Activation of PPARδ in bone marrow endothelial progenitor cells improves their hematopoiesis-supporting ability after myelosuppressive injury.

Dysfunctional bone marrow (BM) endothelial progenitor cells (EPCs) with high levels of reactive oxygen species (ROS) are responsible for defective hematopoiesis in poor graft function (PGF) patients with acute leukemia or myelodysplastic neoplasms post-allotransplant. However, the underlying mechanism by which BM EPCs regulate their intracellular ROS levels and the capacity to support hematopoiesis have not been well clarified. Herein, we demonstrated decreased levels of peroxisome proliferator-activated receptor delta (PPARδ), a lipid-activated nuclear receptor, in BM EPCs of PGF patients compared with those with good graft function (GGF). In vitro assays further identified that PPARδ knockdown contributed to reduced and dysfunctional BM EPCs, characterized by the impaired ability to support hematopoiesis, which were restored by PPARδ overexpression. Moreover, GW501516, an agonist of PPARδ, repaired the damaged BM EPCs triggered by 5-fluorouracil (5FU) in vitro and in vivo. Clinically, activation of PPARδ by GW501516 benefited the damaged BM EPCs from PGF patients or acute leukemia patients in complete remission (CR) post-chemotherapy. Mechanistically, we found that increased expression of NADPH oxidases (NOXs), the main ROS-generating enzymes, may lead to elevated ROS level in BM EPCs, and insufficient PPARδ may trigger BM EPC damage via ROS/p53 pathway. Collectively, we found that defective PPARδ contributes to BM EPC dysfunction, whereas activation of PPARδ in BM EPCs improves their hematopoiesis-supporting ability after myelosuppressive therapy, which may provide a potential therapeutic target not only for patients with leukemia but also for those with other cancers.

Repair of dysfunctional bone marrow endothelial cells alleviates aplastic anemia.

Aplastic anemia (AA) is a life-threatening disease characterized by bone marrow (BM) failure and pancytopenia. As an important component of the BM microenvironment, endothelial cells (ECs) play a crucial role in supporting hematopoiesis and regulating immunity. However, whether impaired BM ECs are involved in the occurrence of AA and whether repairing BM ECs could improve hematopoiesis and immune status in AA remain unknown. In this study, a classical AA mouse model and VE-cadherin blocking antibody that could antagonize the function of ECs were used to validate the role of BM ECs in the occurrence of AA. N-acetyl-L-cysteine (NAC, a reactive oxygen species scavenger) or exogenous EC infusion was administered to AA mice. Furthermore, the frequency and functions of BM ECs from AA patients and healthy donors were evaluated. BM ECs from AA patients were treated with NAC in vitro, and then the functions of BM ECs were evaluated. We found that BM ECs were significantly decreased and damaged in AA mice. Hematopoietic failure and immune imbalance became more severe when the function of BM ECs was antagonized, whereas NAC or EC infusion improved hematopoietic and immunological status by repairing BM ECs in AA mice. Consistently, BM ECs in AA patients were decreased and dysfunctional. Furthermore, dysfunctional BM ECs in AA patients led to their impaired ability to support hematopoiesis and dysregulate T cell differentiation toward proinflammatory phenotypes, which could be repaired by NAC in vitro. The reactive oxygen species pathway was activated, and hematopoiesis- and immune-related signaling pathways were enriched in BM ECs of AA patients. In conclusion, our data indicate that dysfunctional BM ECs with impaired hematopoiesis-supporting and immunomodulatory abilities are involved in the occurrence of AA, suggesting that repairing dysfunctional BM ECs may be a potential therapeutic approach for AA patients.

The glycolytic enzyme PFKFB3 determines bone marrow endothelial progenitor cell damage after chemotherapy and irradiation.

