Cytoophidia: a conserved yet promising mode of enzyme regulation in nucleotide metabolism.

Nucleotide biosynthesis encompasses both de novo and salvage synthesis pathways, each characterized by significant material and procedural distinctions. Despite these differences, cells with elevated nucleotide demands exhibit a preference for the more intricate de novo synthesis pathway, intricately linked to modes of enzyme regulation. In this study, we primarily scrutinize the biological importance of a conserved yet promising mode of enzyme regulation in nucleotide metabolism-cytoophidia. Cytoophidia, comprising cytidine triphosphate synthase or inosine monophosphate dehydrogenase, is explored across diverse biological models, including yeasts, Drosophila, mice, and human cancer cell lines. Additionally, we delineate potential biomedical applications of cytoophidia. As our understanding of cytoophidia deepens, the roles of enzyme compartmentalization and polymerization in various biochemical processes will unveil, promising profound impacts on both research and the treatment of metabolism-related diseases.

Development of an integrated and project-based laboratory course in upper-level biochemistry and molecular biology.

An integrated and projected-based laboratory course was described, integrating interconnected knowledge points and biochemistry and molecular biology techniques on a research project-based system. The program, which served as an essential extension of theoretical courses to practice, was conducted with a sophomore of basic medical science who had completed the course in medical biochemistry and molecular biology. This course engaged students in learning "genetic manipulation" and "recombinant DNA technology" to understand the target gene's role in disease mechanics, thus altering evaluation and treatment for clinical disease. Students could master applied and advanced techniques, such as cell culture, transfection, inducing exogenous fusion protein expression, purifying protein and its concentration assay, quantitative polymerase chain reaction, and western bot analysis. This laboratory exercise links laboratory practices with the methods of current basic research. Students need to complete the experimental design report and laboratory report, which could be advantageous for improving their ability to write lab summaries and scientific papers in the future. The reliability and validity analyses were conducted on the questionnaire, and we examined students' satisfaction with the course and their gains from the course. The student feedback was generally positive, indicating that the exercise helped consolidate theoretical knowledge, increase scientific research enthusiasm, and provide a powerful tool to be a better person and make informed decisions.

Identification and validation of lactate metabolism-related genes in oxygen-induced retinopathy.

Retinopathy of Prematurity (ROP) is a multifactorial disease characterized by abnormal retinal vascular growth in premature infants, which is one of the leading causes of childhood blindness. Lactic acid metabolism may play an imperative role in the development of ROP, but there are still few relevant studies. Our team use a dataset GSE158799 contained 284 genes in 3 P17_OIR mice and 3 P30_OIR mice to identify 41 potentially differentially expressed lactate metabolism-related genes (LMRGs) related to ROP. Then through bioinformatics analysis, we strive to reveal the interaction, the enriched pathways and the immune cell infiltration among these LMRGs, and predict their functions and internal mechanisms. These DEGs may regulate lactate metabolism, leading to the changes of metabolism and immunity, thereby inducing the development of ROP. Our results will expand our understanding of the intrinsic mechanism of ROP and may be helpful for the directions for treatment of ROP in the future.

Circadian pattern subtyping unveiling distinct immune landscapes in breast cancer patients for better immunotherapy.

BACKGROUND: While epidemiological studies have established a firm link between circadian disruption and tumorigenesis, the role and mechanism are not fully understood, complicating the design of therapeutic targets related to circadian rhythms (CR). Here, we aimed to explore the intertumoral heterogeneity of CR and elucidate its impact on the tumor microenvironment (TME), drug sensitivity, and immunotherapy. METHODS: Based on unsupervised clustering of 28 CR genes, two distinct CR subtypes (cluster-A and cluster-B) were identified in the TCGA cohort. We further constructed a circadian rhythm signature (CRS) based on the CR genes primarily responsible for clustering to quantify CR activity and to distinguish CR subtypes of individual patients from external datasets. CR subtypes were evaluated by TME characteristics, functional annotation, clinical features, and therapeutic response. RESULTS: The cluster-B (low-CRS) group was characterized by highly enriched immune-related pathways, high immune cell infiltration, and high anti-tumor immunity, while the cluster-A (high-CRS) group was associated with immunosuppression, synaptic transmission pathways, EMT activation, poor prognosis, and drug resistance. Immunohistochemistry (IHC) results demonstrated that high CD8+ T cell infiltration was associated with low-CR-protein expression. Importantly, patients with low CRS were more likely to benefit from immune checkpoint blockade (ICB) treatment, possibly due to their higher tumor mutation burden (TMB), increased immune checkpoint expression, and higher proportion of "hot" immunophenotype. CONCLUSION: In a nutshell, the cross talk in CR could reflect the TME immunoreactivity in breast cancer. Besides providing the first comprehensive pathway-level analysis of CR in breast cancer, this work highlights the potential clinical utility of CR for immunotherapy.

