell carcinoma (KIRC), which is one of the major malignancies, whether APA functions in KIRC remains unknown. Herein, we found that chromatin modifier MORC2 gained oncogenic potential in KIRC among the genes with APA reprogramming, and moreover, its oncogenic potential was enhanced by 3'UTR shortening through stabilization of MORC2 mRNA. MORC2 was found to function in KIRC by downregulating tumor suppressor DAPK1 via DNA methylation. Mechanistically, MORC2 recruited DNMT3A to facilitate hypermethylation of the DAPK1 promoter, which was strengthened by 3'UTR shortening of MORC2. Furthermore, loss of APA regulator NUDT21, which was induced by DNMT3B-mediated promoter methylation, was identified as responsible for 3'UTR shortening of MORC2 in KIRC. Additionally, NUDT21 was confirmed to act as a tumor suppressor mainly depending on downregulation of MORC2. Finally, we designed an antisense oligonucleotide (ASO) to enhance NUDT21 expression and validated its antitumor effect in vivo and in vitro. This study uncovers the DNMT3B/NUDT21/APA/MORC2/DAPK1 regulatory axis in KIRC, disclosing the role of APA in KIRC and the crosstalk between DNA methylation and APA.

Alternative polyadenylation writer CSTF2 forms a positive loop with FGF2 to promote tubular epithelial-mesenchymal transition and renal fibrosis.

Effective therapies for renal fibrosis, the common endpoint for most kidney diseases, are lacking. We previously reported that alternative polyadenylation (APA) drives transition from acute kidney injury to chronic kidney disease, suggesting a potential role for APA in renal fibrogenesis. Here, we found that among canonical APA writers, CSTF2 expression was upregulated in tubular epithelial cells (TEC) of fibrotic kidneys. CSTF2 was also identified as a TGF-ß-inducible pro-fibrotic gene. Further analysis revealed that CSTF2 promoted epithelial-mesenchymal transition (EMT) and extracellular matrix (ECM) overproduction in TEC by inducing 3'UTR shortening and upregulation of the expression of basic fibroblast growth factor 2 (FGF2). Additionally, 3'UTR shortening stabilised FGF2 mRNA through miRNA evasion. Interestingly, FGF2 enhanced CSTF2 expression, leading to the forming of a CSTF2-FGF2 positive loop in TEC. Furthermore, CSTF2 knockdown alleviated unilateral ureteral obstruction-induced renal fibrosis in vivo. Finally, we developed a CSTF2-targeted antisense oligonucleotide (ASO) and validated its effectiveness in vitro. These results indicate that the expression of the APA writer, CSTF2, is upregulated by TGF-ß and CSTF2 facilitates TGF-ß-induced FGF2 overexpression, forming a TGF-ß-CSTF2-FGF2 pro-fibrotic axis in TEC. CSTF2 is a potentially promising target for renal fibrosis that does not directly disrupt TGF-ß.

NAT10 regulates mitotic cell fate by acetylating Eg5 to control bipolar spindle assembly and chromosome segregation.

Cell fate of mitotic cell is controlled by spindle assembly. Deficient spindle assembly results in mitotic catastrophe leading to cell death to maintain cellular homeostasis. Therefore, inducing mitotic catastrophe provides a strategy for tumor therapy. Nucleolar acetyltransferase NAT10 has been found to regulate various cellular processes to maintain cell homeostasis. Here we report that NAT10 regulates mitotic cell fate by acetylating Eg5. NAT10 depletion results in multinuclear giant cells, which is the hallmark of mitotic catastrophe. Live-cell imaging showed that knockdown of NAT10 dramatically prolongs the mitotic time and induces defective chromosome segregation including chromosome misalignment, bridge and lagging. NAT10 binds and co-localizes with Eg5 in the centrosome during mitosis. Depletion of NAT10 reduces the centrosome loading of Eg5 and impairs the poleward movement of centrosome, leading to monopolar and asymmetrical spindle formation. Furthermore, NAT10 stabilizes Eg5 through its acetyltransferase function. NAT10 acetylates Eg5 at K771 to control Eg5 stabilization. We generated K771-Ac specific antibody and showed that Eg5 K771-Ac specifically localizes in the centrosome during mitosis. Additionally, K771 acetylation is required for the motor function of Eg5. The hyper-acetylation mimic Flag-Eg5 K771Q but not Flag-Eg5 rescued the NAT10 depletion-induced defective spindle formation and mitotic catastrophe, demonstrating that NAT10 controls mitosis through acetylating Eg5 K771. Collectively, we identify Eg5 as an important substrate of NAT10 in the control of mitosis and provide K771 as an essential acetylation site in the stabilization and motor function of Eg5. Our findings reveal that targeting the NAT10-mediated Eg5 K771 acetylation provides a potential strategy for tumor therapy.

