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Acral melanoma (AM) exhibits a high incidence in Asian patients with melanoma, and it is not well treated with immunotherapy. However, little attention has been paid to the characteristics of the immune microenvironment in AM. Therefore, in this study, we collected clinical samples from Chinese patients with AM and conducted single-cell RNA sequencing to analyze the heterogeneity of its tumor microenvironments (TMEs) and the molecular regulatory network. Our analysis revealed that genes, such asAcral melanoma is a type of cancer that affects the hands and feet. It tends to form on the palms, soles, and under the nails. It is rare in people of European descent, but in Asian populations it makes up more than half of all melanoma cases. Unlike other types of skin cancer, it does not respond well to immunotherapy, but scientists did not understand why. Historically, cancer research has focused on the genetics of whole tumors. But cancer is complicated. Malignant cells recruit other cells to help them survive and grow, and to protect them from attacks by the immune system. Together, they create their own ecosystem, called the tumor microenvironment. The exact makeup of the tumor microenvironment differs depending on the type of cancer and on the genetics of the individual. Investigating the cells that ‘support’ the tumor could help to explain how acral melanoma develops and why it does not respond to treatment. To address these questions, He et al. collected samples from six patients with acral melanoma and examined the genes used by more than 60,000 individual cells. This revealed nine different types of cells in the tumor microenvironment. Most were cancer cells, but there were also immune cells, blood vessel cells, skin cells, and a type of cell that makes connective tissue. He et al. also identified four genes that most likely shape the tumor microenvironment, and two gene pairs that may control some of the interactions between the cells. Investigating these early findings in more detail could open new treatment avenues for acral melanoma. The number of samples in this study was small, but it provides a starting point for future investigation. With more data, researchers could start to develop treatments that target the unique tumor microenvironment of this type of cancer.

Erythropoiesis is a complex and sophisticated multi-stage process regulated by a variety of factors, including the transcription factor GATA1 and non-coding RNA. GATA1 is regarded as an essential transcriptional regulator promoting transcription of erythroid-specific genes—such as long non-coding RNAs (lncRNA). Here, we comprehensively screened lncRNAs that were potentially regulated by GATA1 in erythroid cells. We identified a novel lncRNA—PCED1B-AS1—and verified its role in promoting erythroid differentiation of K562 erythroid cells. We also predicted a model in which PCED1B-AS1 participates in erythroid differentiation via dynamic chromatin remodeling involving GATA1. The relationship between lncRNA and chromatin in the process of erythroid differentiation remains to be revealed, and in our study we have carried out preliminary explorations.

Background Heart failure with preserved ejection fraction (HFpEF), affected collectively by genetic and environmental factors, is the common subtype of chronic heart failure. Although the available risk assessment methods for HFpEF have achieved some progress, they were based on clinical or genetic features alone. Here, we have developed a deep learning framework, HFmeRisk, using both 5 clinical features and 25 DNA methylation loci to predict the early risk of HFpEF in the Framingham Heart Study Cohort. Results The framework incorporates Least Absolute Shrinkage and Selection Operator and Extreme Gradient Boosting-based feature selection, as well as a Factorization-Machine based neural network-based recommender system. Model discrimination and calibration were assessed using the AUC and Hosmer–Lemeshow test. HFmeRisk, including 25 CpGs and 5 clinical features, have achieved the AUC of 0.90 (95% confidence interval 0.88–0.92) and Hosmer–Lemeshow statistic was 6.17 (P = 0.632), which outperformed models with clinical characteristics or DNA methylation levels alone, published chronic heart failure risk prediction models and other benchmark machine learning models. Out of them, the DNA methylation levels of two CpGs were significantly correlated with the paired transcriptome levels (R P Conclusion Our study proposes an early risk assessment framework for HFpEF integrating both clinical and epigenetic features, providing a promising path for clinical decision making.