The SMYD3-MTHFD1L-formate metabolic regulatory axis mediates mitophagy to inhibit M1 polarization in macrophages.

BACKGROUND: SMYD3 (protein 3 containing SET and MYND structural domains) belongs to the SMYD methylesterase family and is a histone lysine methyltransferase that promotes gene transcription mainly by catalysing the trimethylation of lysine at position 4 of histone subunit 3 (H3K4me3). Studies have shown that SMYD3 plays a key role in tumour cell proliferation and differentiation; however, its role in macrophage polarization is unclear. METHODS: We screened the M1- and M2-polarized macrophage differential histone modifying enzyme using bioinformatics analysis. The SMYD3 overexpression plasmid was transfected into M1 macrophages, and the SMYD3-regulated target gene was analysed by RNA-seq and ChIP-Seq. The effect of knocking down MTHFD1L on M1 polarization and the change of the intracellular metabolite formic acid content were investigated. M1 macrophages were stimulated with different concentrations of formic acid (2, 10 and 40 mM) to detect the expression of M1-related genes, ROS production, and changes in the expression of the mitophagy-related proteins LC3, PINK1 and p-Parkin. RESULTS: Here, we used bioinformatics to analyse SMYD3, a histone methyltransferase associated with M1 polarization; overexpression of SMYD3 significantly suppressed the LPS/IFN-γ-induced M1 phenotype in macrophages. RNA-seq analysis demonstrated that SMYD3 significantly activated the one-carbon folate metabolic pathway in M1 macrophages. In addition, we used ChIP-seq analysis to identify methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) as the target gene of the transcriptional activation by SMYD3 through H3K4me3 histone modification. Activation of MTHFD1L causes the accumulation of the intracellular metabolite formate. Exogenous stimulation with different concentrations of formate increased the expression of key genes involved in the mitochondrial respiratory chain complex, ROS production, and the expression of autophagy-related proteins LC3, PINK1, and p-Parkin and suppressed the expression of M1-related genes. CONCLUSIONS: Our study demonstrates that SMYD3 regulates the activity of the mitochondrial metabolic enzyme MTHFD1L through H3K4me3 histone methylation modification, promotes formate synthesis and induces mitophagy, which inhibits M1 polarization in macrophages.

Tyrosine hydroxylase involved in cuticle tanning and reproduction in the 28-spotted potato ladybeetle, Henosepilachna vigintioctopunctata.

