Cell Cycle-related Genes Research Articles

The regulatory role of BMP4 in testicular Sertoli cells of Tibetan sheep.

This study aimed to determine the regulatory mechanism of bone morphogenetic protein 4 (BMP4) gene in the testes of Tibetan sheep and its role in the blood-testis barrier (BTB). First, we cloned BMP4 gene for bioinformatics analysis, and detected the mRNA and protein expression levels of BMP4 in the testes of Tibetan sheep pre-puberty (3-mo-old), during sexual maturity (1-yr-old), and in adulthood (3-yr-old) by qRT-PCR and Western blot. In addition, the subcellular localization of BMP4 was analyzed by immunohistochemical staining. Next, BMP4 overexpression and silencing vectors were constructed and transfected into primary Sertoli cells (SCs) to promote and inhibit the proliferation of BMP4, respectively. Then, CCK-8 was used to detect the proliferation effect of SCs. The expression of BMP4 and downstream genes, pathway receptors, tight junction-related proteins, and cell proliferation and apoptosis-related genes in SCs were studied using qRT-PCR and Western blot. The results revealed that the relative expression of BMP4 mRNA and protein in testicular tissues of 1Y group and 3Y group was dramatically higher than that of 3M group (P < 0.01), and BMP4 protein is mainly located in SCs and Leydig cells at different development stages. The CDS region of the Tibetan sheep BMP4 gene was 1,229 bp. CCK-8 results demonstrated that the proliferation rate of BMP4 was significantly increased in the overexpression group (pc-DNA-3.1(+)-BMP4; P < 0.05). In addition, the mRNA and protein expressions of SMAD5, BMPR1A, and BMPR1B and tight junction-related proteins Claudin11, Occludin, and ZO1 were significantly increased (P < 0.05). The mRNA expression of cell proliferation-related gene Bcl2 was significantly enhanced (P < 0.05), and the expression of GDNF was enhanced (P > 0.05). The mRNA expression of apoptosis-related genes Caspase3 and Bax decreased significantly (P < 0.05), while the mRNA expression of cell cycle-related genes CyclinA2 and CDK2 increased significantly (P < 0.05). It is worth noting that the opposite results were observed after transfection with si-BMP4. In summary, what should be clear from the results reported here is that BMP4 affects testicular development by regulating the Sertoli cells and BTB, thereby modulating the spermatogenesis of Tibetan sheep.

Combined effects of temperature and humidity on the interaction between tomato and Botrytis cinerea revealed by integration of histological characteristics and transcriptome sequencing.

The environment significantly impacts the interaction between plants and pathogens, thus remarkably affecting crop disease occurrence. However, the detailed combined mechanisms of temperature and humidity influencing this interaction remain unclear. In this study, the interaction between tomato and Botrytis cinerea in various temperature and humidity conditions was analyzed by histological observation and a dual RNA-seq approach. Results showed that low humidity was not favorable for mycelial growth, resulting in infection failure. Both high and low temperatures at high humidity successfully inhibited pathogenic infection and disease incidence in the tomato plants, thus enhancing their resistance to B. cinerea. The high temperature and high humidity (HH) treatment induced the upregulation of light reaction genes, increased the net photosynthetic rate, and expanded the chloroplast morphology of infected tomatoes. The HH treatment also inhibited the expression of cell cycle-related genes of B. cinerea, interfered with conidial germination and mycelial growth, and damaged mycelial cell structure. Low temperature and high humidity (LH) treatment induced the expression of cell wall modification genes and remodeled the cell wall morphology of tomatoes in response to B. cinerea. In addition, the downregulated fungal catabolic genes and the abnormal increase in electron density of mycelial cells under LH treatment subsequently reduced the infection ability of B. cinerea. These results further explain the coupled effects of temperature and humidity on plant defenses and pathogen virulence, and provide a potential means to control gray mold.

Biphasic transcriptional and posttranscriptional regulation of MYB by androgen signaling mediates its growth control in prostate cancer

MYB, a proto-oncogene, is overexpressed in prostate cancer (PCa) and promotes its growth, aggressiveness, and resistance to androgen-deprivation therapy. Here, we examined the effect of androgen signaling on MYB expression and delineated the underlying molecular mechanisms. Paralleling a dichotomous effect on growth, low-dose androgen induced MYB expression at both transcript and protein levels, whereas it was suppressed in high-dose androgen-treated PCa cells. Interestingly, treatment with both low- and high-dose androgen transcriptionally upregulated MYB by increasing the binding of androgen receptor to the MYB promoter. In a time-course assay, androgen induced MYB expression at early time points followed by a sharp decline in high-dose androgen-treated cells due to decreased stability of MYB mRNA. Additionally, profiling of MYB-targeted miRNAs demonstrated significant induction of miR-150 in high-dose androgen-treated PCa cells. We observed a differential binding of androgen receptor on miR-150 promoter with significantly greater occupancy recorded in high-dose androgen-treated cells than those treated with low-dose androgen. Functional inhibition of miR-150 relieved MYB suppression by high-dose androgen, while miR-150 mimic abolished MYB induction by low-dose androgen. Furthermore, MYB-silencing or miR-150 mimic transfection suppressed PCa cell growth induced by low-dose androgen, whereas miR-150 inhibition rescued PCa cells from growth repression by high-dose androgen. Similarly, we observed that MYB silencing suppressed the expression of androgen-responsive, cell cycle-related genes in low-dose androgen-treated cells, while miR-150 inhibition increased their expression in cells treated with high-dose androgen. Overall, these findings reveal novel androgen-mediated mechanisms of MYB regulation that support its biphasic growth control in PCa cells.