Myeloid leukemias are highly diverse diseases and have been shown to be associated with microRNA (miRNA) expression aberrations. The present study involved an in-depth miRNome analysis of two human acute myeloid leukemia (AML) cell lines, HL-60 and THP-1, and one human chronic myeloid leukemia (CML) cell line, K562, via massively parallel signature sequencing. mRNA expression profiles of these cell lines that were established previously in our lab facilitated an integrative analysis of miRNA and mRNA expression patterns. miRNA expression profiling followed by differential expression analysis and target prediction suggested numerous miRNA signatures in AML and CML cell lines. Some miRNAs may act as either tumor suppressors or oncomiRs in AML and CML by targeting key genes in AML and CML pathways. Expression patterns of cell type-specific miRNAs could partially reflect the characteristics of K562, HL-60 and THP-1 cell lines, such as actin filament-based processes, responsiveness to stimulus and phagocytic activity. miRNAs may also regulate myeloid differentiation, since they usually suppress differentiation regulators. Our study provides a resource to further investigate the employment of miRNAs in human leukemia subtyping, leukemogenesis and myeloid development. In addition, the distinctive miRNA signatures may be potential candidates for the clinical diagnosis, prognosis and treatment of myeloid leukemias.

To explore the mechanisms controlling erythroid differentiation and development, we analyzed the genome-wide transcription dynamics occurring during the differentiation of human embryonic stem cells (HESCs) into the erythroid lineage and development of embryonic to adult erythropoiesis using high throughput sequencing technology. HESCs and erythroid cells at three developmental stages: ESER (embryonic), FLER (fetal), and PBER (adult) were analyzed. Our findings revealed that the number of expressed genes decreased during differentiation, whereas the total expression intensity increased. At each of the three transitions (HESCs–ESERs, ESERs–FLERs, and FLERs–PBERs), many differentially expressed genes were observed, which were involved in maintaining pluripotency, early erythroid specification, rapid cell growth, and cell–cell adhesion and interaction. We also discovered dynamic networks and their central nodes in each transition. Our study provides a fundamental basis for further investigation of erythroid differentiation and development, and has implications in using ESERs for transfusion product in clinical settings.

Carbon monoxide (CO) is a vasoactive molecule that is generated by vascular cells as a byproduct of heme catabolism and it plays an important physiological role in circulation system. In order to investigate whether exogenous CO can mediate the growth and proliferation of vascular cells, in this study, we used 250 parts per million (ppm) of CO to treat human umbilical artery smooth muscle cell (hUASMC) and human umbilical vein endothelial cell (HuVEC) and further evaluated the growth and apoptosis status of SMC and HuVEC. After SMC and HuVEC were exposed to CO for 7-day, the growth of SMC and HuVEC was significantly inhibited by CO in vitro on day 5 of CO exposure. And CO blocked cell cycle progress of SMC and HuVEC, more SMC and HuVEC stagnated at G0/G1 phase by flow cytometric analysis. Moreover, CO treatment inhibited SMC and HuVEC apoptosis caused by hydrogen peroxide through decreasing caspase 3 and 9 activities. To confirm the molecular mechanism of CO effect on SMC and HuVEC growth, we compared the gene expression profile in SMC and CO-treated SMC, HuVEC and CO-treated HuVEC. By microarray analysis, we found the expression level of some genes which are related to cell cycle regulation, cell growth and proliferation, and apoptosis were changed during CO exposure. We further identified that the down-regulated CDK2 contributed to arresting cell growth and the down-regulated Caspase 3 (CASP3) and Caspase 9 (CASP9) were associated with the inhibition of cell apoptosis. Therefore, CO exerts a certain growth arrest on SMC and HuVEC by inhibiting cell cycle transition from G0/G1 phase to S phase and has regulatory effect on cell apoptosis by regulating the expression of apoptosis-associated genes.

