Erythroid Krüppel-Like Factor (KLF1): A Surprisingly Versatile Regulator of Erythroid Differentiation.

Erythroid Krüppel-like factor (KLF1), first discovered in 1992, is an erythroid-restricted transcription factor (TF) that is essential for terminal differentiation of erythroid progenitors. At face value, KLF1 is a rather inconspicuous member of the 26-strong SP/KLF TF family. However, 30 years of research have revealed that KLF1 is a jack of all trades in the molecular control of erythropoiesis. Initially described as a one-trick pony required for high-level transcription of the adult HBB gene, we now know that it orchestrates the entire erythroid differentiation program. It does so not only as an activator but also as a repressor. In addition, KLF1 was the first TF shown to be directly involved in enhancer/promoter loop formation. KLF1 variants underlie a wide range of erythroid phenotypes in the human population, varying from very mild conditions such as hereditary persistence of fetal hemoglobin and the In(Lu) blood type in the case of haploinsufficiency, to much more serious non-spherocytic hemolytic anemias in the case of compound heterozygosity, to dominant congenital dyserythropoietic anemia type IV invariably caused by a de novo variant in a highly conserved amino acid in the KLF1 DNA-binding domain. In this chapter, we present an overview of the past and present of KLF1 research and discuss the significance of human KLF1 variants.

Creg1 Regulates Erythroid Development via TGF-ß/Smad2-Klf1 Axis in Zebrafish.

Understanding the regulation of normal erythroid development will help to develop new potential therapeutic strategies for disorders of the erythroid lineage. Cellular repressor of E1A-stimulated genes 1 (CREG1) is a glycoprotein that has been implicated in the regulation of tissue homeostasis. However, its role in erythropoiesis remains largely undefined. In this study, it is found that CREG1 expression increases progressively during erythroid differentiation. In zebrafish, creg1 mRNA is preferentially expressed within the intermediate cell mass (ICM)/peripheral blood island (PBI) region where primitive erythropoiesis occurs. Loss of creg1 leads to anemia caused by defective erythroid differentiation and excessive apoptosis of erythroid progenitors. Mechanistically, creg1 deficiency results in reduced activation of TGF-ß/Smad2 signaling pathway. Treatment with an agonist of the Smad2 pathway (IDE2) could significantly restore the defective erythroid development in creg1-/- mutants. Further, Klf1, identified as a key target gene downstream of the TGF-ß/Smad2 signaling pathway, is involved in creg1 deficiency-induced aberrant erythropoiesis. Thus, this study reveals a previously unrecognized role for Creg1 as a critical regulator of erythropoiesis, mediated at least in part by the TGF-ß/Smad2-Klf1 axis. This finding may contribute to the understanding of normal erythropoiesis and the pathogenesis of erythroid disorders.

Long-term hematopoietic transfer of the anti-cancer and lifespan-extending capabilities of a genetically engineered blood system by transplantation of bone marrow mononuclear cells.

A causal relationship exists among the aging process, organ decay and disfunction, and the occurrence of various diseases including cancer. A genetically engineered mouse model, termed Klf1K74R/K74R or Klf1(K74R), carrying mutation on the well-conserved sumoylation site of the hematopoietic transcription factor KLF1/EKLF has been generated that possesses extended lifespan and healthy characteristics, including cancer resistance. We show that the healthy longevity characteristics of the Klf1(K74R) mice, as exemplified by their higher anti-cancer capability, are likely gender-, age-, and genetic background-independent. Significantly, the anti-cancer capability, in particular that against melanoma as well as hepatocellular carcinoma, and lifespan-extending property of Klf1(K74R) mice, could be transferred to wild-type mice via transplantation of their bone marrow mononuclear cells at a young age of the latter. Furthermore, NK(K74R) cells carry higher in vitro cancer cell-killing ability than wild-type NK cells. Targeted/global gene expression profiling analysis has identified changes in the expression of specific proteins, including the immune checkpoint factors PDCD and CD274, and cellular pathways in the leukocytes of the Klf1(K74R) that are in the directions of anti-cancer and/or anti-aging. This study demonstrates the feasibility of developing a transferable hematopoietic/blood system for long-term anti-cancer and, potentially, for anti-aging.