Redox Metabolism-Associated Molecular Classification of Clear Cell Renal Cell Carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma. Redox metabolism has been recognized as the hallmark of cancer. But the concrete role of redox-related genes in patient stratification of ccRCC remains unknown. Herein, we aimed to characterize the molecular features of ccRCC based on the redox gene expression profiles from The Cancer Genome Atlas. Differentially expressed redox genes (DERGs) and vital genes in metabolism regulation were identified and analyzed in the ccRCC. Consensus clustering was performed to divide patients into three clusters (C1, C2, and C3) based on 139 redox genes with median FPKM value > 1. We analyzed the correlation of clusters with clinicopathological characteristics, immune infiltration, gene mutation, and response to immunotherapy. Subclass C1 was metabolic active with moderate prognosis and associated with glucose, lipid, and protein metabolism. C2 had intermediate metabolic activity with worse prognosis and correlated with more tumor mutation burden, neoantigen, and aneuploidy, indicating possible drug sensitivities towards immune checkpoint inhibitors. Metabolic exhausted subtype C3 showed high cytolytic activity score, suggesting better prognosis than C1 and C2. Moreover, the qRT-PCR was performed to verify the expression of downregulated DERGs including ALDH6A1, ALDH1L1, GLRX5, ALDH1A3, and GSTM3, and upregulated SHMT1 in ccRCC. Overall, our study provides an insight into the characteristics of molecular classification of ccRCC patients based on redox genes, thereby deepening the understanding of heterogeneity of ccRCC and allowing prediction of prognosis of ccRCC patients.

A Cross-Sectional Survey of Family Care Behaviors for Children with Upper Respiratory Tract Infections in China: Are There Opportunities for Improvement?

OBJECTIVE: To describe family care behaviors for children with upper respiratory tract infections (URTIs) and explore related factors. DESIGN AND METHODS: Parents of children with URTIs were included in this cross-sectional study. Family care behaviors, disease-related knowledge, and parental self-efficacy were evaluated with validated measures. RESULTS: Among the 419 participants, 73.80% recognized diseases based on their children's abnormal presentation. Self-medication was the main home care measure (36.28% used only self-medication; 27.92% used both self-medication and physical cooling methods), and 36.5% received suggestions from medical professionals. All the participants took their children to the hospital, and 28.20% did so two or three times. The proportions of visits to level II or III hospitals were 49.64% and 83.87% for first and third hospital visits, respectively. Parents who had less disease knowledge and assessed children' diseases as more serious took their children to the hospital more often (p < 0.05); those whose nearest medical institution was a community health center were more likely to visit such centers (p < 0.001). CONCLUSIONS: Most of the parents recognized symptoms of URTIs and provided home care but lacked enough knowledge and professional support to take reasonable measures. Hospital visits were their primary choice. PRACTICAL IMPLICATIONS: Family care behaviors for children with URTIs could be improved through health education, and an internet nursing service or family doctor system is suggested. A hierarchical medical system is necessary to reduce hospital visits, as are more community health centers with pediatric services.

Alternative polyadenylation trans-factor FIP1 exacerbates UUO/IRI-induced kidney injury and contributes to AKI-CKD transition via ROS-NLRP3 axis.

NLRP3, a decisive role in inflammation regulation, is obviously upregulated by oxidative stress in kidney injury. The NLRP3 upregulation leads to unsolved inflammation and other pathological effects, contributing to aggravation of kidney injury and even transition to chronic kidney disease (CKD). However, the mechanism for NLRP3 upregulation and further aggravation of kidney injury remains largely elusive. In this study, we found NLRP3 3'UTR was shortened in response to kidney injury in vivo and oxidative stress in vitro. Functionally, such NLRP3 3'UTR shortening upregulated NLRP3 expression and amplified inflammation, fibrogenesis, ROS production and apoptosis, depending on stabilizing NLRP3 mRNA. Mechanistically, FIP1 was found to bind to pPAS of NLRP3 mRNA via its arginine-rich domain and to induce NLRP3 3'UTR shortening. In addition, FIP1 was upregulated in CKD specimens and negatively associated with renal function of CKD patients. More importantly, we found FIP1 was upregulated by oxidative stress and required for oxidative stress-induced NLRP3 upregulation, inflammation activation, cell damage and apoptosis. Finally, we proved that FIP1 silencing attenuated the inflammation activation, fibrogenesis, ROS production and apoptosis induced by UUO or IRI. Taken together, our results demonstrated that oxidative stress-upregulated FIP1 amplified inflammation, fibrogenesis, ROS production and apoptosis via inducing 3'UTR shortening of NLRP3, highlighting the importance of crosstalk between oxidative stress and alternative polyadenylation in AKI-CKD transition, as well as the therapeutic potential of FIP1 in kidney injury treatment.