Identification of a novel cell cycle-related risk signature predicting prognosis in patients with pancreatic adenocarcinoma.

Growing evidence have indicated that cell cycle-related genes (CRGs) play an essential role in the progression of pancreatic adenocarcinoma (PAAD). Nevertheless, the application of CRGs in estimating the prognosis of PAAD patients is still lacking. This study aimed to establish a risk signature based on CRGs that can predict patients' overall survival for PAAD. The expression and corresponding clinical data of PAAD patients from The Cancer Genome Atlas database and 200 cell cycle-related genes from the MSigDB were used for the generation and validation of the signature. LASSO Cox regression was applied to build the prediction model. The diagnostic value of signature was evaluated by receiver operating characteristic curves. Univariate and multivariate regression was used to construct the nomogram providing the clinicians a useful tool. A total of 103 CRGs were identified. Seven genes (RBM14, SMAD3, CENPA, KIF23, NUSAP1, INCENP, SMC4) with non-zero coefficients in LASSO analysis were used to construct the prognostic signature. The 7-gene signature significantly stratified patients into high- and low-risk groups in terms of overall survival, and the area under the receiver operating characteristic curve of 5-year survival reached 0.749. Multivariate analysis showed that the signature is an independent prognostic factor. We then mapped a nomogram to predict 1-, 3-, and 5-year survival for PAAD patients. The calibration curves indicated that the model was reliable. Finally, we discovered that TP53 and KRAS mutated most frequently in low and high-risk groups, respectively. Our findings suggested that the seven genes identified in this study are valuable prognostic predictors for patients with PAAD. These findings provided us with a novel insight that it is useful for understanding cell cycle mechanisms and for identifying patients with PAAD with poor prognosis.

Exploring the mechanisms underlying the therapeutic effect of the Radix Bupleuri-Rhizoma Cyperi herb pair on hepatocellular carcinoma using multilevel data integration and molecular docking.

Traditional Chinese medicine (TCM) is a promising and effective treatment for cancer with minimal side effects through a multi-active ingredient multitarget network. Radix Bupleuri and Rhizoma Cyperi are listed as herbs dispersing stagnated liver Qi in China. They have been used clinically to treat liver diseases for many years and recent pharmacological studies have shown that they inhibit the proliferation of hepatocellular carcinoma (HCC). However, the pharmacological mechanisms, potential targets, and clinical value of the Radix Bupleuri-Rhizoma Cyperi herb pair (CXP) for suppressing HCC growth have not been fully elucidated. We identified 44 CXP targets involved in the treatment of HCC using the GEO dataset and HERB database. An analysis of the Traditional Chinese Medicine System Pharmacology Database (TCMSP) showed that CXP exerts synergistic effects through 4 active ingredients, including quercetin, stigmasterol, isorhamnetin, and kaempferol. GO and KEGG analyses revealed that CXP mainly regulates HCC progression through metabolic pathways, the p53 signaling pathway, and the cell cycle. Additionally, we applied The Cancer Genome Atlas (TCGA)-liver hepatocellular carcinoma (LIHC) database to perform the expression patterns, clinical features, and prognosis of 6 genes (CCNB1, CDK1, CDK4, MYC, CDKN2A, and CHEK1) in cell cycle pathways to reveal that CXP suppresses HCC clinical therapeutic value. Moreover, based on molecular docking, we further verified that CXP exerts its anti-HCC activity through the interaction of multiple active components with cell cycle-related genes. We systematically revealed the potential pharmacological mechanisms and targets of CXP in HCC using multilevel data integration and molecular docking strategies.