Proteomic analysis reveals a potential role for extracellular vesicles within the erythroblastic island niche.

Introduction: Erythroblastic island (EBI) macrophages play an essential role in the production and maturation of the vast numbers of red blood cells (RBCs) that are produced throughout life. Their location within the bone marrow makes it difficult to study the cellular and molecular interactions associated with their action so we have used an in vitro model of the EBI niche using macrophages derived from human induced pluripotent stem cells (hiPSCs). We previously demonstrated that the activation of the transcription factor KLF1 enhanced the activity of hiPSC-derived EBI macrophages. Methods: To elucidate the mechanisms associated with EBI-like activity we carried out a quantitative proteomic analysis and assessed the role of extracellular vesicles using Nanosight Tracking analyses and media filtration. Results and Discussion: Gene ontology analysis showed that many of the proteins upregulated by KLF1 were protein-binding factors, some of which were associated with the cell membrane or extracellular vesicles We demonstrated that filtration of macrophage-conditioned media resulted in a reduction in the supportive effects on erythroid cell viability and maturation implying a role for extracellular vesicles but this was not KLF1 dependent. Pathway analyses of the proteomic data revealed that proteins upregulated by KLF1 were associated with the citric acid cycle, pyruvate metabolism and ATP synthesis indicating that KLF1-activated macrophages had a metabolic profile comparable to a pro-reparative phenotype. This study has generated a proteomic dataset that could provide new insights into the role of macrophages within the EBI niche and has indicated a potential role for extracellular vesicles in the differentiation and maturation of RBCs in vitro. Further research will aid in the production of RBCs in vitro for use in disease modelling and cell therapy.

Impact of transcription factors KLF1 and GATA1 on red blood cell antigen expression: a review.

KLF transcription factor 1 (KLF1) and GATA binding protein 1 (GATA1) are transcription factors (TFs) that initiate and regulate transcription of the genes involved in erythropoiesis. These TFs possess DNA-binding domains that recognize specific nucleotide sequences in genes, to which they bind and regulate transcription. Variants in the genes that encode either KLF1 or GATA1 can result in a range of hematologic phenotypes-from benign to severe forms of thrombocytopenia and anemia; they can also weaken the expression of blood group antigens. The Lutheran (LU) blood group system is susceptible to TF gene variations, particularly KLF1 variants. Individuals heterozygous for KLF1 gene variants show reduced Lutheran antigens on red blood cells that are not usually detected by routine hemagglutination methods. This reduced antigen expression is referred to as the In(Lu) phenotype. For accurate blood typing, it is important to distinguish between the In(Lu) phenotype, which has very weak antigen expression, and the true Lunull phenotype, which has no antigen expression. The International Society of Blood Transfusion blood group allele database registers KLF1 and GATA1 variants associated with modified Lutheran expression. Here, we review KLF1 and recent novel gene variants defined through investigating blood group phenotype and genotype discrepancies or, for one report, investigating cases with unexplained chronic anemia. In addition, we include a review of the GATA1 TF, including a case report describing the second GATA1 variant associated with a serologic Lu(a-b-) phenotype. Finally, we review both past and recent reports on variations in the DNA sequence motifs on the blood group genes that disrupt the binding of the GATA1 TF and either remove or reduce erythroid antigen expression. This review highlights the diversity and complexity of the transcription process itself and the need to consider these factors as an added component for accurate blood group phenotyping.

Targeted next-generation sequencing and long-read HiFi sequencing provide novel insights into clinically significant KLF1 variants.