A kindergarten-based, family-involved intervention to improve children's hand hygiene behavior: A cluster-randomized controlled trial.

OBJECTIVE: The present study determined the feasibility and initial efficacy of a kindergarten-based, family-involved intervention in improving children's hand hygiene (HH) behaviors. DESIGN: A cluster-randomized controlled trial was performed, with a cluster defined as a kindergarten class. SAMPLE: Participants were recruited from 20 classes in six kindergartens. A total of 289 children and their families were enrolled in the intervention group, and 293 children and their families were enrolled in the control group. MEASUREMENTS: HH behavior and a related knowledge survey, as well as data on absences due to infection, were collected. INTERVENTION: An 8-week training session on HH for children and an education program combining a seminar and WeChat groups for parents were provided to participants in the intervention group. RESULTS: Two HH behaviors of children, namely, HH after playing outside and 7-stage HH compliance, were significantly different between the two groups after the intervention. The two HH behaviors and knowledge of infections of parents/legal guardians in the intervention group were better than those in the control group after the intervention. The number of absences due to infections in children was lower in the intervention group than in the control group. CONCLUSIONS: Kindergarten-based, family-involved interventions effectively improved the HH behavior of kindergarten children and decreased absences due to infections.

Loss of nucleolar localization of NAT10 promotes cell migration and invasion in hepatocellular carcinoma.

NAT10, a nucleolar acetyltransferase, participates in a variety of cellular processes including ribosome biogenesis and DNA damage response. Immunohistochemistry staining showed that cytoplasmic and membranous NAT10 is related to the clinical pathologic characteristics in human cancer tissues. However, the mechanism about how NAT10 translocates from the nucleolus to cytoplasm and membrane is unclear. Here, we obtain a NAT10 deletion mutant localizing in cytoplasm and membrane. Bioinformatics analysis showed that residues 68-75 and 989-1018 are two potential nuclear localization signals (NLS) of NAT10. GFP-NAT10 deletion mutant (Δ989-1018) predominantly translocates into cytoplasm with faint signal retained in the nucleolus, while GFP-NAT10(Δ68-75) still remains in the nucleolus and nucleoplasm, indicating residues 989-1018 is the main nucleolar localization signal (NuLS). GFP-NAT10-D3, with both fragments (residues 68-75 and 989-1018) deleted, completely excludes from the nucleolus and translocates to cytoplasm and membrane. Therefore, complete NuLSs of NAT10 should include residues 68-75 and 989-1018. The cytoplasmic and membranous NAT10 mutant (Flag-NAT10-D3) colocalizes with α-tubulin in cytoplasm and with integrin on cell membrane. Importantly, Flag-NAT10-D3 promotes α-tubulin acetylation and stabilizes microtubules. Consequently, Flag-NAT10-D3 promotes migration and invasion in hepatocellular carcinoma (HCC) cells. Statistical analysis of immunohistochemistry staining of NAT10 in HCC tissues demonstrates that the cytoplasmic NAT10 is correlated with poorer prognosis compared with nuclear NAT10, while the membranous NAT10 predicts the poorest clinical outcome of the patients. We thus provide the evidence for the function of cytoplasmic and membranous NAT10 in the metastasis and prognosis of HCC patients.

NAT10 regulates p53 activation through acetylating p53 at K120 and ubiquitinating Mdm2.

As a genome guardian, p53 maintains genome stability by arresting cells for damage repair or inducing cell apoptosis to eliminate the damaged cells in stress response. Several nucleolar proteins stabilize p53 by interfering Mdm2-p53 interaction upon cellular stress, while other mechanisms by which nucleolar proteins activate p53 remain to be determined. Here, we identify NAT10 as a novel regulator for p53 activation. NAT10 acetylates p53 at K120 and stabilizes p53 by counteracting Mdm2 action. In addition, NAT10 promotes Mdm2 degradation with its intrinsic E3 ligase activity. After DNA damage, NAT10 translocates to nucleoplasm and activates p53-mediated cell cycle control and apoptosis. Finally, NAT10 inhibits cell proliferation and expression of NAT10 decreases in human colorectal carcinomas. Thus, our data demonstrate that NAT10 plays a critical role in p53 activation via acetylating p53 and counteracting Mdm2 action, providing a novel pathway by which nucleolar protein activates p53 as a cellular stress sensor.