BACKGROUND: Tyrosine hydroxylase (TH), a melanin synthesis pathway enzyme hydroxylating tyrosine into 3,4-dihydroxyphenylalanine, is involved in the pigmentation and sclerotization of insect cuticles. However, the role of TH in 28-spotted potato ladybeetle (Henosepilachna vigintioctopunctata), an emerging pest of the solanaceous crops has been explored to a limited extent. In this study, we integrated dietary RNA interference (RNAi) and hematoxylin and eosin (H&E) staining with various bioassays to analyze the role of tyrosine hydroxylase (HvTH) throughout the developmental processes of Henosepilachna vigintioctopunctata. RESULTS: The results revealed that ingestion of dsHvTH led to cuticle tanning impairment, arrested larval feeding in the first and second instars of Henosepilachna vigintioctopunctata, and subsequently resulted in 100% mortality. The H&E staining assays revealed that dsHvTH prevented new abdominal cuticle formation. A pharmacological study using 3-iodo-tyrosine (3-IT), a HvTH inhibitor, disrupted larval-larval-pupal cuticle tanning during the third-fourth instar larval development and eventually failed to pupate. Similarly, dsHvTH fed to fourth instars hindered larval-pupal-adult cuticle tanning, and the eclose adults were 100% malformed. Ingestion of dsHvTH or 3-IT significantly down-regulated HvTH, HvDDC, Hvebony, and Hvlaccase2 expression and reduced dopamine levels. Finally, HvTH silencing in adult females substantially reduced the offspring hatching rates. CONCLUSIONS: The collective results of the study suggested that HvTH plays conserved roles in larval-pupal-adult cuticle melanization and sclerotization while exhibiting a novel function in Henosepilachna vigintioctopunctata reproduction. © 2022 Society of Chemical Industry.

RNAi suppression of the nuclear receptor FTZ-F1 impaired ecdysis, pupation, and reproduction in the 28-spotted potato ladybeetle, Henosepilachna vigintioctopunctata.

Fushi-tarazu factor 1 (FTZF1) is an ecdysone-inducible transcription factor that plays a vital role during the metamorphosis in insects. In this study, we functionally characterized HvFTZ-F1 in H. vigintioctopunctata, a dreadful solanaceous crop pest, by using a dietary RNA interference technique. The HvFTZ-F1 expression levels were elevated in the 1st and 2nd-instars before molting and declined immediately after ecdysis. The HvFTZ-F1 silencing led to high mortality in the 1st instars, while the expression of the osmosis-regulative gene, HvAQPAn.G, was significantly increased in the 1st instars. HvFTZ-F1 silencing downregulated the Halloween and 20E-related genes, decreased the ecdysteroids titer, suppressed the expression of pigmentation-related genes, and reduced the catecholamines titer. In the 4th instars, HvFTZ-F1 silencing caused 100% mortality by arresting the development at the prepupal stage and preventing new abdominal cuticle formation. In the female adults, HvFTZ-F1 silencing caused an evident decrease in fecundity, prolonged the pre-oviposition period, reduced the number of eggs and hatching rate, severely atrophied the ovaries. Moreover, the 20E-related genes and the dopamine synthesis genes were suppressed in the dsHvFTZ-F1-treated females. Overall, our results revealed that HvFTZ-F1 regulates ecdysis, pupation, and reproduction in H. vigintioctopunctata, thereby could be a promising molecular target for the development of RNAi-based biopesticides to control H. vigintioctopunctata.

H3K9me3 represses G6PD expression to suppress the pentose phosphate pathway and ROS production to promote human mesothelioma growth.

The role of glucose-6-phosphate dehydrogenase (G6PD) in human cancer is incompletely understood. In a metabolite screening, we observed that inhibition of H3K9 methylation suppressed aerobic glycolysis and enhances the PPP in human mesothelioma cells. Genome-wide screening identified G6PD as an H3K9me3 target gene whose expression is correlated with increased tumor cell apoptosis. Inhibition of aerobic glycolysis enzyme LDHA and G6PD had no significant effects on tumor cell survival. Ablation of G6PD had no significant effect on human mesothelioma and colon carcinoma xenograft growth in athymic mice. However, activation of G6PD with the G6PD-selective activator AG1 induced tumor cell death. AG1 increased tumor cell ROS production and the resultant extrinsic and intrinsic death pathways, mitochondrial processes, and unfolded protein response in tumor cells. Consistent with increased tumor cell death in vitro, AG1 suppressed human mesothelioma xenograft growth in a dose-dependent manner in vivo. Furthermore, AG1 treatment significantly increased tumor-bearing mouse survival in an intra-peritoneum xenograft athymic mouse model. Therefore, in human mesothelioma and colon carcinoma, G6PD is not essential for tumor growth. G6PD acts as a metabolic checkpoint to control metabolic flux towards the PPP to promote tumor cell apoptosis, and its expression is repressed by its promotor H3K9me3 deposition.