BACKGROUND: Krüppel-like factor 1 (KLF1), a crucial erythroid transcription factor, plays a significant role in various erythroid changes and haemolytic diseases. The rare erythrocyte Lutheran inhibitor (In(Lu)) blood group phenotype serves as an effective model for identifying KLF1 hypomorphic and loss-of-function variants. In this study, we aimed to analyse the genetic background of the In(Lu) phenotype in a population-based sample group by high-throughput technologies to find potentially clinically significant KLF1 variants. RESULTS: We included 62 samples with In(Lu) phenotype, screened from over 300,000 Chinese blood donors. Among them, 36 samples were sequenced using targeted Next Generation Sequencing (NGS), whereas 19 samples were sequenced using High Fidelity (HiFi) technology. In addition, seven samples were simply sequenced using Sanger sequencing. A total of 29 hypomorphic or loss-of-function variants of KLF1 were identified, 21 of which were newly discovered. All new variants discovered by targeted NGS or HiFi sequencing were validated through Sanger sequencing, and the obtained results were found to be consistent. The KLF1 haplotypes of all new variants were further confirmed using clone sequencing or HiFi sequencing. The lack of functional KLF1 variants detected in the four samples indicates the presence of additional regulatory mechanisms. In addition, some samples exhibited BCAM polymorphisms, which encodes antigens of the Lutheran (LU) blood group system. However, no BCAM mutations which leads to the absence of LU proteins were detected. CONCLUSIONS: High-throughput sequencing methods, particularly HiFi sequencing, were introduced for the first time into genetic analysis of the In(Lu) phenotype. Targeted NGS and HiFi sequencing demonstrated the accuracy of the results, providing additional advantages such as simultaneous analysis of other blood group genes and clarification of haplotypes. Using the In(Lu) phenotype, a powerful model for identifying hypomorphic or loss-of-function KLF1 variants, numerous novel variants have been detected, which have contributed to the comprehensive understanding of KLF1. These clinically significant KLF1 mutations can serve as a valuable reference for the diagnosis of related blood cell diseases.

A 6-Year Follow-up of a Chinese Child with Homozygous ß0-Thalaasemia and a Heterozygous KLF1 Mutation.

Patients with the genotype of ß0/ß0 for ß-thalassemia (ß-thal) usually behave as ß-thal major (ß-TM) phenotype which is transfusion-dependent. The pathophysiology of ß-thal is the imbalance between α/ß-globin chains. The degree of α/ß-globin imbalance can be reduced by the more effective synthesis of γ-globin chains, and increased Hb F levels, modifying clinical severity of ß-TM. We report a Chinese child who had homozygous ß0-thal and a heterozygous KLF1 mutation. The patient had a moderate anemia since 6 months old, keeping a baseline Hb value of 8.0-9.0 g/dL. She had normal development except for a short stature (3rd percentile) until 6 years old, when splenomegaly and facial bone deformities occurred. Although genetic alteration of KLF1 expression in ß0/ß0 patients can result in some degree of disease alleviation, our case shows that it is insufficient to ameliorate satisfactorily the presentation. This point should be borne in mind for physicians who provide the genetic counseling and prenatal diagnosis to at-risk families.

Schistosoma japonicum EKLF/KLF1 is a potential immune target to tackle schistosomiasis.