Carbon monoxide (CO) is a vasoactive molecule that is generated by vascular cells as a byproduct of heme catabolism and it plays an important physiological role in circulation system. In order to investigate whether exogenous CO can mediate the growth and proliferation of vascular cells, in this study, we used 250 parts per million (ppm) of CO to treat human umbilical artery smooth muscle cell (hUASMC) and human umbilical vein endothelial cell (HuVEC) and further evaluated the growth and apoptosis status of SMC and HuVEC. After SMC and HuVEC were exposed to CO for 7-day, the growth of SMC and HuVEC was significantly inhibited by CO in vitro on day 5 of CO exposure. And CO blocked cell cycle progress of SMC and HuVEC, more SMC and HuVEC stagnated at G0/G1 phase by flow cytometric analysis. Moreover, CO treatment inhibited SMC and HuVEC apoptosis caused by hydrogen peroxide through decreasing caspase 3 and 9 activities. To confirm the molecular mechanism of CO effect on SMC and HuVEC growth, we compared the gene expression profile in SMC and CO-treated SMC, HuVEC and CO-treated HuVEC. By microarray analysis, we found the expression level of some genes which are related to cell cycle regulation, cell growth and proliferation, and apoptosis were changed during CO exposure. We further identified that the down-regulated CDK2 contributed to arresting cell growth and the down-regulated Caspase 3 (CASP3) and Caspase 9 (CASP9) were associated with the inhibition of cell apoptosis. Therefore, CO exerts a certain growth arrest on SMC and HuVEC by inhibiting cell cycle transition from G0/G1 phase to S phase and has regulatory effect on cell apoptosis by regulating the expression of apoptosis-associated genes.

Sp1-like and Kruppel-like factors (Sp1/KLFs) are a family of zinc-finger proteins that are important components of transcriptional machinery in eukaryotic cells. Sp1/KLFs have highly conserved DNA binding domains at the carboxyl terminus that have three tandem zinc-finger motifs. Their amino terminal regions vary greatly and contain transcription regulatory domains that interact with co-regulators. By regulating the expression of a large number of genes containing GC-rich or CACCC promoters in a tissue-, cell- and developmental stage-specific manner, Sp1/KLFs may be involved in many biological processes, including cell proliferation, differentiation, apoptosis and neoplastic transformation. In this article, we reviewed the structure, molecular mechanisms and biological functions of Sp1/KLFs.

Background Non-sentinel lymph node (NSLN) status prediction with molecular biomarkers may make some sentinel lymph node (SLN) positive breast cancer patients avoid the axillary lymph node dissection, but the available markers remain limited. Methods SLN positive patients with and without NSLN invasion were selected, and genes differentially expressed or fused in SLN metastasis were screened by next-generation RNA sequencing. Results Six candidates (all ER/PR+, HER2−, Ki-67

Expression levels of 98 down-regulated genes and 62 up-regulated genes in the NSLN positive group. A table showing the expression values of regulated genes.

The schematic diagram of other fused genes. A figure showing how the rest of gene fusions occurred. The two genes that fused together were shown in blue and green. There fused point was shown in a vertical red line.

Classification of genes based on the expression levels. A table showing how the gene expression levels were classified.

The results of RNA extraction. A table listing the quality of RNA extraction.

Specifically expressed genes in the NSLN negative and â positive groups. A table showing the FPKM values of specifically expressed genes in two groups.

Statistic information of sequencing reads in selected patients. A table showing how the sequencing reads mapped.

Melanoma is the most malignant cutaneous cancer and causes over 9000 deaths annually. Because fatality rates from malignant melanoma (MM) increase dramatically upon metastasis, we investigated tumorigenesis and metastasis of MM in transcriptome analyses of three distinct cell lines that correspond with the stages of MM pathogenesis: the normal stage (HEMn-LP), the onset of MM (A375), and the metastasis stage (A2058). Using next-generation sequencing (NGS) technology, we detected asymmetrical expression of genes among the three cell lines, notably on chromosomes 9, 11, 12, and 14, suggesting their involvement in tumorigenesis and metastasis of MM. These genes were clustered into 41 categories based on their expression patterns, and their biological functions were analyzed using Ingenuity Pathway Analysis. In the top cancer-associated category, HIF1A, IL8, TERT, ONECUT1, and FOXA1 directly interacted with either transcription factors or cytokines that are known to be involved in the tumorigenesis or metastasis of other malignant tumors. The present data suggest that cytokine regulatory pathways in macrophages predominate over other pathways during the pathogenesis of MM. This study provides new targets for the downstream mechanistic studies of the tumorigenesis and metastasis of MM and demonstrates a new strategy for studies of the progression of other malignant cancers.