Oral RNAi toxicity assay suggests clathrin heavy chain as a promising molecular target for controlling the 28-spotted potato ladybird, Henosepilachna vigintioctopunctata.

BACKGROUND: Use of RNA interference (RNAi) technology in effective pest management has been explored for decades. Henosepilachna vigintioctopunctata is a major solanaceous crop pest in Asia. In this study, the effects of the RNAi-mediated silencing of clathrin heavy chain in H. vigintioctopunctata were investigated. RESULTS: Feeding either the in vitro-synthesized or the bacterially expressed double-stranded RNAs (dsRNAs) significantly impaired the normal physiology of H. vigintioctopunctata instars and adults. However, the bacterially expressed dsHvChc caused higher mortality than the in vitro-synthesized ones in the larvae and adults. Moreover, on evaluating the potential risk of dsHvChc on Propylea japonica, significant transcriptional effects of dsHvChc1 were observed, while the organismal level effects were not significant. On the contrary, dsHvChc2 did not affect P. japonica at either level. A similar test revealed significant transcriptional effects of dsPjChc1 on H. vigintioctopunctata, while staying ineffective at the organismal levels. Conversely, dsPjChc2 did not affect H. vigintioctopunctata at either level. Importantly, no effect of dsPjChc1 exposure on H. vigintioctopunctata suggested that other factors besides the 21-nucleotide (nt) matches between sequences were responsible. Finally, ingestion of dsHvmChc1 derived from H. vigintioctomaculata, containing 265-nt matches with dsHvChc1, caused 100% mortality in H. vigintioctopunctata. CONCLUSIONS: We conclude that (i) species with numerous 21-nt matches in homologous genes are more likely to be susceptible to dsRNA; (ii) dsRNA can be safely designed to avoid negative effects on non-target organisms at both transcriptional and organismal levels; (iii) HvChc can be used as an efficient RNAi target gene to effectively manage H. vigintioctopunctata. © 2021 Society of Chemical Industry.

Toxicity of fluralaner against vegetable pests and its sublethal impact on a biocontrol predatory ladybeetle.

Fluralaner, a systemic pesticide, was originally registered with the US Food and Drug Administration in 2014 under the trade name Bravecto for flea treatment for pets. As a GABA antagonist, the footprint of fluralaner has expended beyond medical and veterinary pests in recent years. In this study, we examined the acute toxicity of fluralaner against three pests of Henosepilachna vigintioctopunctata, Megalurothrips usitatus, and Phyllotreta striolata in the Solanaceae, Fabaceae, and Cruciferae families, respectively, and the sublethal impact of fluralaner on Propylaea japonica, a widely distributed predatory ladybeetle. Based on LC50, fluralaner was effective against H. vigintioctopunctata (0.098 mg a.i. L-1 for the second instar larvae), M. usitatus (0.134 mg a.i. L-1 for adult females), and P. striolata (0.595 mg a.i. L-1 for adults). For P. japonica, however, fluralaner was substantially less effective (1.177 mg a.i. L-1 for the third instar larvae). Furthermore, the LC10 and LC30 of P. japonica were also consistently higher than the LC50 of the three pests. In addition, we did not observe any significant impacts of fluralaner at LC10 and LC30 on the life history traits, including body weight, developmental time, pre-oviposition period, and fecundity of P. japonica. Based on our results from acute toxicities and sublethal impacts, fluralaner is effective against vegetable pests, while potentially friendly to P. japonica when employed as a biological control agent.

WDR5-H3K4me3 epigenetic axis regulates OPN expression to compensate PD-L1 function to promote pancreatic cancer immune escape.