Bone marrow (BM) endothelial progenitor cell (EPC) damage of unknown mechanism delays the repair of endothelial cells (EC) and recovery of hematopoiesis after chemo-radiotherapy. We found increased levels of the glycolytic enzyme PFKFB3 in the damaged BM EPC of patients with poor graft function, a clinical model of EPC damage-associated poor hematopoiesis after allogeneic hematopoietic stem cell transplantation. Moreover, in vitro the glycolysis inhibitor 3-(3-pyridinyl)- 1-(4-pyridinyl)-2-propen-1-one (3PO) alleviated the damaged BM EPC from patients with poor graft function. Consistently, PFKFB3 overexpression triggered BM EPC damage after 5-fluorouracil treatment and impaired hematopoiesis-supporting ability in vitro. Mechanistically, PFKFB3 facilitated pro-apoptotic transcription factor FOXO3A and expression of its downstream genes, including p21, p27, and FAS, after 5-fluorouracil treatment in vitro. Moreover, PFKFB3 induced activation of NF-κB and expression of its downstream adhesion molecule E-selectin, while it reduced hematopoietic factor SDF-1 expression, which could be rescued by FOXO3A silencing. High expression of PFKFB3 was found in damaged BM EC of murine models of chemo-radiotherapy-induced myelosuppression. Furthermore, a murine model of BM EC-specific PFKFB3 overexpression demonstrated that PFKFB3 aggravated BM EC damage, and impaired the recovery of hematopoiesis after chemotherapy in vivo, effects which could be mitigated by 3PO, indicating a critical role of PFKFB3 in regulating BM EC damage. Clinically, PFKFB3-induced FOXO3A expression and NF-κB activation were confirmed to contribute to the damaged BM EPC of patients with acute leukemia after chemotherapy. 3PO repaired the damaged BM EPC by reducing FOXO3A expression and phospho-NF-κB p65 in patients after chemotherapy. In summary, our results reveal a critical role of PFKFB3 in triggering BM EPC damage and indicate that endothelial-PFKFB3 may be a potential therapeutic target for myelosuppressive injury.

Dysfunctional bone marrow endothelial progenitor cells are involved in patients with myelodysplastic syndromes.

BACKGROUND: Myelodysplastic syndromes (MDS) are a group of heterogeneous myeloid clonal disorders characterized by ineffective haematopoiesis and immune deregulation. Emerging evidence has shown the effect of bone marrow (BM) endothelial progenitor cells (EPCs) in regulating haematopoiesis and immune balance. However, the number and functions of BM EPCs in patients with different stages of MDS remain largely unknown. METHODS: Patients with MDS (N = 30), de novo acute myeloid leukaemia (AML) (N = 15), and healthy donors (HDs) (N = 15) were enrolled. MDS patients were divided into lower-risk MDS (N = 15) and higher-risk MDS (N = 15) groups according to the dichotomization of the Revised International Prognostic Scoring System. Flow cytometry was performed to analyse the number of BM EPCs. Tube formation and migration assays were performed to evaluate the functions of BM EPCs. In order to assess the gene expression profiles of BM EPCs, RNA sequencing (RNA-seq) were performed. BM EPC supporting abilities of haematopoietic stem cells (HSCs), leukaemia cells and T cells were assessed by in vitro coculture experiments. RESULTS: Increased but dysfunctional BM EPCs were found in MDS patients compared with HDs, especially in patients with higher-risk MDS. RNA-seq indicated the progressive change and differences of haematopoiesis- and immune-related pathways and genes in MDS BM EPCs. In vitro coculture experiments verified that BM EPCs from HDs, lower-risk MDS, and higher-risk MDS to AML exhibited a progressively decreased ability to support HSCs, manifested as elevated apoptosis rates and intracellular reactive oxygen species (ROS) levels and decreased colony-forming unit plating efficiencies of HSCs. Moreover, BM EPCs from higher-risk MDS patients demonstrated an increased ability to support leukaemia cells, characterized by increased proliferation, leukaemia colony-forming unit plating efficiencies, decreased apoptosis rates and apoptosis-related genes. Furthermore, BM EPCs induced T cell differentiation towards more immune-tolerant cells in higher-risk MDS patients in vitro. In addition, the levels of intracellular ROS and the apoptosis ratios were increased in BM EPCs from MDS patients, especially in higher-risk MDS patients, which may be therapeutic candidates for MDS patients. CONCLUSION: Our results suggest that dysfunctional BM EPCs are involved in MDS patients, which indicates that improving haematopoiesis supporting ability and immuneregulation ability of BM EPCs may represent a promising therapeutic approach for MDS patients.