To identify the mechanisms controlling chronic myeloid leukemia (CML) and acute myeloid leukemia (AML) in humans, we analyzed genome-wide transcription dynamics in three myeloid leukemia cell lines (K562, HL-60, and THP1) using high-throughput sequencing technology. Using KEGG analysis, we found that the ERK/MAPK, JAK-STAT and ErbB pathways promoted proliferation and metabolism in CML. However, in AML, differentiation and apoptosis blocking resulted in the accumulation of blast cells in marrow. In addition, each cell type had unique characteristics. K562 cells are an ideal model for studying erythroid differentiation and globin gene expression. The chemokine signaling pathway and Fc gamma R-mediated phagocytosis were markedly upregulated in HL-60 cells. In THP1 cells, highly expressed genes ensured strong phagocytosis by monocytes. Further, we provide a new insight into myeloid development. The abundant data sets and well-defined analysis methods will provide a resource and strategy for further investigation of myeloid leukemia.

PDCD2 was first identified in human in 1995, which is highly homologous to a rat programmed cell death related gene Rp8. Nonetheless, up to now, there have been only 43 papers about PDCD2 published. Therefore, the role of PDCD2 in erythroid differentiation remains largely unknown. TF-1 cell is a erythroleukemia erythroblast, which is a good system for investigating the proliferation and differentiation of myeloid progenitor cells for its responsiveness to multiple cytokines.To study the function of PDCD2 in erythroid differentiation, we performed PDCD2 knockdown in TF-1 cells. The expression of PDCD2 was significantly decreased at both mRNA and protein level (Fig.1A, B). The expression of globin genes were detected by real-time qPCR, and the results indicated that ebsilon-, gamma-, beta-globin genes expression increased after PDCD2 knockdown in TF-1 cells (Fig.1C). Also, the expression of CD71 and CD235a at cell surface was measured by FACS, and our data showed that the percentage of CD71+CD235a+ cells was significantly increased by PDCD2 knockdown in TF-1 cells (Fig.1D). Furthermore, we induced TF-1 cells to erythroid differentiation by removing GM-CSF and adding EPO in culture medium. Both globin genes expression at mRNA level and CD71+CD235a+ expression at cell surface were measured at different time points during induction. With the progression of induction, expression of globin genes and the percentage of CD71+CD235a+ cells were increased in both control and PDCD2 knockdown cells (Fig.2). However, at same time points, increased expression of globin genes were detected in PDCD2 knockdown cells, compared to control cells (Fig.2A, B). Similar pattern was also observed in terms of the expression of CD71+CD235a+ at cell surface (Fig.2C). GATA-1 and KLF1 are the two most important transcription factors during erythroid differentiation. We detected the expression of the two TFs after PDCD2 knockdown in TF-1 cells. We found that expression of GATA-1 and KLF1 was augmented after PDCD2 knockdown (Fig.3A, B), and the augmentation remained during the induced differentiation (Fig.3C, D). To get a further insight of how PDCD2 regulates erythrioid differentiation, we performed a PDCD2 ChIP assay in normal TF-1 cells. Our data showed that there was an enrichment of PDCD2 at GATA-1 promoter region (data not shown). Since there is no evidence indicating that PDCD2 is a transcription factor, we assumed that PDCD2 bind at DNA sequence indirectly through interacting with other proteins. We thus performed co-IP assay of HDAC8 and HDAC1 in TF-1 cells and found that PDCD2 interacted with HDAC8 (data not shown). These data indicate that PDCD2 knockdown promoted induced erythroid differentiation in TF-1 cells. The promotion may be partially explained by increased expression of GATA-1 and KLF1. PDCD2 interacting with HDAC8 and binding at GATA-1 promoter region may play a role during erythroid differentiation. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures No relevant conflicts of interest to declare.