Functional and Molecular Heterogeneity of Aging Hematopoietic Stem Cells

Background: Aging hematopoiesis is characterized by increased numbers of hematopoietic stem cells (HSCs) that exhibit impaired self-renewal and long-term reconstitution potential. We previously demonstrated that differences in surface expression of c-Kit in young mouse HSCs could identify distinct functional HSC subsets, with c-Kithi HSCs exhibiting reduced self-renewal and megakaryocytic biased differentiation. We therefore sought to determine if similar functional and molecular differences may be observed in old HSCs. Methods: We evaluated c-Kithi and c-Kitlo subsets of HSCs (CD34-CD150+LSK) from young (8-12 wk), and old (18-20 mo) C57BL/6J mice. For competitive transplantation assays, 250 double sorted c-Kithi or c-Kitlo HSCs from aged CD45.2 mice were mixed with 5x105 unfractionated bone marrow mononuclear cells (BMMCs) from young CD45.1 mice. In parallel, we generated the chromatin accessibility and transcriptional profiles of c-Kithi and c-Kitlo HSCs from young and old mice. Results: We first performed competitive transplants of old c-Kithi and c-Kitlo HSCs into young recipients. These studies demonstrated age-related myeloid bias exhibited by both HSC subsets. In addition, similar to young HSCs, old c-Kithi HSCs exhibit significantly reduced long-term reconstitution capacity compared to old c-Kitlo HSCs. Next, to evaluate age-related functional differences, we performed equal competitive transplants in which equal numbers of c-Kithi or c-Kitlo old and young HSCs were transplanted into the same young recipient. These studies demonstrated that c-Kithi HSCs exhibit similar reconstituting capacity independent of age. In contrast, old c-Kitlo HSCs exhibited significantly reduced long-term reconstitution compared to their young counterparts, though with better-preserved self-renewal capacity, as reflected in their self-renewal quotients (SRQ). However, when we quantified the capacity of transplanted HSCs to give rise to downstream committed progenitors and mature hematopoietic cells, calculated as their differentiation quotient (DQ), old c-Kitlo HSCs exhibited markedly reduced DQ compared to their young counterparts. In contrast, c-kithi recipients showed that old c-Kithi HSCs gave rise to mature cell lineages more efficiently. To investigate molecular mechanisms driving heterogeneity in HSC aging, we evaluated the transcriptional profiles of young and old HSCs. Old c-Kitlo HSCs showed significant enrichment of genes associated with inflammation, interferon responses, and the quiescent state in comparison to old c-Kithi HSCs. On the other hand, old c-Kithi HSCs were enriched for cell cycle related and MYC target genes compared to old c-Kitlo HSCs and young HSCs. Old c-Kithi HSCs also exhibited increased expression of genes associated with high cell output state and high mitochondrial membrane potential, which is associated with a more active HSC cell state. To investigate the potential role of chromatin accessibility in determining the observed transcriptional changes, we performed ATAC-seq of young and old HSC subsets. Old HSCs exhibited more open chromatin peaks than young HSCs, with the number of peaks decreasing in the order of old c-Kitlo > old c-Kithi > young c-Kithi > young c-Kitlo HSCs. Evaluation of predicted transcription factor binding sites showed enrichment for CTCF and PU.1 sites in young c-Kitlo and c-Kithi HSCs, respectively, with PU.1 associated with high cell output states. In contrast, old c-Kitlo and c-Kithi HSCs were enriched for ERG and CTCF sites, respectively, with predicted binding sites enriched in genes associated with low and high cell output, respectively. These data suggest that differential ERG binding of quiescence and cellular output-related gene signatures regulate functional differences between old c-Kitlo and old c-Kithi HSCs. Conclusion: Overall, our studies demonstrate functional heterogeneity among old HSCs and identify a novel strategy to identify old HSCs with preserved self-renewal and long-term reconstitution capacity. Identifying and prospectively fractionating old HSCs offers a novel approach for investigating the molecular mechanisms underlying HSC aging.

Acute Anemic Stress Is Modulated By Intercellular Mitochondria Transport from Erythroblastic Island Macrophages to Early Erythroblasts Via CD47-Sirpα Interactions

Stress erythropoiesis can be modulated by multiple cellular and molecular mediators from early stages of erythroblasts and precursors via cell intrinsic and extrinsic mechanisms. To investigate novel cell autonomous and non-autonomous mechanisms to overcome erythroid stress, we adopted phenylhydrazine (PHZ) induced acute anemic stress in mice and analyzed bulk RNA-seq on splenic early erythroblasts (Ter119+CD44high), and found significant enrichment of the early erythroid progenitor populations, as well as up-regulation of cell-cycle, DNA replication, proliferation and mitochondrial metabolism related genes (Gata1, Tal1, Sox6, E2Fs, Lin54, Foxo3 etc), suggesting that the most prominent erythroblastic cell-autonomous responses to stress include proliferation and energy metabolism. Next, we aim to explore the cell extrinsic pathways to mediate stress erythropoiesis from the perspective of the erythroid niche, i.e., erythroblastic islands (EBIs) macrophages. Intercellular mitochondria transfer is an emerging topic with implications in multiple biological processes, while it remains elusive whether mitochondria can be transported intercellularly within the EBIs, and if such a phenomenon is functionally relevant. In our study, we infused EBI macrophages into PHZ or serial bleeding induced anemic mice, and demonstrated that exogenous EBI macrophage infusion accelerated the erythroid regeneration in both modes of acute anemic stressors in a dynamic manner. More interestingly, we infused EBI macrophage isolated from mito-Dendra2 mice that express trackable mitochondria, and observed that mitochondria are transferred from EBI macrophages to early stages of erythroblasts. We conducted in vitro co-culture of EBI macrophages isolated from mito-Dendra2 mice and early erythroblasts isolated from PHZ treated tdTomato-Vav-Cre mice, and observed that mitochondria transfer mainly occurs via cell-cell contact mediated direct uptake during erythroid stress. Single cell RNA-sequencing (scRNA-seq) revealed that early erythroblasts receiving mitochondria exhibit up-regulation of a myriad of of proliferation signatures that are enriched in RNA binding (Rps2, Rpl28, Rpl10a, etc.), translation/protein synthesis (Eif5a, Eef1g, Ybx1, etc.), ATP metabolism (Atp5o, Atp5d, Atp6v0e, etc.) related molecular functions. The result pointed to the functional importance of intercellular mitochondria transfer to mediate the bioenergetic profiles of recipient erythroblasts by enhancing their protein/ATP synthesis and proliferation. Furthermore, scRNA-seq analysis revealed a subset of metabolically active erythroid populations marked by CD47 that receive higher abundance of mitochondria, as well as exhibiting enrichment in proliferation signatures, suggesting a potential role of the CD47 labelled erythroblasts in mitochondria transfer mediated erythroid regeneration. Notably, CD47 or Sirpα blockade in vivo leads to remarkable decline in both mitochondria transfer events as well as their mediated erythroid recovery. Thus, we postulate that there is a clear association between CD47 and mitochondria transfer mediated stress erythropoiesis. Collectively, our study explored the cell intrinsic and extrinsic pathways of early erythroblasts' responses to overcome stress, and shed light on the regulatory mechanisms of mitochondria transfer through CD47-SIRPα mediated cell-cell contacts and/or their downstream signaling in the context of stress erythropoiesis.