M2 macrophages, but not M1 macrophages, support megakaryopoiesis by upregulating PI3K-AKT pathway activity.

Dysfunctional megakaryopoiesis hampers platelet production, which is closely associated with thrombocytopenia (PT). Macrophages (MФs) are crucial cellular components in the bone marrow (BM) microenvironment. However, the specific effects of M1 MФs or M2 MФs on regulating megakaryocytes (MKs) are largely unknown. In the current study, aberrant BM-M1/M2 MФ polarization, characterized by increased M1 MФs and decreased M2 MФs and accompanied by impaired megakaryopoiesis-supporting abilities, was found in patients with PT post-allotransplant. RNA-seq and western blot analysis showed that the PI3K-AKT pathway was downregulated in the BM MФs of PT patients. Moreover, in vitro treatment with PI3K-AKT activators restored the impaired megakaryopoiesis-supporting ability of MФs from PT patients. Furthermore, we found M1 MФs suppress, whereas M2 MФs support MK maturation and platelet formation in humans. Chemical inhibition of PI3K-AKT pathway reduced megakaryopoiesis-supporting ability of M2 MФs, as indicated by decreased MK count, colony-forming unit number, high-ploidy distribution, and platelet count. Importantly, genetic knockdown of the PI3K-AKT pathway impaired the megakaryopoiesis-supporting ability of MФs both in vitro and in a MФ-specific PI3K-knockdown murine model, indicating a critical role of PI3K-AKT pathway in regulating the megakaryopoiesis-supporting ability of M2 MФs. Furthermore, our preliminary data indicated that TGF-ß released by M2 MФs may facilitate megakaryopoiesis through upregulation of the JAK2/STAT5 and MAPK/ERK pathways in MKs. Taken together, our data reveal that M1 and M2 MФs have opposing effects on MKs in a PI3K-AKT pathway-dependent manner, which may lead to new insights into the pathogenesis of thrombocytopenia and provide a potential therapeutic strategy to promote megakaryopoiesis.

Autophagy in endothelial cells regulates their haematopoiesis-supporting ability.

BACKGROUND: Endothelial cells (ECs) function as an instructive platform to support haematopoietic stem cell (HSC) homeostasis. Our recent studies found that impaired bone marrow (BM) ECs are responsible for the defective haematopoiesis in patients with poor graft function (PGF), which is characterised by pancytopenia post-allotransplant. Although activated autophagy was reported to benefit ECs, whether EC autophagy plays a critical role in supporting HSCs and its effect on PGF patients post-allotransplant remain unclear. METHODS: To evaluate whether the autophagy status of ECs modulates their ability to support haematopoiesis, human umbilical vein endothelial cells (HUVECs) and primary BM ECs derived from healthy donors were subjected to knockdown or overexpression of Beclin-1 (an autophagy-related protein). Moreover, BM ECs derived from PGF patients were studied. FINDINGS: Beclin-1 knockdown significantly reduced the haematopoiesis-supporting ability of ECs by suppressing autophagy, which could be restored by activating autophagy via Beclin-1 upregulation. Moreover, autophagy positively regulated haematopoiesis-related genes in HUVECs. Subsequently, a prospective case-control study demonstrated that defective autophagy reduced Beclin-1 expression and the colony-forming unit (CFU) plating efficiency in BM ECs from PGF patients compared to matched patients with good graft function. Rapamycin, an autophagy activator, quantitatively and functionally improved BM ECs from PGF patients in vitro and enhanced their ability to support HSCs by activating the Beclin-1 pathway. INTERPRETATION: Our results suggest that the autophagy status of ECs modulates their ability to support haematopoiesis by regulating the Beclin-1 pathway. Defective autophagy in BM ECs may be involved in the pathogenesis of PGF post-allotransplant. Rapamycin provides a promising therapeutic approach for PGF patients. FUNDING: Please see funding sources.

PPARγ maintains the metabolic heterogeneity and homeostasis of renal tubules.

