LEF1 Research Articles

CLL-016: WNT3 and LEF1 Expression Levels and Outcome of Chronic Lymphocytic Leukemia in Egyptian Patients

Context Chronic lymphocytic leukemia (CLL), a hematologic malignancy, characterized by progressive accumulation of mature B lymphocytes. The role of Wnt signaling pathway has been reported in CLL biogenesis. Lymphoid enhancer-binding factor-1 (LEF1), a mediator of this canonical pathway has been identified in CLL. Objective To analyze Wnt signaling pathway members (WNT3) and its key mediator LEF1 expression levels in CLL patients and to assess their potential role as markers of CLL outcome. Design An observational prospective study from February 2017 till February 2018. Setting It was carried out in the Hematology unit at Oncology Center Mansoura University (OCMU). Patients This study was carried out on 35 newly diagnosed CLL patients in comparison with 15 adult controls, they were M/F 14/16 (47/53%), median age 63. Interventions The patients were subjected to history taking, physical examination and laboratory investigations of CLL. Wnt3 gene and LEF1 expression levels in peripheral blood of untreated CLL mononuclear cells and normal healthy controls were determined by RT-PCR. Main outcome measures Our study demonstrated significant upregulation of both Wnt3 and LEF1 in CLL PBMNC in comparison to healthy controls (P Results WNT3 and LEF1 were significantly decreased in CLL patients with ECOG 2&3 P=0.023 and 0.007, respectively. Untreated CLL patients harboring 17p deletion expressed significantly low LEF1 (P=0.033). A significant positive association was observed between WNT3 and LEF1 levels (Spearman r=0.74, P Conclusions WNT3 and its key mediator LEF1 were shown to be overexpressed and activated in CLL cells in comparison to healthy persons. Raising the interest that antagonizing Wnt signaling in CLL may represent an effective treatment potential. Interestingly, low expression of both WNT3 and LEF1 were associated with shortened survival in CLL patients which trigger more studies on a larger patient cohort.

Prenatal Exposure to Electronic-Cigarette Aerosols Leads to Sex-Dependent Pulmonary Extracellular-Matrix Remodeling and Myogenesis in Offspring Mice.

Electronic-cigarette (e-cig) vaping is a serious concern, as many pregnant women who vape consider it safe. However, little is known about the harmful effects of prenatal e-cig exposure on adult offspring, especially on extracellular-matrix (ECM) deposition and myogenesis in the lungs of offspring. We evaluated the biochemical and molecular implications of maternal exposure during pregnancy to e-cig aerosols on the adult offspring of both sexes, with a particular focus on pulmonary ECM remodeling and myogenesis. Pregnant CD-1 mice were exposed to e-cig aerosols with or without nicotine, throughout gestation, and lungs were collected from adult male and female offspring. Compared with the air-exposed control group, female mice exposed to e-cig aerosols, with or without nicotine, demonstrated increased lung protein abundance of LEF-1 (lymphoid enhancer-binding factor 1), fibronectin, and E-cadherin, whereas altered E-cadherin and PPARγ (peroxisome proliferator-activated receptor γ) levels were observed only in males exposed to e-cig aerosols with nicotine. Moreover, lipogenic and myogenic mRNAs were dysregulated in adult offspring in a sex-dependent manner. PAI-1 (plasminogen activator inhibitor-1), one of the ECM regulators, was significantly increased in females exposed prenatally to e-cig aerosols with nicotine and in males exposed to e-cig aerosols compared with control animals exposed to air. MMP9 (matrix metalloproteinase 9), a downstream target of PAI-1, was downregulated in both sexes exposed to e-cig aerosols with nicotine. No differences in lung histology were observed among any of the treatment groups. Overall, adult mice exposed prenatally to e-cig aerosols could be predisposed to developing pulmonary disease later in life. Thus, these findings suggest that vaping during pregnancy is unsafe and increases the propensity for later-life interstitial lung diseases.

2,4-Diamino-Quinazoline, a Wnt Signaling Inhibitor, Suppresses Gastric Cancer Progression and Metastasis.