EVI1 Promotes Immune-Evasive Microenvironment Via Cyclin D1 in Acute Myeloid Leukemia

Inappropriate expression of Ecotropic viral integration site 1 (EVI1) has been associated with dismal clinical outcomes in acute myeloid leukemia (AML), while EVI1high (EVI1+) AML cells do not show clear resistance to cytotoxic agents in vitro. Since EVI1 is essential for normal hematopoietic stem cells (HSCs), we performed chromatin-immunoprecipitation (ChIP)- and RNA-sequencing of murine EVI1+ AML cells and normal hematopoietic cells to identify factors involved explicitly in AML, and thereby to elucidate molecular cues to refractory EVI1+ AML. We firstly employed a murine EVI1-transformed AML (EVI1-AML) model, where murine hematopoietic cells exogenously expressing 3×FLAG-tagged EVI1 were transplanted into syngeneic mice. Using EVI1-AML cells, we next performed ChIP coupled to next-generation sequencing (ChIP-seq) by anti-FLAG tag antibody and transcriptome analysis by RNA-seq. We further generated another murine EVI1+ AML model by retrovirally transducing the KMT2A-MLLT1 fusion gene into Lineage- Sca-1+ c-kit+ cells from EVI1-GFP knock-in mice, where Evi1-IRES-GFP allele was inserted into the Evi1 locus to enable real-time monitoring of Evi1 expression by GFP, followed by transplantation into lethally irradiated syngeneic mice. Intriguingly, GFPhigh L-GMPs from KMT2A-MLLT1 AML mice showed distinct expression profiles from GFPlow L-GMPs, including high NF-kB activity and low stem cell activity, along with the lower frequency of leukemia-initiating cells in GFPhigh fraction. Consistently, an exogenous expression of EVI1 in KMT2A-MLLT1 AML cells without Evi1 expression decreased colony-forming and leukemia-initiating activity, suggesting a differential role of EVI1 in AML from normal HSCs, where EVI1 is essential in maintaining stem cell function. Combined with functional screening by cell proliferation-related short hairpin RNAs (shRNAs), we identified that cyclin D1 (CCND1) was a downstream target, commonly implied in the above analyses, and a potential therapeutic vulnerability of EVI1-AML. EVI1 bound to the region 3.8 kb upstream of the transcription start site of Ccnd1, through which EVI1 demonstrated strong transcriptional activity in a luciferase reporter assay. Genetic silencing of Ccnd1 and pharmacologic inhibition of Cyclin D1 in EVI1- and EVI1+ KMT2A-MLLT1 AML cells negatively affected proliferation and colony-forming activity in vitro. Moreover, in vivo knockdown of Ccnd1 strikingly impaired the leukemogenic capacity of murine EVI1-AML models. It was surprising that transcriptomic analysis of EVI1-AML cells with Ccnd1 silencing showed the downregulation of genes involved in chemokine production and response to interferon instead of cell cycle-related genes. The expression of Cd274 (PD-L1) as well as Stat1, the primary signaling mediator for type I and II IFN, was decreased in EVI1-AML cells with Ccnd1 knockdown. EVI1 directly bound to the promoter region of Stat1 and activated its transcription in line with a previous report, and indirectly up-regulated STAT1 expression via Cyclin D1. Accordingly, infiltrating CD4 and CD8 T cells from EVI1-shCcnd1 AML mice demonstrated decreased exhaustion markers, including LAG3, PDCD1, and TIGIT. Silencing of Stat1 and Ifngr, the receptor for IFN-γ, impaired the leukemogenic capacity of EVI1-AML in vivo, with a decrease in T and NK cells with exhaustion phenotypes, while Ifnar, the receptor for IFN-α, was not involved in this process. Genetic inhibition of the IFN-γ-STAT1 axis in non-EVI1+ AML models did not affect AML development, nor the frequency of immune cells expressing exhaustion-related molecules, suggesting this axis is specifically important in EVI1+ AML. By using human AML datasets, overexpression of EVI1 and CCND1 were associated with IFN-γ signature, including up-regulation of STAT1 and CD274, and elevated expression of chemokines, with increased exhaustion molecules in T cells. These data collectively suggest that cyclin D1 is implicated in the development of EVI1-AML through the formation of IFN-γ signature and exhausted T cell phenotypes, which could be potentially targeted.