BACKGROUND: The renal tubules, which have distant metabolic features and functions in different segments, reabsorb >99% of approximately 180 l of water and 25,000 mmol of Na + daily. Defective metabolism in renal tubules is involved in the pathobiology of kidney diseases. However, the mechanisms underlying the metabolic regulation in renal tubules remain to be defined. METHODS: We quantitatively compared the proteomes of the isolated proximal tubules (PT) and distal tubules (DT) from C57BL/6 mouse using tandem mass tag (TMT) labeling-based quantitative mass spectrometry. Bioinformatics analysis of the differentially expressed proteins revealed the significant differences between PT and DT in metabolism pathway. We also performed in vitro and in vivo assays to investigate the molecular mechanism underlying the distant metabolic features in PT and DT. FINDINGS: We demonstrate that the renal proximal tubule (PT) has high expression of lipid metabolism enzymes, which is transcriptionally upregulated by abundantly expressed PPARα/γ. In contrast, the renal distal tubule (DT) has elevated glycolytic enzyme expression, which is mediated by highly expressed c-Myc. Importantly, PPARγ transcriptionally enhances the protease iRhom2 expression in PT, which suppresses EGF expression and secretion and subsequent EGFR-dependent glycolytic gene expression and glycolysis. PPARγ inhibition reduces iRhom2 expression and increases EGF and GLUT1 expression in PT in mice, resulting in renal tubule hypertrophy, tubulointerstitial fibrosis and damaged kidney functions, which are rescued by 2-deoxy-d-glucose treatment. INTERPRETATION: These findings delineate instrumental mechanisms underlying the active lipid metabolism and suppressed glycolysis in PT and active glycolysis in DT and reveal critical roles for PPARs and c-Myc in maintaining renal metabolic homeostasis. FUND: This work was supported by the National Natural Science Foundation of China (grants 81572076 and 81873932; to Q.Z.), the Applied Development Program of the Science and Technology Committee of Chongqing (cstc2014yykfB10003; Q.Z.), the Program of Populace Creativities Workshops of the Science and Technology Committee of Chongqing (Q.Z.), the special demonstration programs for innovation and application of techniques (cstc2018jscx-mszdX0022) from the Science and Technology Committee of Chongqing (Q.Z.).

Impairment of bone marrow endothelial progenitor cells in acute graft-versus-host disease patients after allotransplant.

Graft-versus-host disease (GVHD) is a major complication after allogeneic haematopoietic stem cell transplantation (allo-HSCT) that is frequently associated with bone marrow (BM) suppression, and clinical management is challenging. BM endothelial progenitor cells (EPCs) play crucial roles in the regulation of haematopoiesis and thrombopoiesis. However, little is known regarding the functional roles of BM EPCs in acute GVHD (aGVHD) patients. In the current prospective case-control study, reduced and dysfunctional BM EPCs, characterized by decreased migration and angiogenesis capacities and increased levels of reactive oxygen species (ROS) and apoptosis, were found in aGVHD patients compared with those without aGVHD. Moreover, lower frequency and increased levels of ROS, apoptosis and DNA damage, but reduced colony-forming unit-plating efficiency were found in BM CD34+ cells of aGVHD patients compared with those without aGVHD. The severity of aGVHD and GVHD-mediated cytopenia was associated with BM EPC impairment in aGVHD patients. In addition, the EPC impairment positively correlated with ROS level. Taken together, our results suggest that reduced and dysfunctional BM EPCs may be involved in the pathogenesis of aGVHD. Although these findings require validation, our data indicate that improvement of BM EPCs may represent a promising therapeutic approach for aGVHD patients.

An unbalanced monocyte macrophage polarization in the bone marrow microenvironment of patients with poor graft function after allogeneic haematopoietic stem cell transplantation.