BACKGROUND: Despite PD-L1 (Programmed death receptor ligand-1) expression on tumor cells and cytotoxic T lymphocytes tumor infiltration in the tumor microenvironment, human pancreatic cancer stands out as one of the human cancers that does not respond to immune checkpoint inhibitor (ICI) immunotherapy. Epigenome dysregulation has emerged as a major mechanism in T cell exhaustion and non-response to ICI immunotherapy, we, therefore, aimed at testing the hypothesis that an epigenetic mechanism compensates PD-L1 function to render pancreatic cancer non-response to ICI immunotherapy. METHODS: Two orthotopic pancreatic tumor mouse models were used for chromatin immunoprecipitation-Seq and RNA-Seq to identify genome-wide dysregulation of H3K4me3 and gene expression. Human pancreatic tumor and serum were analyzed for osteopontin (OPN) protein level and for correlation with patient prognosis. OPN and PD-L1 cellular location were determined in the tumors using flow cytometry. The function of WDR5-H3K4me3 axis in OPN expression were determined by Western blotting. The function of H3K4me3-OPN axis in pancreatic cancer immune escape and response to ICI immunotherapy was determined in an orthotopic pancreatic tumor mouse model. RESULTS: Mouse pancreatic tumors have a genome-wide increase in H3K4me3 deposition as compared with normal pancreas. OPN and its receptor CD44 were identified being upregulated in pancreatic tumors by their promoter H3K4me3 deposition. OPN protein is increased in both tumor cells and tumor-infiltrating immune cells in human pancreatic carcinoma and is inversely correlated with pancreatic cancer patient survival. OPN is primarily expressed in tumor cells and monocytic myeloid-derived suppressor cells (M-MDSCs), whereas PD-L1 is expressed in tumor cells, M-MDSCs, polymorphonuclear MDSCs and tumor-associated macrophages. WDR5 is essential for H3K4me3-specific histone methyltransferase activity that regulates OPN expression in tumor cells and MDSCs. Inhibition of WDR5 significantly decreased OPN protein level. Inhibition of WDR5 or knocking out of OPN suppressed orthotopic mouse pancreatic tumor growth. Inhibition of WDR5 also significantly increased efficacy of anti-PD-1 immunotherapy in suppression of mouse pancreatic tumor growth in vivo. CONCLUSIONS: OPN compensates PD-L1 function to promote pancreatic cancer immune escape. Pharmacological inhibition of the WDR5-H3K4me3 epigenetic axis is effective in suppressing pancreatic tumor immune escape and in improving efficacy of anti-PD-1 immunotherapy in pancreatic cancer.

Innovations in education of the medical molecular biology curriculum during the COVID-19 pandemic in China.

The COVID-19 pandemic is a huge challenge to education systems. Most governments around the world have temporarily closed schools, universities, and colleges. At the same time, teachers and students are encouraged to use the online and distance learning programs and platforms as an alternative. In the present study, we proposed a series of innovative solutions in Medical Molecular Biology education during the COVID-19 pandemic in China, including a flipped classroom model, live streaming course, chat Apps, and scientific papers on COVID-19 as additional learning material. Our results demonstrated that these innovations not only help teachers to maintain the teaching process as usual but also be useful for protecting students from psychological trauma. Our study indicates that online education with a well-designed workflow for conducting provides an alternative approach for teachers to maintain quality education during the onset of the emerging crisis.

Macrophage balance fraction determines the degree of immunosuppression and metastatic ability of breast cancer.

Macrophages are important immune cells in the tumor microenvironment and can be divided into two polarized subtypes, M1 and M2. M1 type macrophages have anti-tumor effects, while M2 type macrophages have pro-tumor effect. Most of the current researches are limited to the effect of M1 or M2 macrophages on tumors, while ignoring the overall balance of macrophages. Our research suggests that the macrophage balance fraction (MBF) can more effectively and comprehensively reflect the balance of tumor associated macrophages. Using bioinformatics analysis and in vitro experiments, we found that MBF is also an effective indicator of the degree of immunosuppression and metastatic ability of breast cancer, and different MBF environment can impact the migration and invasion ability of breast cancer cells. Finally, we also found that the mechanism of MBF changes in breast cancer may be affected by breast cancer-derived exosomes. In summary, MBF was proposed and validated as a novel indicator of macrophage balance state. Using this indicator, we found that the balance of macrophages can affect the degree of immunosuppression and metastatic ability of breast cancer.