ΔNp63α transcriptionally represses p53 target genes involved in the radiation-induced DNA damage response

BackgroundThe DNA damage response (DDR) is a mechanism that protects cells against radiation-induced oxidative DNA damage by causing cell cycle arrest and apoptosis. TP63 is a member of the tumour suppressor TP53 gene family, and ΔNp63α, a TP63 splicing variant, is constitutively expressed in the stem cell-containing basal layer of stratified epithelial tissues, including the mammary gland, where it plays a critical role in stemness and tissue development. ΔNp63α has been reported to transcriptionally inhibit the tumour suppression protein p53. This p53-repressive activity may cause genomic instability in epithelial stem cells exposed to radiation. In this study, we analysed the inhibitory effect of ΔNp63α on radiation-induced DDR.MethodsTo elucidate the role of the p53-repressive effect of ΔNp63α in radiation response, we performed a p63-siRNA knockdown experiment using human mammary epithelial cells (HMECs) expressing ΔNp63α and then performed ectopic and entopic expression experiments using human induced pluripotent stem cells (hiPSCs). After irradiation, the expression of DDR-related genes and proteins in ΔNp63α-expressing and control cells was analysed by RT–qPCR, Western blotting, and flow cytometry.ResultsThe mRNA/protein expression levels of BAX and p21 were significantly increased in p63-siRNA-treated HMECs (sip63) after X-ray irradiation (4 Gy, 0.7 Gy/min) but not in scramble-siRNA treated HMECs (scr). Transcriptomic analysis showed decreased RNA expression of cell cycle-related genes and increased expression of programmed cell death-related genes in sip63 cells compared to scr cells. Furthermore, flow cytometric analysis revealed an increase in apoptotic cells and a decrease in 5-ethynyl-2´-deoxyuridine uptake in sip63 cells compared to scr cells. On the other hand, both the ectopic and entopic expression of ΔNp63α in apoptosis-sensitive hiPSCs reduced the expression levels of BAX after irradiation and significantly decreased the number of apoptotic cells induced by radiation.ConclusionTaken together, these results indicate that ΔNp63α represses p53-related radiation-induced DDR, thereby potentially causing genomic instability in epithelial stem cells.

The Expression of Cell Cycle-Related Genes in USP8-Mutated Corticotroph Neuroendocrine Pituitary Tumors and Their Possible Role in Cell Cycle-Targeting Treatment.

Protein deubiquitinases USP8 and USP48 are known driver genes in corticotroph pituitary neuroendocrine tumors (PitNETs). USP8 mutations have pleiotropic effects that include notable changes in genes' expression. Genes involved in cell cycle regulation were found differentially expressed in mutated and wild-type tumors. This study aimed to verify difference in the expression level of selected cell cycle-related genes and investigate their potential role in response to cell cycle inhibitors. Analysis of 70 corticotroph PitNETs showed that USP8-mutated tumors have lower CDKN1B, CDK6, CCND2 and higher CDC25A expression. USP48-mutated tumors have lower CDKN1B and CCND1 expression. A lower p27 protein level in mutated than in wild-type tumors was confirmed that may potentially influence the response to small molecule inhibitors targeting the cell cycle. We looked for the role of USP8 mutations or a changed p27 level in the response to palbociclib, flavopiridol and roscovitine in vitro using murine corticotroph AtT-20/D16v-F2 cells. The cells were sensitive to each agent and treatment influenced the expression of genes involved in cell cycle regulation. Overexpression of mutated Usp8 in the cells did not affect the expression of p27 nor the response to the inhibitors. Downregulating or upregulating p27 expression in AtT-20/D16v-F2 cells also did not affect treatment response.