Poor graft function (PGF) is a severe complication of allogeneic haematopoietic stem cell transplantation (allo-HSCT). Murine studies have demonstrated that effective haematopoiesis depends on the specific bone marrow (BM) microenvironment. Increasing evidence shows that BM macrophages (MФs), which constitute an important component of BM immune microenvironment, are indispensable for the regulation of haematopoietic stem cells (HSCs) in the BM. However, little is known about the number and function of BM MФs or whether they directly interact with HSCs in PGF patients. In the current prospective case-control study, PGF patients showed a significant increase in classically activated inflammatory MФs (M1; 2·18 ± 0·11% vs. 0·82 ± 0·06%, P < 0·0001), a striking reduction in alternatively activated anti-inflammatory MФs (M2; 3·02 ± 0·31% vs. 21·89 ± 0·90%, P < 0·0001), resulting in a markedly increased M1/M2 ratio (0·82 ± 0·06 vs. 0·06 ± 0·002; P < 0·0001) in the BM compared with good graft function patients. Meanwhile, standard monocyte subsets were altered in PGF patients. Dysfunctional BM MФs, which were characterized by reduced proliferation, migration and phagocytosis, were evident in PGF patients. Furthermore, BM MФs from PGF patients with high tumour necrosis factor-α and interleukin 12 levels and low transforming growth factor-ß levels, led to impaired BM CD34+ cell function. In summary, our data indicate that an unbalanced BM M1/M2 ratio and dysfunctional MФs may contribute to the occurrence of PGF following allo-HSCT.

Dysfunctional Bone Marrow Mesenchymal Stem Cells in Patients with Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation.

Poor graft function (PGF) is a life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is characterized by defective hematopoiesis. Mesenchymal stem cells (MSCs) have been shown to support hematopoiesis, but little is known about the role of MSCs in the pathogenesis of PGF. In the current prospective case-control study, we evaluated whether the number and function of bone marrow (BM) MSCs in PGF patients differed from those in good graft function (GGF) patients. We found that BM MSCs from PGF patients expanded more slowly and appeared flattened and larger, exhibiting more apoptosis and senescence than MSCs from GGF patients. Furthermore, increased intracellular reactive oxygen species, p-p53, and p21 (but not p38) levels were detected in MSCs from PGF patients. Moreover, the ability of MSCs to sustain hematopoiesis was significantly reduced in PGF patients, as evaluated by cell number, apoptosis, and the colony-forming unit-plating efficiency of CD34+ cells. In summary, the biologic characteristics of PGF MSCs are different from those of GGF MSCs, and the in vitro hematopoiesis-supporting ability of PGF MSCs is significantly lower. Although requiring further validation, our study indicates that reduced and dysfunctional BM MSCs may contribute to deficient hematopoiesis in PGF patients. Therefore, improvement of BM MSCs may represent a promising therapeutic approach for PGF patients after allo-HSCT.

Apobec-1 complementation factor regulates cell migration and apoptosis through Dickkopf1 by acting on its 3' untranslated region in MCF7 cells.

A1CF (apobec-1 complementation factor) acts as a component of the apolipoprotein-B messenger RNA editing complex. Previous researches mainly focused on its post-transcriptional cytidine to uridine RNA editing. However, few study reported its role in progression of breast carcinoma cells. Wound healing assay and flow cytometry were applied to detect the migration and apoptosis; western blot, real-time polymerase chain reaction, and dual-luciferase assays were applied to investigate the potential regulation mechanism of A1CF-mediated cell migration and apoptosis. Knockdown of A1CF decreased cell migration and enhanced cell apoptosis in MCF7 cells in vitro. Western blot analysis showed that knockdown of A1CF decreased Dickkopf1 but increased c-Myc and ß-catenin expression, and overexpression of A1CF can get opposite results. Knockdown of Dickkopf1 in A1CF-overexpressed cells decreased cell migration and enhanced cell apoptosis compared with A1CF-overexpressed cells. Luciferase-fused 3' untranslated region of human Dickkopf1 activity was highly upregulated in A1CF-overexpressed MCF7 cells, but this upregulation can be inhibited by mutating conserved binding motifs of Dickkopf1 3' untranslated region. A1CF played a crucial role in cell migration and survival through affecting 3' untranslated region of Dickkopf1 to upregulate its expression in MCF7 cells.

TßRII Regulates the Proliferation of Metanephric Mesenchyme Cells through Six2 In Vitro.