RNA interference-mediated silencing of vATPase subunits A and E affect survival and development of the 28-spotted ladybeetle, Henosepilachna vigintioctopunctata.

RNA interference (RNAi) has emerged as a powerful tool for developing novel management strategies for controlling insect pests. The 28-spotted ladybeetle, Henosepilachna vigintioctopunctata is one of the most important pests attacking solanaceous plants in Asia. In this study, the potential of dietary RNAi to manage H. vigintioctopunctata was investigated using both in vitro synthesized and bacterially expressed double-stranded RNAs (dsRNAs) of HvvATPase A and HvvATPase E. The expression levels of HvvATPase A and HvvATPase E were higher in Malpighian tubules than in other tissue types. The silencing of HvvATPase A and HvvATPase E led to significant mortality in H. vigintioctopunctata larvae. In addition, the ingestion of HvvATPase A and HvvATPase E significantly deterred feeding behavior and subsequently arrested the development of H. vigintioctopunctata. Notably, the bacterially expressed dsRNAs consistently caused higher mortality in larvae and adults. Finally, the nontarget effects of the dsRNAs of H. vigintioctopunctata on the predatory ladybeetle Propylaea japonica were evaluated. P. japonica 1st instar larvae were administered vATPase A and vATPase E dsRNAs from H. vigintioctopunctata and P. japonica under the worst-case scenario, in which dsGFP served as negative control. There were significant effects of dsHvvATPase A on P. japonica at the transcriptional level but not at the organismal level, whereas dsHvvATPase E did not effect P. japonica at either the transcriptional or the organismal level. Collectively, the results of the study suggest that HvvATPase A and HvvATPase E can act as novel molecular targets for the control of H. vigintioctopunctata.

Oral delivery of dsHvlwr is a feasible method for managing the pest Henosepilachna vigintioctopunctata (Coleoptera: Coccinellidae).

RNA interference (RNAi) techniques have emerged as powerful tools that facilitate development of novel management strategies for insect pests, such as Henosepilachna vigintioctopunctata (Coleoptera: Coccinellidae), which is a major pest of solanaceous plants in Asia. In this study, the potential of oral delivery of in vitro-synthesized and bacterially expressed double-stranded H. vigintioctopunctata lesswright (lwr) gene (dsHvlwr) to manage of H. vigintioctopunctata was investigated. Our results showed that the gene Hvlwr had a 480-bp open reading frame and encoded a 160-amino acid protein. Hvlwr expression levels were greater in the fat body than other tissue types. Hvlwr silencing led to greater H. vigintioctopunctata mortality rates and appeared to be time- and partially dose-dependent, likely as a result of the number of hemocytes increasing with dsRNA concentration, but decreasing with time. Bacterially expressed dsHvlwr that was applied to leaf discs caused 88%, 66%, and 36% mortality in 1st instars, 3rd instars, and adults after 10, 10, and 14 d, respectively; when applied to living plants, there was greater mortality in 1st and 3rd instars, but there was no effect on adults. Furthermore, dsHvlwr led to improved plant protection against H. vigintioctopunctata. Our study shows an effective dietary RNAi response in H. vigintioctopunctata and that Hvlwr is a promising RNAi target gene for control of this pest species.

Persistent STAT5 activation reprograms the epigenetic landscape in CD4+ T cells to drive polyfunctionality and antitumor immunity.