Gastric cancer (GC) is among the most treatment-refractory epithelial malignancies. Aberrant activation of Wnt/β-catenin-signaling has been implicated in a variety of human cancers, including gastric cancer. Here we report that the elevated expression of lymphoid enhancer binding factor 1 (Lef1) is associated with the TNM (tumor– node–metastasis) stage of gastric cancer. Subsequently, 2,4-diamino-quinazoline (2,4-DAQ), a selective inhibitor of Lef1, was identified to suppress the expression of Wnt/β-catenin target genes such as AXIN2, MYC and LGR5 and result in the suppression of gastric cancer cell growth through the apoptotic pathway. The 2,4-DAQ also exhibited an inhibitory effect on the migration/invasion of gastric cancer cells. Importantly, the treatment of human gastric tumor xenograft with 2,4-DAQ suppressed tumor growth in a nude mouse model. Furthermore, 2,4-DAQ appears effective on patient-derived organoids (PDOs). Transcriptome sequencing analysis also revealed that 2,4-DAQ are more effective on the gastric cancers that exhibit higher expression levels of Wnt-signaling pathway-related genes than their adjacent normal gastric tissues.

LEF1-AS1 accelerates tumorigenesis in glioma by sponging miR-489-3p to enhance HIGD1A

Long non-coding (lncRNA) lymphoid enhancer-binding factor 1 antisense RNA 1 (LEF1-AS1) has been validated to be implicated in manifold cancers, whereas its function in glioma has not been understood thoroughly. Hence, in this study, we tested that LEF1-AS1 expression was significantly upregulated in glioma tissues and cell lines. Besides, knockdown of LEF1-AS1 repressed cell proliferation while activated apoptosis in glioma cells in vitro, and also suppressed tumor growth in vivo. RNA pull-down and luciferase reporter assays affirmed that LEF1-AS1 could bind with miR-489-3p. In addition, miR-489-3p expression was downregulated in glioma cells. Moreover, miR-489-3p depletion partly offset LEF1-AS1 knockdown-mediated function on proliferation and apoptosis. Further, HIGD1A identified as the target gene of miR-489-3p was upregulated in glioma cells. HIGD1A silence could restrict the process of glioma. In rescue assays, upregulation of HIGD1A remedied the inhibitory impacts of LEF1-AS1 silence on glioma cell growth. In summary, our studies corroborated the regulatory mechanism of LEF1-AS1/miR-489-3p/HIGD1A axis in glioma, suggesting that targeting LEF1-AS1 might be a promising method for glioma therapy in the future.

Lymphoid enhancer-binding factor 1 (LEF-1): a favorable prognostic factor in adult acute myeloid leukemia in Egyptian patients

BackgroundCanonical wingless-type (Wnt) signaling is a crucial pathway involved in normal hematopoiesis and the self-renewal process of hematopoietic stem cells. Deregulation of this pathway has been associated with different subtypes of leukemia. Lymphoid enhancer-binding factor 1 (LEF-1) is a major transcription factor of this pathway and plays a pivotal role in lymphoid differentiation and granulopoiesis. High LEF-1 expression has been reported as a prognostic marker in several types of adult hematological malignancies. We aimed to assess the prognostic utility of LEF-1 expression in adult de novo acute myeloid leukemia (AML) Egyptian patients in continuation of our previous work. LEF-1 expression was analyzed by real-time polymerase chain reaction (PCR) in 30 adults with newly diagnosed AML and remeasured at day 28 after induction therapy with the assessment of remission status.ResultsPatients were classified according to median expression level into high and low LEF-1 expression groups. LEF-1 levels were dramatically decreased following successful induction therapy. Also, high LEF-1 expression patients had a better response to therapy with better overall survival. ROC curve analysis of LEF-1 expression yielded a cutoff value of < 10.11 log10 (sensitivity of 90.48% and specificity of 100%) for predicting poor outcome. Univariate logistic regression analysis showed that for every log10 increase in the LEF-1 expression level, the chance of the patient to achieve hematological remission was increased by 2.29 folds.ConclusionOur study showed preliminary results that overexpression of LEF-1 is a favorable prognostic factor in newly diagnosed adult AML patients. The prognostic value of LEF-1 could suggest its utility for further risk classifications of AML and potentiality for being a target for therapy.

An oncogenic viral interferon regulatory factor upregulates CUB domain-containing protein 1 to promote angiogenesis by hijacking transcription factor lymphoid enhancer-binding factor 1 and metastasis suppressor CD82.