TMIC-75. TUMOR ENDOTHELIAL CELL-DERIVED ASPORIN DIFFERENTIALLY REGULATES GLIOBLASTOMA GROWTH

Abstract Gliomas are the most prevalent and malignant brain tumors. A major histopathological feature that distinguishes the high-grade (IV) glioblastoma (GBM) from slow-growing low-grade (II and III) glioma is microvascular hyperproliferation. Although gliomas are highly vascularized tumors, anti-angiogenic therapies have failed to prevent recurrence and significantly improve patient survival. To determine if tumor endothelial cells (TEC) display molecular heterogeneity, we performed bulk RNA-sequencing on cultured CD31+ TEC from low-grade (II/III), and high-grade (IV, primary and recurrent) gliomas. Low- and high-grade TEC exhibited significant molecular heterogeneity, with increased enrichment of extracellular matrix and cell cycle-related gene sets in low-grade and enrichment of cytokine and immune response-related gene sets in high-grade TEC. We also found significant functional differences in proliferation capacity, and resistance to anti-angiogenic therapies between low-and high-grade TEC. Co-transplantation of low-grade TEC with tumor cells significantly inhibited GBM growth and enhanced survival, whereas high-grade TEC promoted growth of tumors in orthotopic xenografts in vivo. Differential gene expression analysis revealed several proteoglycans distinctly expressed by low-grade TEC. Of the highly enriched molecules in low-grade TEC, Asporin (ASPN), a small leucine-rich repeat proteoglycan (SLRP) significantly inhibited growth and proliferation of IDH1-wt GBM cells but did not affect IDH1-mutant tumor cells. Co-transplantation of TEC-lacking ASPN with IDH1-wt GBM partially rescued the growth-inhibitory effect of low-grade TEC on tumor growth and reduced survival. Ongoing experiments are aimed at determining the molecular mechanism underlying the growth-inhibitory effects of TEC-derived Asporin on glioma.

Parasympathetic-macrophages-ductal epithelial cells axis promotes female rat submandibular gland regeneration after excretory duct ligation/deligation

ObjectiveSubmandibular gland (SMG) regeneration depends on parasympathetic signals that modulate this process primarily by promoting ductal epithelial cell proliferation. The purpose of this study was to elucidate the mechanism underlying of parasympathetic regulation on salivary gland regeneration. Materials and methodsWe performed duct ligation/deligation with or without chorda lingual innervation in female Sprague-Dawley rats. Clodronate liposomes were administered retroductally and intravenously into the SMG to deplete macrophages. The proliferative ability and cell cycle-related genes of SMG were assessed using western blotting, quantitative real-time PCR, and immunohistochemical staining. ResultsParasympathetic stimulation boosted Interleukin-6 (IL-6) release by macrophage via muscarinic signalling. This process was suppressed by denervation, muscarinic blockade, or SMG macrophage depletion. SMG IL-6 release led to signal transducer and activator of transcription 3 (STAT3) phosphorylation in ductal epithelial cells to promote proliferation and regeneration by upregulating the expression of cell cycle-related genes. SMG parasympathetic denervation and macrophage depletion suppressed cell cycle-related genes upregulation and ductal cells proliferation. ConclusionsThese data indicated that the parasympathetic-macrophage-ductal epithelial cells axis promote SMG regeneration after duct ligation and deligation.

Abstract 9501: Single-Cell Transcriptomic Analysis and Patient-Specific iPSCs Reveal Dysfunctional Coronary Arterial Endothelial Cells in Hypoplastic Left Heart Syndrome

Introduction: Hypoplastic left heart syndrome (HLHS) is a severe form of single ventricle congenital heart disease characterized by the underdeveloped left ventricle. Early serial postmortem examinations revealed a high rate of coronary artery abnormalities in HLHS fetal hearts (e.g., thickened wall and kinking arteries). However, the intrinsic defect in HLHS coronary vessels and its genetic basis remain unclear. Methods: We profiled human fetal heart with an underdeveloped left ventricle (ULV) and induced pluripotent stem cells derived endothelial cells (iPSC-ECs) from HLHS patients at single-cell resolution. CD144 + / NPR3 - vascular ECs were selected and classified as venous, arterial, and late arterial populations. To study the arterial EC phenotypes, we generated iPSC-arterial ECs (AECs, CDH5 + CXCR4 + NT5E -/low ) derived from 3 HLHS patients and 3 age-matched controls, and evaluated their functionalities including cell cycle regulation, angiogenesis, and inflammatory response. Results: Revealed by single cell RNA-seq and subsequent gene ontology analysis, ULV late arterial EC population showed significant defects in EC development, proliferation, angiogenesis, and Notch signaling compared to the control. Consistently, HLHS iPSCs exhibited impaired AEC differentiation judged by the reduced CXCR4 + NT5E -/low AEC progenitors. Mature HLHS iPSC-AECs showed reduced angiogenesis and enhanced G0/G1 cell cycle arrest with downregulated cell cycle-related genes (e.g., Ki67, CCND1/2 ). Healthy human aortic smooth muscle cells exhibited abnormal proliferation and synthetic phenotypes when co-cultured with HLHS iPSC-AECs. Additionally, NOTCH pathway genes (e.g., DLL4, HEY1, GJA5 ) were suppressed in both ULV AECs and HLHS iPSC-AECs. HLHS de novo variant KMT2D directly regulated the transcription of NOTCH targeted genes involved in arterial development and proliferation via H3K4me2. Intriguingly, the treatment of NOTCH ligands (Jag1, Dll1) significantly improved the proliferation of HLHS AECs. Conclusions: Our study revealed that HLHS coronary AECs were dysfunctional in angiogenesis, proliferation, and EC-SMC interaction. KMT2D-NOTCH signaling may contribute to the impaired development and proliferation of HLHS AECs.