The presence of polyfunctional CD4+ T cells is often associated with favorable antitumor immunity. We report here that persistent activation of signal transducer and activator of transcription 5 (STAT5) in tumor-specific CD4+ T cells drives the development of polyfunctional T cells. We showed that ectopic expression of a constitutively active form of murine STAT5A (CASTAT5) enabled tumor-specific CD4+ T cells to undergo robust expansion, infiltrate tumors vigorously, and elicit antitumor CD8+ T cell responses in a CD4+ T cell adoptive transfer model system. Integrated epigenomic and transcriptomic analysis revealed that CASTAT5 induced genome-wide chromatin remodeling in CD4+ T cells and established a distinct epigenetic and transcriptional landscape. Single-cell RNA sequencing analysis further identified a subset of CASTAT5-transduced CD4+ T cells with a molecular signature indicative of progenitor polyfunctional T cells. The therapeutic significance of CASTAT5 came from our finding that adoptive transfer of T cells engineered to coexpress CD19-targeting chimeric antigen receptor (CAR) and CASTAT5 gave rise to polyfunctional CD4+ CAR T cells in a mouse B cell lymphoma model. The optimal therapeutic outcome was obtained when both CD4+ and CD8+ CAR T cells were transduced with CASTAT5, indicating that CASTAT5 facilitates productive CD4 help to CD8+ T cells. Furthermore, we provide evidence that CASTAT5 is functional in primary human CD4+ T cells, underscoring its potential clinical relevance. Our results implicate STAT5 as a valid candidate for T cell engineering to generate polyfunctional, exhaustion-resistant, and tumor-tropic antitumor CD4+ T cells to potentiate adoptive T cell therapy for cancer.

Autocrine IL6-Mediated Activation of the STAT3-DNMT Axis Silences the TNFα-RIP1 Necroptosis Pathway to Sustain Survival and Accumulation of Myeloid-Derived Suppressor Cells.

Although accumulation of myeloid-derived suppressor cells (MDSC) is a hallmark of cancer, the underlying mechanism of this accumulation within the tumor microenvironment remains incompletely understood. We report here that TNFα-RIP1-mediated necroptosis regulates accumulation of MDSCs. In tumor-bearing mice, pharmacologic inhibition of DNMT with the DNA methyltransferease inhibitor decitabine (DAC) decreased MDSC accumulation and increased activation of antigen-specific cytotoxic T lymphocytes. DAC-induced decreases in MDSC accumulation correlated with increased expression of the myeloid cell lineage-specific transcription factor IRF8 in MDSCs. However, DAC also suppressed MDSC-like cell accumulation in IRF8-deficient mice, indicating that DNA methylation may regulate MDSC survival through an IRF8-independent mechanism. Instead, DAC decreased MDSC accumulation by increasing cell death via disrupting DNA methylation of RIP1-dependent targets of necroptosis. Genome-wide DNA bisulfite sequencing revealed that the Tnf promoter was hypermethylated in tumor-induced MDSCs in vivo. DAC treatment dramatically increased TNFα levels in MDSC in vitro, and neutralizing TNFα significantly increased MDSC accumulation and tumor growth in tumor-bearing mice in vivo. Recombinant TNFα induced MDSC cell death in a dose- and RIP1-dependent manner. IL6 was abundantly expressed in MDSCs in tumor-bearing mice and patients with human colorectal cancer. In vitro, IL6 treatment of MDSC-like cells activated STAT3, increased expression of DNMT1 and DNMT3b, and enhanced survival. Overall, our findings reveal that MDSCs establish a STAT3-DNMT epigenetic axis, regulated by autocrine IL6, to silence TNFα expression. This results in decreased TNFα-induced and RIP1-dependent necroptosis to sustain survival and accumulation. SIGNIFICANCE: These findings demonstrate that targeting IL6 expression or function represent potentially effective approaches to suppress MDSC survival and accumulation in the tumor microenvironment.

An integrative bioinformatics analysis identified miR-375 as a candidate key regulator of malignant breast cancer.

MicroRNAs (miRNAs) are key regulators that play important biological roles in carcinogenesis and are promising biomarkers for cancer diagnosis and therapy. hsa-miR-375-3p (miR-375) has been suggested to serve as a tumor suppressor or oncogene in various tumor types; however, its specific expression and potential regulatory role in malignant breast cancer remain unclear. In this study, the results from noncoding RNA microarray analysis indicated that the miR-375 expression level is significantly decreased in malignant basal-like breast cancer compared with luminal-like breast cancer. A total of 1895 co-downregulated and 1645 co-upregulated genes were identified in miR-375 mimic-transfected basal-like breast cancer cell lines. Predicted miR-375 targets were obtained from the online databases TargetScan and DIANA-microT-CDS. Combined KEGG enrichment analysis for coregulated genes and predicted miR-375 targets provided information and revealed differences in potential dynamic signaling pathways regulated by miR-375 and also indicated specific regulatory pathways, such as RNA transport and processing, in basal-like breast cancer. Additionally, gene expression microarray analysis accompanied by UALCAN analysis was performed to screen upregulated genes in the basal-like subtype. Four potential key genes, including LDHB, CPNE8, QKI, and EIF5A2, were identified as candidate target genes of miR-375. Therefore, the present study demonstrated that miR-375 may be a potential key regulator and provide a promising direction for diagnostic and therapeutic developments for malignant breast cancer.