Kaposi's sarcoma (KS), a highly angiogenic and invasive vascular tumor, is the most common AIDS-associated cancer caused by KS-associated herpesvirus (KSHV) infection. We have recently shown that KSHV-encoded viral interferon regulatory factor 1 (vIRF1) contributes to KSHV-induced cell motility (PLoS Pathog. 15:e1007578, 2019). However, the role of vIRF1 in KSHV-induced angiogenesis remains unknown. Here, using two in vivo angiogenesis models including the chick chorioallantoic membrane assay (CAM) and the matrigel plug angiogenesis assay in mice, we show that vIRF1 promotes angiogenesis by upregulating CUB domain (for complement C1r/C1s, Uegf, Bmp1) containing protein 1 (CDCP1). Mechanistically, vIRF1 enhances the expression of transcription factor lymphoid enhancer-binding factor 1 (Lef1) and binds to Lef1 to promote CDCP1 transcription. Meanwhile, vIRF1 degrades metastasis suppressor CD82 through an ubiquitin-proteasome pathway by recruiting E3 ubiquitin ligase AMFR to CD82, which protects CDCP1 from CD82-mediated, palmitoylation-dependent degradation. CDCP1 activates AKT signaling, which is required for vIRF1-induced cell motility but not angiogenesis. Our results illustrate that, by hijacking Lef1 and CD82, vIRF1 upregulates CDCP1 to promote angiogenesis and cell invasion. These novel findings demonstrate the vIRF1 targets multiple cellular proteins and pathways to promote the pathogenesis of KS, which could be attractive therapeutic targets for KSHV-induced malignancies.

Diagnostic Utility of LEF1 Immunohistochemistry in Differentiating Deep Penetrating Nevi From Histologic Mimics.

Deep penetrating nevi (DPNs) are intermediate grade lesions which have the capacity to recur, metastasize, or progress to melanoma. Differentiating DPN from other melanocytic lesions including blue and cellular blue nevi can be diagnostically challenging, and markers to distinguish these entities can be useful. Mutations of the β-catenin and mitogen-activated protein kinase pathways have recently been elucidated as distinctive of DPN. This pathway can subsequently activate lymphoid enhancer-binding factor 1 (LEF1), a transcription factor shown to facilitate the epithelial-mesenchymal transition to propagate tumorigenesis. Seventy-two cases in total were examined on hematoxylin and eosin sections and with β-catenin and LEF1 immunohistochemistry. This included: DPN (14), cellular blue nevi (19), blue nevi (15), congenital melanocytic nevi (12), and melanoma (12). Nuclear expression of LEF1, present throughout the entire depth of the lesion, was noted in 13/14 (93%) of DPN, 0/19 (0%) of cellular blue nevi, 0/15 (0%) of blue nevi, 1/12 (8%) of congenital melanocytic nevi, and 9/12 (75%) of melanoma cases. Nuclear expression of β-catenin, present throughout the entire depth of the lesion, was noted in 14/14 (100%) of DPN, 0/18 (0%) of cellular blue nevi, 0/15 (0%) of blue nevi, 1/12 (8%) of congenital melanocytic nevi, and 1/12 (8%) of melanoma cases. A majority of congenital melanocytic nevi demonstrated a gradient of LEF1 and β-catenin expression with more intense staining superficially and loss of staining with increasing depth. Deep, uniform nuclear LEF1 combined with β-catenin immunohistochemical staining can be useful in distinguishing DPN from histologic mimics.

MicroRNA-381 inhibits metastasis and epithelial-mesenchymal transition of glioblastoma cells through targeting LEF1.

Glioblastoma is a common intracranial malignancy that is extremely harmful to human life and health. Various microRNAs (miRNAs) have been reported to be involved in the progression of glioblastoma, except miR-381. Therefore, the purpose of this study is to investigate the role of miR-381 in glioblastoma. The expression of miR-381 and LEF1 (lymphoid enhancer-binding factor-1) was quantified using quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot analysis. Transwell and Dual-Luciferase reporter assays were used to investigate the regulatory mechanism of miR-381/LEF1 in glioblastoma. Downregulation of miR-381 was observed in A172 cells. In addition, the overexpression of miR-381 restrained migration and invasion of glioblastoma cells. Furthermore, overexpression of miR-381 inhibited epithelial-mesenchymal transition (EMT) in A172 cells. Further, miR-381 was confirmed to directly target LEF1 and negatively regulates its expression in glioblastoma cells. Downregulation of LEF1 also inhibited cell migration, invasion, and EMT in glioblastoma cells. More importantly, the upregulation of LEF1 abolished the inhibitory effect of miR-381 in glioblastoma cells. MiR-381 inhibits cell metastasis and EMT in glioblastoma by suppressing LEF1 expression.