Buffy Coat Transcriptomic Analysis Reveals Alterations in Host Cell Protein Synthesis and Cell Cycle in Severe COVID-19 Patients.

Transcriptome studies have reported the dysregulation of cell cycle-related genes and the global inhibition of host mRNA translation in COVID-19 cases. However, the key genes and cellular mechanisms that are most affected by the severe outcome of this disease remain unclear. For this work, the RNA-seq approach was used to study the differential expression in buffy coat cells of two groups of people infected with SARS-CoV-2: (a) Mild, with mild symptoms; and (b) SARS (Severe Acute Respiratory Syndrome), who were admitted to the intensive care unit with the severe COVID-19 outcome. Transcriptomic analysis revealed 1009 up-regulated and 501 down-regulated genes in the SARS group, with 10% of both being composed of long non-coding RNA. Ribosome and cell cycle pathways were enriched among down-regulated genes. The most connected proteins among the differentially expressed genes involved transport dysregulation, proteasome degradation, interferon response, cytokinesis failure, and host translation inhibition. Furthermore, interactome analysis showed Fibrillarin to be one of the key genes affected by SARS-CoV-2. This protein interacts directly with the N protein and long non-coding RNAs affecting transcription, translation, and ribosomal processes. This work reveals a group of dysregulated processes, including translation and cell cycle, as key pathways altered in severe COVID-19 outcomes.

Identification of the HAK gene family reveals their critical response to potassium regulation during adventitious root formation in apple rootstock

Adventitious root formation is a bottleneck during vegetative proliferation. Potassium (K+) is an essential macronutrient for plants. K+ accumulation from the soil and its distribution to the different plant organs is mediated by K+ transporters named K+ transporter (KT), K+ uptake (KUP), or high-affinity K+ (HAK). This study aimed to identify members of the HAK gene family in apples and to characterize the effects of K+ supply on adventitious root formation and on the expression of HAK genes and the genes that putatively control auxin transport, signaling, and cell fate during adventitious root formation. In this study, 34 HAK genes (MdHAKs) were identified in the apple (Malus × domestica ‘Golden Delicious’) genome. A phylogenetic analysis divided MdHAKs into four clusters (I, II, III, and IV), comprising 16, 1, 4, and 13 genes, respectively. The syntenic relationships revealed that 62.5% of the total MdHAK genes arise from genomic duplication events. Chromosome location, domain structure, motif analysis, and physico-chemical characteristics were subsequently investigated. Furthermore, the application of K+ indicated the emergence of adventitious roots at 8 d and produced more adventitious roots at 16 d than the K+-free control (CK) treatment. In addition, various MdHAKs showed root-specific expression in B9 apple rootstock stem cuttings and enhanced expression during the initiation and emergence stages of adventitious root formation in response to K+ treatment. Additionally, K+ treatment enhanced the expression levels of MdPIN1, MdPIN2, and MdAUX1. Further data indicated that a higher expression of MdWOX11, MdLBD16, and MdLBD29 and of cell cycle-related genes contributed to the auxin-stimulated adventitious root formation in response to K+.

DJ-1 promotes osteosarcoma progression through activating CDK4/RB/E2F1 signaling pathway

Osteosarcoma (OS) is a primary malignant tumor of the bone characterized by poor prognosis due to chemotherapy resistance and high recurrence rates. DJ-1 (PARK7) is known as an oncogene and its abnormal expression is related to the poor prognosis of various types of malignant tumors. It was found in this study that upregulated expression of DJ-1 was closely correlated with the prognosis of OS patients by promoting the proliferation, migration and chemotherapy resistance of OS cells in vitro through regulating the activity of CDK4 but not through the oxidation mechanism or AKT pathway. The combination of DJ-1 and CDK4 promoted RB phosphorylation, leading to the dissociation of E2F1 into the nucleus to regulate the expression of cell cycle-related genes. The tumor xenograft mouse model demonstrated that DJ-1 knockout suppressed tumor growth in vivo. All these findings indicate that DJ-1 can affect the occurrence and progression of OS by regulating the CDK/RB/E2F1axis, suggesting a novel therapeutic opportunity for OS patients.

E2F4 regulates cell cycle to mediate embryonic development in pigs

In mammals, E2 factor (E2F) acts as a cell cycle regulator. E2F transcription factor 4 (E2F4) is a member of the E2F family of transcription factors and usually represents predominant E2F activity in cells. The E2F4 gene has been extensively studied in animals and is associated with multiple functions, such as cell cycle regulation and apoptosis; however, little is known about its role during embryonic development. In this study, we investigated the function of E2F4 and its mechanism of action in porcine embryo development. For this purpose, we knocked down E2F4 by microinjecting double-stranded RNA of E2F4 at the 1-cell stage. The results showed that E2F4 knockdown in porcine embryos led to a significant decrease in the blastocyst rate and total cell number. Defective E2F4 expression reduced the level of G1/S checkpoints (cyclin E-cyclin-dependent kinase 2) and cell cycle-related gene expression at the 4-cell embryo stage and blastocyst. Moreover, a decrease in E2F4 expression increased phosphorylated H2A.X variant histones and activated ataxia telangiectasia mutated (ATM) and p53-p21 pathway. In addition, E2F4 depletion caused a significant decrease in histone acetylation. Taken together, E2F4 plays a critical role as a transcriptional activator in the development of porcine embryos, an observation that contradicts its well-established role as a transcription repressor.