Protective Roles and Mechanisms of Taurine on Myocardial Hypoxia/Reoxygenation-Induced Apoptosis.

BACKGROUND: This study aimed to investigate the protective roles and mechanisms of taurine on myocardial ischemia/reperfusion (I/R)-induced cell apoptosis, and thus provide evidence for the treatment of myocardial I/R injury and the development of related drugs. METHODS: The cardiomyocytes of neonatal rats were used to prepare the hypoxia/reoxygenation (H/R) injury model; gene transfection and small interfering RNA (siRNA) target gene silencing techniques were performed, along with methyl thiazolyl tetrazolium (MTT) assay to detect cell survival, flow cytometry to detect cell apoptosis, and Western blot to measure protein expressions. RESULTS: Compared with the H/R group, the apoptosis rates of cardiomyocytes in the three taurine (TAU)-protection groups were significantly decreased (p < 0.05). As the TAU concentration increased, the expression of Bcl-2 protein in H/R cardiomyocytes also gradually increased (p < 0.05), while the protein expressions of p53 up-regulated modulator of apoptosis (PUMA), C/EBP homologous protein (CHOP), Bax, glucose-regulated protein 78kD (GRP78), and caspase-3 gradually decreased (p < 0.01). TAU strongly downregulated the expression of PUMA-transfected cardiomyocytes. After targeted silencing of PUMA, the apoptosis rate was significantly decreased, while the expression of Bcl-2 protein was increased, and that of Bax protein was decreased (p < 0.05). CONCLUSIONS: TAU significantly inhibited myocardial H/R-induced apoptosis, and the mechanism may be related to a downregulated expression of PUMA.

Screening of immunosuppressive factors for biomarkers of breast cancer malignancy phenotypes and subtype-specific targeted therapy.

We aimed to screen and validate immunosuppressive factors in luminal- and basal-like breast cancer cell lines and tissue samples associated with malignant phenotypes. The mRNA microarray datasets, GSE40057 and GSE1561, were downloaded and remodeled, and differentially expressed genes were identified. Weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) and KEGG pathway enrichment analysis were performed to explore the immune-related events related to the basal-like breast cancer. The online resources, GOBO, Kaplan-Meier Plotter and UALCAN, were employed to screen for immunosuppressive factors associated with breast cancer malignant phenotypes. Immunohistochemistry was used to evaluate VEGFA and MIF levels in breast tumors and normal breast tissues; qPCRs and western blots were used to validate the expression of clinical immuno-oncology (IO) therapeutic targets CD274 (PD-L1) and IL8 in cell lines. The results showed that various immune-related events contribute to basal-like breast cancer. First, TGFß1 and IL8 had higher average expression levels in more malignant cell lines; second, MIF and VEGFA had higher average expression levels in more malignant breast cancer tissues, and the high expression levels were associated with poor survival rate. Third, IO targets CD274 and IL8 which were confirmed to be more suitable for the treatment of basal-like breast cancer. In view of the above, during the formation and development of breast cancer, immune-related genes are always activated, and immunosuppressive factors, IL8, TGFß1, MIF, and VEGFA are up-regulated. Such molecules could be used as biomarkers for breast cancer prognosis. However, because individual immune-related factors can play several biological roles, the mechanistic relationship between immunosuppressive factors and breast cancer malignant phenotypes and the feasibility of their application as drug targets require further investigation.