Integrated miRNA-mRNA network revealing the key molecular characteristics of ossification of the posterior longitudinal ligament

Background:Ossification of the posterior longitudinal ligament (OPLL) refers to an ectopic ossification disease originating from the posterior longitudinal ligament of the spine. Pressing on the spinal cord or nerve roots can cause limb sensory and motor disorders, significantly reducing the patient's quality of life. At present, the pathogenesis of OPLL is still unclear. The purpose of this study is to integrate microRNA (miRNA)-mRNA biological information data to further analyze the important molecules in the pathogenesis of OPLL, so as to provide targets for future OPLL molecular therapy.Methods:miRNA and mRNA expression profiles of GSE69787 were downloaded from Gene Expression Omnibus database and analyzed by edge R package. Funrich software was used to predict the target genes and transcription factors of de-miRNA. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis of differentially expressed genes (DEGs) were carried out based on CLUEGO plug-in in Cytoscape. Using data collected from a search tool for the retrieval of interacting genes online database, a protein-protein interaction (PPI) network was constructed using Cytoscape. The hub gene selection and module analysis of PPI network were carried out by cytoHubba and molecular complex detection, plug-ins of Cytoscape software respectively.Results:A total of 346 genes, including 247 up-regulated genes and 99 down-regulated genes were selected as DEGs. SP1 was identified as an upstream transcription factor of de-miRNAs. Notably, gene ontology enrichment analysis shows that up- and down-regulated DEGs are mainly involved in BP, such as skeletal structure morphogenesis, skeletal system development, and animal organ morphogenesis. Kyoto Encyclopedia of Genes and Genomes enrichment analysis indicated that only WNT signaling pathway was associated with osteogenic differentiation. Lymphoid enhancer binding factor 1 and wingless-type MMTV integration site family member 2 Wingless-Type MMTV Integration site family member 2 were identified as hub genes, miR-520d-3p, miR-4782-3p, miR-6766-3p, and miR-199b-5p were identified as key miRNAs. In addition, 2 important network modules were obtained from PPI network.Conclusions:In this study, we established a potential miRNA-mRNA regulatory network associated with OPLL, revealing the key molecular mechanism of OPLL and providing targets for future treatment or prevent its occurrence.

Low LEF1 expression is a biomarker of early T-cell precursor, an aggressive subtype of T-cell lymphoblastic leukemia.

Early T-cell precursor (ETP) is the only subtype of acute T-cell lymphoblastic leukemia (T-ALL) listed in the World Health Organization (WHO) classification of myeloid neoplasms and acute leukemia. Patients with ETP tend to have worse disease outcomes. ETP is defined by a series of immune markers. The diagnosis of ETP status can be vague due to the limitation of the current measurement. In this study, we performed unsupervised clustering and supervised prediction to investigate whether a molecular biomarker can be used to identify the ETP status in order to stratify risk groups. We found that the ETP status can be predicted by the expression level of Lymphoid enhancer binding factor 1 (LEF1) with high accuracy (AUC of ROC = 0.957 and 0.933 in two T-ALL cohorts). The patients with ETP subtype have a lower level of LEF1 comparing to the those without ETP. We suggest that incorporating the biomarker LEF1 with traditional immune-phenotyping will improve the diagnosis of ETP.

CREB1-induced lncRNA LEF1-AS1 contributes to colorectal cancer progression via the miR-489/DIAPH1 axis

Long non-coding RNAs (lncRNAs) have been identified as new regulatory factors in tumor progression. Lymphoid enhancer-binding factor 1 antisense RNA 1 (LEF1-AS1) was a recently identified lncRNA. This research aimed to investigate the roles and mechanisms of LEF1-AS1 in colorectal cancer (CRC). We firstly showed that LEF1-AS1 expression was upregulated in human CRC tissues and cell lines. LEF1-AS1 upregulation was demonstrated to be induced by CREB1. Clinical study revealed that high LEF1-AS1 expression was positively associated with histological grade, lymph nodes metastasis, and decreased survivals of CRC patients. Functionally, down-regulation of LEF1-AS1 using si-LEF1-AS1 decreased cell growth, migration and invasion, as well as increased apoptosis in CRC cells. Mechanically, LEF1-AS1 functioned as competing endogenous RNA (ceRNA) for miR-489 to positively recover DIAPH1, thus playing an oncogenic role in CRC pathogenesis. Overall, our observations identified a novel CRC-related lncRNA LEF1-AS1 and discovered a critical role for this lncRNA as a ceRNA in CRC pathogenesis, suggesting that it may serve as a novel biomarker for prognosis and act as a therapeutic target for CRC treatment.