BACKGROUND: Interruption of parasite reproduction by targeting migrating schistosomula is a promising strategy for managing schistosomiasis. Hepatic schistosomula proteins previously identified based on second-generation schistosome DNA sequencing were found to hold excellent potential for schistosomiasis japonica diagnosis and as vaccine candidates. However, there are still many unknown schistosomula proteins that warrant further investigations. Herein, a novel schistosomula protein, the Schistosoma japonicum erythroid Krüppel-like factor (SjEKLF/KLF1), was explored. METHODS: Sequence alignment was carried out to detect the amino acid sequence characteristics of SjEKLF. The expression profile of SjEKLF was determined by western blot and immunofluorescence analysis. Enzyme-linked immunosorbent assay was used to determine the antigenicity of SjEKLF in hosts. Mice immunised with recombinant SjEKLF were challenged to test the potential value of the protein as an immunoprotective target. RESULTS: SjEKLF is defined as EKLF/KLF1 for its C-terminal DNA-binding domain. SjEKLF is mainly expressed in hepatic schistosomula and male adults and located within the intestinal intima of the parasites. Notably, high levels of SjEKLF-specific antibodies were detected in host sera and SjEKLF exhibited outstanding sensitivity and specificity for schistosomiasis japonica immunodiagnosis but failed to distinguish between ongoing infection and previous exposure. In addition, SjEKLF immunisation reduced the infection in vivo, resulting in decreased worm and egg counts, and alleviated body weight loss and hepatomegaly in infected mice. CONCLUSIONS: Overall, these findings demonstrate that SjEKLF is critical for the infection of S. japonicum and may be a potential target to help control S. japonicum infection and transmission.

Identification of Lutheran Blood Groups and Genetic Variants within KLF1 among Thai Blood Donors.

Background: Lua and Lub are inherited as codominant allelic characters resulting from a single nucleotide variant (SNV) of the basal cell adhesion molecule (BCAM) gene. Red cells of the dominantly inherited suppressor of the Lutheran antigens In(Lu) phenotypically appear as Lu(a-b-) by the haemagglutination test. In(Lu) resulted from heterozygosity for mutations within the erythroid-specific Krüppel-like factor 1 (KLF1) gene. This study aimed to determine the frequency of the Lu(a) and Lu(b) phenotypes and genotypes and genetic variants of the distinct In(Lu) among Thai blood donors. Material and Methods: Samples from 334 Thai donors were phenotyped with anti-Lua and anti-Lub. These DNA samples and an additional 1,370 donor DNA samples with unknown Lu(a)/Lu(b) phenotypes were genotyped using an in-house PCR-SSP. In the case of the three Lu(a-b-) donors, the BCAM and KLF1 genes were analysed by PCR and sequencing. Results: A total of 331 of the 334 donors were Lu(a-b+), while the other observed phenotype, appearing as Lu(a-b-), was found among three donors. Of those three Lu(a-b-) donors with the LU*02/02 genotype, we identified KLF1 variant alleles, consisting of two variants: c.[304T>C, 1001C>G] and c.[304T>C, 519_525dupCGGCGCC], leading to the In(Lu) phenotype, and one homozygous variant (c.304T>C) mutation. Also, only one Thai donor was genotyped as LU*01/02, confirmed by serology test and DNA sequencing. Conclusion: In this study, we identified KLF1 variants to be included in Lutheran typing analysis in Thai populations. Therefore, the application of genotyping and phenotyping methods has simultaneously been in use to screen and confirm the rare Lu(a+) and In(Lu) phenotypes.

Regulatory mechanism of RNA binding motif protein 15-mediated N6 methyladenosine modification in proliferation, invasion, and migration of colorectal cancer cells.

This study aims to explore the regulatory mechanism of RNA binding motif protein 15 (RBM15) on the proliferation, invasion, and migration of colorectal cancer (CRC) cells. RBM15, KLF1, or SIN3A expression in CRC tissues and cells was detected by RT-qPCR or Western blot. CRC cell functions were measured by CCK-8, colony formation, and Transwell assays after RBM15 intervention. MeRIP and RIP measured N6 methyladenosine (m6 A) and IGF2BP3 enrichment on KLF1 mRNA. ChIP and dual-luciferase analyzed KLF1 enrichment on SIN3A promoter. Combined experiments verified the effect of KLF1/SIN3A on CRC cell functions. Lung/liver metastasis models were established to validate the effect of RBM15 on CRC in vivo. RBM15, KLF1, and SIN3A were highly expressed in CRC. RBM15 knockdown reduced the proliferation, invasion, and migration of CRC cells in vitro. Mechanistically, RBM15 facilitated KLF1 mRNA stability and expression through IGF2BP3-dependent m6 A modification, thus promoting KLF1 enrichment on the SIN3A promoter and activating SIN3A transcription. Overexpression of KLF1 or SIN3A reversed the inhibitory effect of RBM15 knockdown on CRC cells. In vivo experiments verified that RBM15 promoted tumorigenesis and lung/liver metastasis via KLF1/SIN3A axis. In conclusion, RBM15 stimulated CRC proliferation and metastasis by promoting the KLF1/SIN3A axis through IGF2BP3-dependent m6 A modification.