The transforming growth factor-ß (TGFß) family signaling pathways play an important role in regulatory cellular networks and exert specific effects on developmental programs during embryo development. However, the function of TGFß signaling pathways on the early kidney development remains unclear. In this work, we aim to detect the underlying role of TGFß type II receptor (TßRII) in vitro, which has a similar expression pattern as the crucial regulator Six2 during early kidney development. Firstly, the 5-ethynyl-2'-deoxyuridine (EdU) assay showed knock down of TßRII significantly decreased the proliferation ratio of metanephric mesenchyme (MM) cells. Additionally, real-time Polymerase Chain Reaction (PCR) and Western blot together with immunofluorescence determined that the mRNA and protein levels of Six2 declined after TßRII knock down. Also, Six2 was observed to be able to partially rescue the proliferation phenotype caused by the depletion of TßRII. Moreover, bioinformatics analysis and luciferase assay indicated Smad3 could transcriptionally target Six2. Further, the EdU assay showed that Smad3 could also rescue the inhibition of proliferation caused by the knock down of TßRII. Taken together, these findings delineate the important function of the TGFß signaling pathway in the early development of kidney and TßRII was shown to be able to promote the expression of Six2 through Smad3 mediating transcriptional regulation and in turn activate the proliferation of MM cells.

The expression patterns of Tetratricopeptide repeat domain 36 (Ttc36).

Tetratricopeptide repeat domain 36 (Ttc36), whose coding protein belongs to tetratricopeptide repeat (TPR) motif family, has not been studied extensively. We for the first time showed that Ttc36 is evolutionarily conserved across mammals by bioinformatics. Rabbit anti-mouse Ttc36 polyclonal antibody was generated by injecting synthetic full-length peptides through "antigen intersection" strategy. Subsequently, we characterized Ttc36 expression profile in mouse, showing its expression in liver and kidney both from embryonic day 15.5 (E15.5) until adult, as well as in testis. Immunofluorescence staining showed that Ttc36 is diffusely expressed in liver, however, specifically in kidney cortex. Thus, we further compare Ttc36 with proximal tubules (PT) marker Lotus Tetragonolobus Lectin (LTL) and distal tubules (DT) marker Calbindin-D28k respectively by double immunofluorescence staining. Results showed the co-localization of Ttc36 with LTL rather than Calbindin-D28k. Collectively, on the basis of the expression pattern, Ttc36 is specifically expressed in proximal distal tubules.

Zeb1 Is a Potential Regulator of Six2 in the Proliferation, Apoptosis and Migration of Metanephric Mesenchyme Cells.

Nephron progenitor cells surround around the ureteric bud tips (UB) and inductively interact with the UB to originate nephrons, the basic units of renal function. This process is determined by the internal balance between self-renewal and consumption of the nephron progenitor cells, which is depending on the complicated regulation networks. It has been reported that Zeb1 regulates the proliferation of mesenchymal cells in mouse embryos. However, the role of Zeb1 in nephrons generation is not clear, especially in metanephric mesenchyme (MM). Here, we detected cell proliferation, apoptosis and migration in MM cells by EdU assay, flow cytometry assay and wound healing assay, respectively. Meanwhile, Western and RT-PCR were used to measure the expression level of Zeb1 and Six2 in MM cells and developing kidney. Besides, the dual-luciferase assay was conducted to study the molecular relationship between Zeb1 and Six2. We found that knock-down of Zeb1 decreased cell proliferation, migration and promoted cell apoptosis in MM cells and Zeb1 overexpression leaded to the opposite data. Western-blot and RT-PCR results showed that knock-down of Zeb1 decreased the expression of Six2 in MM cells and Zeb1 overexpression contributed to the opposite results. Similarly, Zeb1 promoted Six2 promoter reporter activity in luciferase assays. However, double knock-down of Zeb1 and Six2 did not enhance the apoptosis of MM cells compared with control cells. Nevertheless, double silence of Zeb1 and Six2 repressed cell proliferation. In addition, we also found that Zeb1 and Six2 had an identical pattern in distinct developing phases of embryonic kidney. These results indicated that there may exist a complicated regulation network between Six2 and Zeb1. Together, we demonstrate Zeb1 promotes proliferation and apoptosis and inhibits the migration of MM cells, in association with Six2.