Abstract 4730 Krüppel-like factors (KLFs) are a conserved family of Cys2His2 zinc finger proteins which are important components of eukaryotic cellular transcriptional machinery that controls many biological processes including erythroid differentiation and development. As a transcriptional activator and a tumor suppressor, KLF6 was also involved in hematopoiesis. Klf6−/− mice is embryonic lethal by embryonic day 12.5 and associated with markedly reduced hematopoiesis as well as poorly organized yolk sac vascularization. Moreover, the expression of erythroid differentiation markers including Klf1, Gata1 and Scl are delayed and hematopoietic differentiation is impaired in klf6−/− ES cells. However, the detailed mechanism that KLF6 regulates hematopoiesis is not fully understood. To characterize the role of KLF6 in hematopoiesis, we firstly detected the dynamic expression pattern of KLF6 during erythroid differentiation by mRNA-seq in undifferentiated human embryonic stem cells (hESC), three primary erythroid cells at different developmental stages including ES-derived erythroid cells (ESER), fetal- and adult-type erythroid cells (FLER, PBER). The transcriptome analysis showed that KLF6 expressed at significantly higher level in ESER cells compared with that in other cells. Meanwhile, chromatin immunoprecipitation (ChIP) studies in human K562 cells demonstrated the enrichment of KLF6 on the promoter region of embryonic epsilon-globin gene. These results probably indicate that KLF6 play an important role in primitive hematopoiesis. To clarify whether the erythroid-specific enhancers in the genomic region of KLF6 participate in the regulation of primitive hematopoiesis, we extensively screened the erythroid-specific DNaseI hypersensitive sites (DHSs) in the KLF6 locus, from 70 kb upstream of the transcription start site to 20 kb downstream of the poly(A) site, from DNase-seq data in four erythroid cells including ESER, FLER, PBER, K562 and seven non-erythroid cells. The enhancer activity of these erythroid-specific DHSs was comprehensively characterized by dual-luciferase reporter assay in K562 cells as well as non-erythroid HeLa and HEK293 cells. Three erythroid-specific enhancers located 18–24 kb upstream of human KLF6 were finally characterized, which not only helps to understand the higher expression of KLF6 in ESER, but also hints that KLF6 could participate in primitive hematopoiesis through erythroid-specific enhancers. In conclusion, we depicted the dynamic expression pattern of KLF6 during erythroid differentiation, characterized three erythroid-specific enhancers in KLF6 gene locus, and disclosed the potential role of KLF6 in primitive hematopoiesis. Next, the overexpression and depletion of KLF6 in K562 cells will be executed to further explore whether the abnormal KLF6 will affect the expression and functions of globin genes as well as erythroid-specific transcription factors. Chromosome conformation capture (3C) analysis will be performed to evaluate the interactions between the erythroid-specific enhancers and the cis-regulatory elements of hematopoiesis related genes. Moreover, we will establish morpholino-based klf6 knockdown zebrafish model and study the target genes, interacting networks and pathways in which KLF6 involved. Collectively, these results will address the detailed cis- and trans- regulatory functions and molecular mechanism of KLF6 in regulating hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 4731 FOX (Forkhead box) proteins are a family of transcription factors that emerged as playing an important role in the embryonic development, cell cycle, carbohydrate and fatty acid metabolism and immune response. It was found that FOXO3A (also known as FOXO3) involved in erythroid differentiation, yet the mechanism for regulating hematopoietic stem cells (HSCs) differentiation is unknown. We analyzed the dynamics of genome-wide transcriptome (mRNA-Seq) of human undifferentiated embryonic stem cells (HESC), erythroid cells derived from ES cells (ESER), human fetal erythroid liver cells (FLER) and peripheral CD34+derived erythroid cells (PBER) using high throughput sequencing technology. The transcriptome analysis showed that FOXO3 was barely expression in HESC while was observably up-regulated in ESER. However, FOXO3 was down-regulated in FLER and PBER compare with ESER, the erythroid cells at early developmental stage. We presumed that FOXO3 plays an important role in primitive erythropoiesis and built up the interactions network in which FOXO3 acts as a central node by Gene Ontology (GO), correlation analysis and Ingenuity Pathways Analysis (IPA). In addition, we analyzed the profiles of histone methylation in the four types of cells by ChIP-Seq to study the chromatin conformation in the vicinity of FOXO3. More histone 3 lysine 4 (H3K4) trimethylation was found near the promoter region of FOXO3 in ESER compared with the other cells, which is coincided with the mRNA-seq results. We performed a series of experiment to identify the roles of FOXO3 in regulating erythroid differentiation. The results showed that the expression level of ε and γ globin were up-regulated in FOXO3-over-expressed 293T and Hela cells and the expression level of FOXO1 and CAT in predicted network were increased by quantitative real-time PCR detection. In addition, when FOXO3 knocked down in K562 cells, the expression level of ε and γ globin were down-regulated. The expression level of CAT, BCL2L1 and other factors in predicted network, were also decreased. These results indicate FOXO3 plays an important role in globin expression and identify the credibility of our predicted networks in which FOXO3 acts as a central node. FOXO3 binding sites (GTAAACA or ATAAACA) were predicted on the upstream of CAT and BCL2L1. We are trying to prove CAT or BCL2L1 is a direct FOXO3 target in vitro and in vivo. In conclusion, we have demonstrated FOXO3 plays a key role in erythroid differentiation and globin expression. We will further determine the enriched profiles of FOXO3 by ChIP-seq in HESC, ESER, FLER and PBER to find more targets of FOXO3. Since the zebrafish is a powerful model system for investigating vertebrate hematopoiesis. We will identify the role of Foxo3b, the homologous gene of human FOXO3, in erythroid differentiation and study the dynamic transcriptomes of Foxo3b morphants in zebrafish. We are trying to make a whole picture to elaborate the molecular mechanism of FOXO3 involved in regulation of erythroid differentiation. Disclosures: No relevant conflicts of interest to declare.