Identification and Functional Analysis of Known and New Mutations in the Transcription Factor KLF1 Linked with ß-Thalassemia-like Phenotypes.

The erythroid transcriptional factor Krüppel-like factor 1 (KLF1) is a master regulator of erythropoiesis. Mutations that cause KLF1 haploinsufficiency have been linked to increased fetal hemoglobin (HbF) and hemoglobin A2 (HbA2) levels with ameliorative effects on the severity of ß-thalassemia. With the aim of determining if KLF1 gene variations might play a role in the modulation of ß-thalassemia, in this study we screened 17 subjects showing a ß-thalassemia-like phenotype with a slight or marked increase in HbA2 and HbF levels. Overall, seven KLF1 gene variants were identified, of which two were novel. Functional studies were performed in K562 cells to clarify the pathogenic significance of these mutations. Our study confirmed the ameliorative effect on the thalassemia phenotype for some of these variants but also raised the notion that certain mutations may have deteriorating effects by increasing KLF1 expression levels or enhancing its transcriptional activity. Our results indicate that functional studies are required to evaluate the possible effects of KLF1 mutations, particularly in the case of the co-existence of two or more mutations that could differently contribute to KLF1 expression or transcriptional activity and consequently to the thalassemia phenotype.

Whole exome sequencing and rare variant association study to identify genetic modifiers, KLF1 mutations, and a novel double mutation in Thai patients with hemoglobin E/beta-thalassemia.

OBJECTIVES: Clinical manifestations of patients with Hemoglobin E/beta-thalassemia vary from mild to severe phenotypes despite exhibiting the same genotype. Studies have partially identified genetic modifiers. We aimed to study the association between rare variants in protein-coding regions and clinical severity in Thai patients. METHODS: From April to November 2018, a case-control study was conducted based on clinical information and DNA samples collected from Thai patients with hemoglobin E/beta-thalassemia over the age of four years. Cases were patients with severe symptoms, while patients with mild symptoms acted as controls. Whole exome sequencing and rare variant association study were used to analyze the data. RESULTS: All 338 unrelated patients were classified into 165 severe and 173 mild cases. Genotypes comprised 81.4% of hemoglobin E/beta-thalassemia, 2.7% of homozygous or compound heterozygous beta-thalassemia, and 0.3% of (뫧)0 thalassemia Hb E while 15.7% of samples were not classified as beta-thalassemia. A novel cis heterozygotes of IVS I-7 (A > T) and codon 26 (G > A) was identified. Six genes (COL4A3, DLK1, FAM186A, PZP, THPO, and TRIM51) showed the strongest associations with severity (observed p-values of <0.05; significance lost after correction for multiplicity). Among known modifiers, KLF1 variants were found in four mild patients and one severe patient. CONCLUSION: No rare variants were identified as contributors to the clinical heterogeneity of hemoglobin E/beta-thalassemia. KLF1 mutations are potential genetic modifiers. Studies to identify genetic factors are still important and helpful for predicting severity and developing targeted therapy.

Congenital dyserythropoietic anemia type IV in the genetic era: A rare neonatal case report of rapid identification with a review of the literature.