EIYMNVPV Motif is Essential for A1CF Nucleus Localization and A1CF (-8aa) Promotes Proliferation of MDA-MB-231 Cells via Up-Regulation of IL-6.

Apobec-1 complementation factor (A1CF) is a heterogeneous nuclear ribonuceloprotein (hnRNP) and mediates apolipoprotein-B mRNA editing. A1CF can promote the regeneration of the liver by post-transcriptionally stabilizing Interleukin-6 (IL-6) mRNA. It also contains two transcriptional variants-A1CF64 and A1CF65, distinguished by the appearance of a 24-nucleotide motif which contributes to the corresponding eight-amino acid motif of EIYMNVPV. For the first time, we demonstrated that the EIYMNVPV motif was essential for A1CF nucleus localization, A1CF deficient of the EIYMNVPV motif, A1CF (-8aa) showed cytoplasm distribution. More importantly, we found that A1CF (-8aa), but not its full-length counterpart, can promote proliferation of MDA-MB-231 cells accompanied with increased level of IL-6 mRNA. Furthermore, silencing of IL-6 attenuated A1CF (-8aa)-induced proliferation in MDA-MB-231 cells. In conclusion, notably, these findings suggest that A1CF (-8aa) promoted proliferation of MDA-MB-231 cells in vitro viewing IL-6 as a target. Thus, the EIYMNVPV motif could be developed as a potential target for basal-like breast cancer therapy.

Apobec-1 Complementation Factor (A1CF) Inhibits Epithelial-Mesenchymal Transition and Migration of Normal Rat Kidney Proximal Tubular Epithelial Cells.

Apobec-1 complementation factor (A1CF) is a member of the heterogeneous nuclear ribonucleoproteins (hnRNP) family, which participates in site-specific posttranscriptional RNA editing of apolipoprotein B (apoB) transcript. The posttranscriptional editing of apoB mRNA by A1CF in the small intestine is required for lipid absorption. Apart from the intestine, A1CF mRNA is also reported to be highly expressed in the kidneys. However, it is remained unknown about the functions of A1CF in the kidneys. The aim of this paper is to explore the potential functions of A1CF in the kidneys. Our results demonstrated that in C57BL/6 mice A1CF was weakly expressed in embryonic kidneys from E15.5dpc while strongly expressed in mature kidneys after birth, and it mainly existed in the tubules of inner cortex. More importantly, we identified A1CF negatively regulated the process of epithelial-mesenchymal transition (EMT) in kidney tubular epithelial cells. Our results found ectopic expression of A1CF up-regulated the epithelial markers E-cadherin, and down-regulated the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA) in NRK52e cells. In addition, knockdown of A1CF enhanced EMT contrary to the overexpression effect. Notably, the two A1CF variants led to the similar trend in the EMT process. Taken together, these data suggest that A1CF may be an antagonistic factor to the EMT process of kidney tubular epithelial cells.

MiR542-3p Regulates the Epithelial-Mesenchymal Transition by Directly Targeting BMP7 in NRK52e.

Accumulating evidence demonstrated that miRNAs are highly involved in kidney fibrosis and Epithelial-Eesenchymal Transition (EMT), however, the mechanisms of miRNAs in kidney fibrosis are poorly understood. In this work, we identified that miR542-3p could promote EMT through down-regulating bone morphogenetic protein 7 (BMP7) expression by targeting BMP7 3'UTR. Firstly, real-time PCR results showed that miR542-3p was significantly up-regulated in kidney fibrosis in vitro and in vivo. Moreover, Western blot results demonstrated that miR542-3p may promote EMT in the NRK52e cell line. In addition, we confirmed that BMP7, which played a crucial role in anti-kidney fibrosis and suppressed the progression of EMT, was a target of miR542-3p through Dual-Luciferase reporter assay, as did Western blot analysis. The effects of miR542-3p on regulating EMT could also be suppressed by transiently overexpressing BMP7 in NRK52e cells. Taken together, miR542-3p may be a critical mediator of the induction of EMT via directly targeting BMP7.