Background Mapping of DNase I hypersensitive sites (DHSs) is a powerful tool to experimentally identify cis-regulatory elements (CREs). Among CREs, enhancers are abundant and predominantly act in driving cell-specific gene expression. Krüppel-like factors (KLFs) are a family of eukaryotic transcription factors. Several KLFs have been demonstrated to play important roles in hematopoiesis. However, transcriptional regulation of KLFs via CREs, particularly enhancers, in erythroid cells has been poorly understood. Results In this study, 23 erythroid-specific or putative erythroid-specific DHSs were identified by DNase-seq in the genomic regions of 17 human KLFs, and their enhancer activities were evaluated using dual-luciferase reporter (DLR) assay. Of the 23 erythroid-specific DHSs, the enhancer activities of 15 DHSs were comparable to that of the classical enhancer HS2 in driving minimal promoter (minP). Fifteen DHSs, some overlapping those that increased minP activities, acted as enhancers when driving the corresponding KLF promoters (KLF-Ps) in erythroid cells; of these, 10 DHSs were finally characterized as erythroid-specific KLF enhancers. These 10 erythroid-specific KLF enhancers were further confirmed using chromatin immunoprecipitation coupled to sequencing (ChIP-seq) data-based bioinformatic and biochemical analyses. Conclusion Our present findings provide a feasible strategy to extensively identify gene- and cell-specific enhancers from DHSs obtained by high-throughput sequencing, which will help reveal the transcriptional regulation and biological functions of genes in some specific cells.