Congenital dyserythropoietic anemia type IV (CDAIV) is a rare inherited hematological disorder, presenting with severe anemia due to altered erythropoiesis and hemolysis, with variable needs for recurrent transfusions. We present a case of a transfusion-dependent male newborn who presented at birth with severe hemolytic anemia, and required an intrauterine transfusion. Genetic testing rapidly identified a Kruppel-like factor 1 (KLF1) pathogenic variant (c.973G>A, p.E325K), known to be causative for CDAIV. This case highlights the advantages of next-generation sequencing testing for congenital hemolytic anemia: diagnostic speed, guidance on natural history, and optimized clinical management and anticipatory guidance for parents and clinicians. Additionally, we reviewed the literature for all CDAIV cases.

EKLF/Klf1 regulates erythroid transcription by its pioneering activity and selective control of RNA Pol II pause-release.

EKLF/Klf1 is a zinc-finger transcription activator essential for erythroid lineage commitment and terminal differentiation. Using ChIP-seq, we investigate EKLF DNA binding and transcription activation mechanisms during mouse embryonic erythropoiesis. We utilize the Nan/+ mouse that expresses the EKLF-E339D (Nan) variant mutated in its conserved zinc-finger region and address the mechanism of hypomorphic and neomorphic changes in downstream gene expression. First, we show that Nan-EKLF limits normal EKLF binding to a subset of its sites. Second, we find that ectopic binding of Nan-EKLF occurs largely at enhancers and activates transcription through pioneering activity. Third, we find that for a subset of ectopic targets, gene activation is achieved in Nan/+ only by Nan-EKLF binding to distal enhancers, leading to RNA polymerase II pause-release. These results have general applicability to understanding how a DNA binding variant factor confers dominant disruptive effects on downstream gene expression even in the presence of its normal counterpart.

Krüppel-Like Factor 1: A Pivotal Gene Regulator in Erythropoiesis.

Krüppel-like factor 1 (KLF1) plays a crucial role in erythropoiesis. In-depth studies conducted on mice and humans have highlighted its importance in erythroid lineage commitment, terminal erythropoiesis progression and the switching of globin genes from γ to ß. The role of KLF1 in haemoglobin switching is exerted by the direct activation of ß-globin gene and by the silencing of γ-globin through activation of BCL11A, an important γ-globin gene repressor. The link between KLF1 and γ-globin silencing identifies this transcription factor as a possible therapeutic target for ß-hemoglobinopathies. Moreover, several mutations have been identified in the human genes that are responsible for various benign phenotypes and erythroid disorders. The study of the phenotype associated with each mutation has greatly contributed to the current understanding of the complex role of KLF1 in erythropoiesis. This review will focus on some of the principal functions of KLF1 on erythroid cell commitment and differentiation, spanning from primitive to definitive erythropoiesis. The fundamental role of KLF1 in haemoglobin switching will be also highlighted. Finally, an overview of the principal human mutations and relative phenotypes and disorders will be described.

Normal and Aberrant TALE-Class Homeobox Gene Activities in Pro-B-Cells and B-Cell Precursor Acute Lymphoblastic Leukemia.