MiR-30a Inhibits the Epithelial--Mesenchymal Transition of Podocytes through Downregulation of NFATc3.

MicroRNAs (miRNAs) possess an important regulating effect among numerous renal diseases, while their functions in the process of epithelial-to-mesenchymal transition (EMT) after podocyte injury remain unclear. The purpose of our study is to identify the potential functions of miR-30a in EMT of podocytes and explore the underlying mechanisms of miR-30a in the impaired podocytes. The results revealed that downregulation of miR-30a in podocyte injury animal models and patients, highly induced the mesenchymal markers of EMT including Collagen I, Fibronectin and Snail. Furthermore, overexpression of miR-30a enhances epithelial markers (E-cadherin) but diminished mesenchymal markers (Collagen I, Fibronectin and Snail) in podocytes. In addition, we established miR-30a target NFATc3, an important transcription factor of Non-canonical Wnt signaling pathway. More importantly, our findings demonstrated that the augmentation of miR-30a level in podocytes inhibits the nuclear translocation of NFATc3 to protect cytoskeleton disorder or rearrangement. In summary, we uncovered the protective function of miR30a targeting NFATc3 in the regulation of podocyte injury response to EMT.

miR181c promotes apoptosis and suppresses proliferation of metanephric mesenchyme cells by targeting Six2 in vitro.

Increasingly recognized importance has been assumed for microRNA (miRNA) in the regulation of the delicate balance of gene expression. In our study, we aimed to explore the regulation role of miR181c towards Six2 in metanephric mesenchyme (MM) cells. Bioinformatics analysis, luciferase assay and semi-quantitative real-time (RT) PCR, subsequently RT PCR, Western blotting, 5-ethynyl-2'-deoxyuridine cell proliferation assay, Cell Counting Kit-8 assay, immunofluorescence and flow cytometry, were employed to verify the modulation function of miR181c on Six2 in the mK3 MM cell line that is one kind of MM cells. miR181c was predicted to bind the 3' untranslated region of Six2 by bioinformatics analysis, which was subsequently validated by the in vitro luciferase reporter assay. Moreover, transfection of miR181c mimic can decrease the expression of Six2 both in mRNA and protein levels in mK3 cells. Still, ectopic expression of miR181c inhibits the proliferation, promotes the apoptosis and even makes the nephron progenitor phenotype lose mK3 cells. These results revealed the ability of a single miRNA-miR181c to downregulate the expression of Six2, restrain the proliferation and promote the apoptosis that even makes the nephron progenitor phenotype lose MM cells, suggesting a potential role of miR181c during the kidney development.

MiR-181b targets Six2 and inhibits the proliferation of metanephric mesenchymal cells in vitro.

MicroRNAs (miRNAs) are small non-coding RNAs that down-regulate gene expression by binding to target mRNA for cleavage or translational repression, and play important regulatory roles in renal development. Despite increasing genes have been predicted to be miRNA targets by bioinformatic analysis during kidney development, few of them have been verified by experiment. The objective of our study is to identify the miRNAs targeting Six2, a critical transcription factor that maintains the mesenchymal progenitor pool via self-renewal (proliferation) during renal development. We initially analyzed the 3'UTR of Six2 and found 37 binding sites targeted by 50 putative miRNAs in the 3'UTR of Six2. Among the 50 miRNAs, miR-181b is the miRNAs predicted by the three used websites. In our study, the results of luciferase reporter assay, realtime-PCR and Western blot demonstrated that miR-181b directly targeted on the 3'UTR of Six2 and down-regulate the expression of Six2 at mRNA and protein levels. Furthermore, EdU proliferation assay along with the Six2 rescue strategy showed that miR-181b suppresses the proliferation of metanephric mesenchymal by targeting Six2 in part. In our research, we concluded that by targeting the transcription factor gene Six2, miR-181b inhibits the proliferation of metanephric mesenchymal cells in vitro and might play an important role in the formation of nephrons.