Hydrogen peroxide is involved in methyl jasmonate-induced adventitious rooting in cucumber under cadmium stress

Cadmium (Cd) is a highly toxic heavy metal and one of the major soil pollution sources. Hydrogen peroxide (H2O2) and methyl jasmonate (Me-JA) act as vital signaling molecules that are involved in various physiological and phytochemical processes in plants. Here, cucumber (Cucumis sativus L. ‘Xinchun NO.4′) explants were used for studying whether H2O2 is involved in Me-JA-induced adventitious root formation under Cd stress. Five-day-old cucumber seedlings with primary roots removed were grown in petri dishes and maintained for 6 days at water (Control) and different reagents [Cd(NO3)2, H2O2, Me-JA, catalase (CAT), diphenylammonium chloride (DPI)]. The results indicate that both H2O2 and Me-JA treatments ameliorated Cd stress in a dose-dependent manner, and 50 μM H2O2 and 0.1 μM Me-JA had the most biological impact on adventitious root formation. In addition, the co-treatment of H2O2 and Me-JA could more effectively promote adventitious rooting under Cd stress. However, H2O2 scavenger CAT and H2O2 synthesis inhibitor DPI inhibited adventitious rooting induced by Me-JA under Cd stress. Thus, H2O2 may be involved in adventitious rooting induced by Me-JA under Cd stress. Meanwhile, H2O2 and Me-JA activated the cell cycle, leading to cell metastasize from G1 phase to S phase, while CAT and DPI could inhibit this metastasis. Both H2O2 and Me-JA up-regulated the expression of cell cycle-related genes (CycA, CycB, CDKA, CDKB), which was inhibited after adding CAT and DPI. Together, H2O2 may be involved in Me-JA-promoted adventitious rooting under Cd stress by activating cell cycle.

The transcriptional terminator XRN2 and the RNA-binding protein Sam68 link alternative polyadenylation to cell cycle progression in prostate cancer.

Alternative polyadenylation (APA) yields transcripts differing in their 3'-end, and its regulation is altered in cancer, including prostate cancer. Here we have uncovered a mechanism of APA regulation impinging on the interaction between the exonuclease XRN2 and the RNA-binding protein Sam68, whose increased expression in prostate cancer is promoted by the transcription factor MYC. Genome-wide transcriptome profiling revealed a widespread impact of the Sam68/XRN2 complex on APA. XRN2 promotes recruitment of Sam68 to its target transcripts, where it competes with the cleavage and polyadenylation specificity factor for binding to strong polyadenylation signals at distal ends of genes, thus promoting usage of suboptimal proximal polyadenylation signals. This mechanism leads to 3' untranslated region shortening and translation of transcripts encoding proteins involved in G1/S progression and proliferation. Thus, our findings indicate that the APA program driven by Sam68/XRN2 promotes cell cycle progression and may represent an actionable target for therapeutic intervention.

Myogenetic Oligodeoxynucleotides as Anti-Nucleolin Aptamers Inhibit the Growth of Embryonal Rhabdomyosarcoma Cells.

Embryonal rhabdomyosarcoma (ERMS) is the muscle-derived tumor retaining myogenic ability. iSN04 and AS1411, which are myogenetic oligodeoxynucleotides (myoDNs) serving as anti-nucleolin aptamers, have been reported to inhibit the proliferation and induce the differentiation of myoblasts. The present study investigated the effects of iSN04 and AS1411 in vitro on the growth of multiple patient-derived ERMS cell lines, ERMS1, KYM1, and RD. RT-PCR and immunostaining revealed that nucleolin was abundantly expressed and localized in nucleoplasm and nucleoli in all ERMS cell lines, similar to myoblasts. Both iSN04 and AS1411 at final concentrations of 10-30 μM significantly decreased the number of all ERMS cells; however, their optimal conditions were different among the cell lines. In all ERMS cell lines, iSN04 at a final concentration of 10 μM markedly reduced the ratio of EdU+ cells, indicating the inhibition of cell proliferation. Quantitative RT-PCR or immunostaining of phosphorylated histone H3 and myosin heavy chain demonstrated that iSN04 suppressed the cell cycle and partially promoted myogenesis but did not induce apoptosis in ERMS cells. Finally, both iSN04 and AS1411 at final concentrations of 10-30 μM disrupted the formation and outgrowth of RD tumorspheres in three-dimensional culture mimicking in vivo tumorigenesis. In conclusion, ERMS cells expressed nucleolin, and their growth was inhibited by the anti-nucleolin aptamers, iSN04 and AS1411, which modulates several cell cycle-related and myogenic gene expression. The present study provides evidence that anti-nucleolin aptamers can be used as nucleic acid drugs for chemotherapy against ERMS.