Homeobox genes encode transcription factors regulating basic developmental processes. They are arranged according to sequence similarities of their conserved homeobox in 11 classes, including TALE. Recently, we have reported the so-called TALE-code. This gene signature describes physiological expression patterns of all active TALE-class homeobox genes in the course of hematopoiesis. The TALE-code allows the evaluation of deregulated TALE homeobox genes in leukemia/lymphoma. Here, we extended the TALE-code to include the stages of pro-B-cells and pre-B-cells in early B-cell development. Detailed analysis of the complete lineage of B-cell differentiation revealed expression of TALE homeobox genes IRX1 and MEIS1 exclusively in pro-B-cells. Furthermore, we identified aberrant expression of IRX2, IRX3 and MEIS1 in patients with B-cell precursor acute lymphoblastic leukemia (BCP-ALL) which originates from early B-cell progenitors. The data showed correlated activities of deregulated TALE-class members with particular BCP-ALL subtype markers, namely IRX2 with TCF3/E2A-fusions, IRX3 with ETV6/TEL-fusions, and MEIS1 with KMT2A/MLL-fusions. These correlations were also detected in BCP-ALL cell lines which served as experimental models. We performed siRNA-mediated knockdown experiments and reporter gene assays to analyze regulatory connections. The results showed mutual activation of IRX1 and TCF3. In contrast, IRX2 directly repressed wild-type TCF3 while the fusion gene TCF3::PBX1 lost the binding site for IRX2 and remained unaltered. IRX3 mutually activated fusion gene ETV6::RUNX1 while activating itself by aberrantly expressed transcription factor KLF15. Finally, KMT2A activated MEIS1 which in turn supported the expression of IRX3. In summary, we revealed normal TALE homeobox gene expression in early B-cell development and identified aberrant activities of IRX2, IRX3 and MEIS1 in particular subtypes of BCP-ALL. Thus, these TALE homeobox genes may serve as novel diagnostic markers and therapeutic targets.

Genetic Disruption of KLF1 K74 SUMOylation in Hematopoietic System Promotes Healthy Longevity in Mice.

The quest for rejuvenation and prolonged lifespan through transfusion of young blood has been studied for decades with the hope of unlocking the mystery of the key substance(s) that exists in the circulating blood of juvenile organisms. However, a pivotal mediator has yet been identified. Here, atypical findings are presented that are observed in a knockin mouse model carrying a lysine to arginine substitution at residue 74 of Krüppel-like factor 1 (KLF1/EKLF), the SUMOylation-deficient Klf1K74R/K74R mouse, that displayed significant improvement in geriatric disorders and lifespan extension. Klf1K74R/K74R mice exhibit a marked delay in age-related physical performance decline and disease progression as evidenced by physiological and pathological examinations. Furthermore, the KLF1(K74R) knockin affects a subset of lymphoid lineage cells; the abundance of tumor infiltrating effector CD8+ T cells and NKT cells is increased resulting in antitumor immune enhancement in response to tumor cell administration. Significantly, infusion of hematopoietic stem cells (HSCs) from Klf1K74R/K74R mice extends the lifespan of the wild-type mice. The Klf1K74R/K74R mice appear to be an ideal animal model system for further understanding of the molecular/cellular basis of aging and development of new strategies for antiaging and prevention/treatment of age-related diseases thus extending the healthspan as well as lifespan.

Knockdown of lncRNA PCAT6 suppresses the growth of non-small cell lung cancer cells by inhibiting macrophages M2 polarization via miR-326/KLF1 axis.

Non-small cell lung cancer (NSCLC) is the most common malignant tumor of lung, which seriously threatens the life of people. It has been reported that lncRNA prostate cancer-associated transcript 6 (PCAT6) could facilitate the metastasis of NSCLC cells. However, whether lncRNA PCAT6 in NSCLC cells could affect the tumor microenvironment (TME) remains unclear. In the present study, the level of PCAT6 in NSCLC cells was detected using RT-qPCR. The effects of PCAT6 knockdown on the viability and apoptosis in NSCLC cells were detected with CCK-8 and flow cytometry assay. NSCLC cell-derived exosomes were isolated with ultracentrifugation. Next, transwell assay was conducted to assess the migration and invasion of NSCLC cells. Dual-luciferase reporter assay was performed to verify the relationship among PCAT6, miR-326, and KLF1 in A549 cells. In addition, nanoparticle tracking analysis (NTA) was applied to detect the particle size of isolated exosomes. Moreover, ELISA assay was performed to detect the levels of IL-1ß and IL-10 in the supernatant of macrophage. We found knockdown of PCAT6 significantly inhibited the viability, migration, and invasion of NSCLC cells. In addition, dual-luciferase reporter assay illustrated that miR-326 was the target of PCAT6 and KLF1 was the target of miR-326 in NSCLC cells. Moreover, NSCLC cells-derived exosomes could promote macrophages M2 polarization by transporting PCAT6. Meanwhile, macrophages M2 polarization was able to promote the metastasis and epithelial-mesenchymal transition (EMT) process of NSCLC cells via regulating PCAT6/miR-326/KLF1 axis. Taken together, knockdown of lncRNA PCAT6 suppressed the growth of NSCLC cells by inhibiting macrophages M2 polarization via miR-326/KLF1 axis.

Thalassemia and erythroid transcription factor KLF1 mutations associated with borderline hemoglobin A2 in the Thai population.

INTRODUCTION: Elevated hemoglobin (Hb) A2 is an important diagnostic marker for ß-thalassemia carriers. However, diagnosis of cases with borderline Hb A2 may be problematic. We described the molecular characteristics found in a large cohort of Thai subjects with borderline Hb A2. MATERIAL AND METHODS: Examination was done on 21,657 Thai subjects investigated for thalassemia at Khon Kaen University, Thailand. A total of 202 subjects with borderline Hb A2 (3.5-4.0%) were selectively recruited and hematological parameters were recorded. DNA variants in α-, ß-, δ-globin, and Krüppel-like factor 1 (KLF1) genes were examined using PCR. RESULTS: Among 202 subjects, DNA analysis identified carriers of α+-thalassemia (n = 48; 23.8%), ß-thalassemia (n = 22; 10.9%) and KLF1 mutations (n = 48; 23.8%). No molecular defect was observed in the remaining 84 (41.5%) subjects. Interaction of KLF1 and α-thalassemia was observed in 10 cases. Of the 22 ß-thalassemia carriers, five ß+-thalassemia mutations were identified with lower MCV and higher Hb A2. Seven KLF1 mutations were detected in 10 genotypes in subjects with higher MCV and Hb F. No ß0-thalassemia, α-globin gene triplication or δ-globin gene mutation was detected. CONCLUSIONS: A large proportion of subjects with borderline Hb A2 are not ß-thalassemia carriers and for those with ß-thalassemia, only mild ß+-thalassemia mutations were detected. Evaluation of the patients using Hb A2, Hb F and MCV values will help in selecting cases for further molecular analysis. The results should explain the unusual phenotype of the cases and facilitate a thalassemia screening program in the region.

Ancestral Stem Cell Reprogramming Genes Active in Hemichordate Regeneration.

Hemichordate enteropneust worms regenerate extensively in a manner that resembles the regeneration for which planaria and hydra are well known. Although hemichordates are often classified as an extant phylogenetic group that may hold ancestral deuterostome body plans at the base of the deuterostome evolutionary line leading to chordates, mammals, and humans, extensive regeneration is not known in any of these more advanced groups. Here we investigated whether hemichordates deploy functional homologs of canonical Yamanaka stem cell reprogramming factors, Oct4, Sox2, Nanog, and Klf4, as they regenerate. These reprogramming factors are not expressed during regeneration of limbs, fins, eyes or other structures that represent the best examples of regeneration in chordates. We first examined Ptychodera flava EST libraries and identified Pf-Pou3, Pf-SoxB1, Pf-Msxlx, and Pf-Klf1/2/4 as most closely related to the Yamanaka factors, respectively. In situ hybridization analyses revealed that all these homologs are expressed in a distinct manner during head regeneration. Furthermore, Pf-Pou3 partially rescued the loss of endogenous Oct4 in mouse embryonic stem cells in maintaining the pluripotency gene expression program. Based on these results, we propose that hemichordates may have co-opted these reprogramming factors for their extensive regeneration or that chordates may have lost the ability to mobilize these factors in response to damage. The robustness of these pluripotency gene circuits in the inner cell mass and in formation of induced pluripotent stem cells from mammalian somatic cells shows that these programs are intact in humans and other mammals and that these circuits may respond to as yet unknown gene regulatory signals, mobilizing full regeneration in hemichordates.