M6A methyltransferase METTL3 promotes the progression of prostate cancer via m6A-modified LEF1.

This study aims to uncover the regulatory effects of METTL3 on promoting the progression of prostate cancer (PCa) through N6-Methyladenosine (m6A) methylation on LEF1 mRNA. The relative levels of METTL3 and LEF1 in 48 paired PCa tissues and adjacent ones were determined by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Their correlation in PCa tissues was analyzed by Spearman correlation test. The survival of PCa patients affected by METTL3 was assessed by introducing Kaplan-Meier method. Wound closure assay was performed to evaluate the potential influence of METTL3 on migratory ability in PC-3 cells. Protein level of LEF1 in PC-3 cells with METTL3 or IGF2BP2 knockdown was examined. The activity of the Wnt pathway was tested through TOP/FOP-Flash. Furthermore, the interaction between LEF1 with METTL3 or IGF2BP2 was verified through RIP (RNA-Binding Protein Immunoprecipitation) assay. At last, the regulatory effects of METTL3/LEF1 axis on the activity of the Wnt pathway and migratory ability in PC-3 cells were determined. METTL3 and LEF1 were upregulated in PCa tissues, and they presented a positive correlation in PCa. A high level of METTL3 predicted poor prognosis in PCa patients. The knockdown of METTL3 suppressed the migratory ability in PC-3 cells. Meanwhile, the knockdown of METTL3 downregulated protein level of LEF1 and decreased the activity of the Wnt pathway. The results of RIP assay indicated that METTL3 methylation sites were present on LEF1 mRNA. Moreover, the silence of METTL3 decreased the enrichment abundance of LEF1 in anti-IGF2BP2. Rescue experiments demonstrated that the overexpression of LEF1 partially reversed the regulatory effects of METTL3 on the Wnt activity and migratory ability in PCa cells. METTL3 is upregulated in PCa tissues. METTL3 influences the activity of the Wnt pathway through m6A methylation on LEF1 mRNA, thereafter, promoting the progression of PCa.

Long Non-Coding RNA LEF1-AS1 Promotes Migration, Invasion and Metastasis of Colon Cancer Cells Through miR-30-5p/SOX9 Axis.

IntroductionAberrant expression of long non-coding RNAs (lncRNAs) has been implicated in the tumorigenesis and progression of colon cancer. Lymphoid enhancer-binding factor 1 antisense RNA 1 (LEF1-AS1), a highly conserved and newly discovered long non-coding RNA, has been reported to be upregulated and correlated with poor prognosis in colon cancer, but the exact role of it remains uncertain.Materials and MethodsIn our study, the biological functions of LEF1-AS1 in colon cancer were analyzed by cell viability assay, colony formation assay, scratch wound healing assay, transwell cell invasion assay, soft agar assay, luciferase reporter assay, pull down assay, tumor xenograft model and Western blot.ResultsWe found that LEF1-AS1 was upregulated in colon cancer patients and correlated with poor overall survival and recurrent-free survival. Besides, enforced expression of LEF1-AS1 in HT29 and T84 cells promoted migration, invasion, anchorage-independent growth, tumor xenograft formation and lung metastasis, while knockdown of LEF1-AS1 in COLO320 cells suppressed cell migration, invasion, anchorage-independent growth and tumor xenograft formation. In addition, LEF1-AS1 was directly interacted and inversely correlated with miR-30-5p in colon cancer, and SOX9 was a downstream target for miR-30-5p. LEF1-AS1 overexpression increased the expression level of SOX9, and restoration of SOX9 attenuated the effects caused by LEF1-AS1 knockdown in cell migration, invasion, anchorage-independent growth and tumor xenograft formation.ConclusionOur results indicated that LEF1-AS1 promoted migration, invasion and metastasis of colon cancer cells partially through miR-30-5p/SOX9 axis. The oncogenic LEF1-AS1 could be a potential prognostic biomarker for colon cancer.

Lef‐1 is Required for Proper Cell Cycle Progression in Airway Basal Cells

The surface airway epithelium (SAE) is a pseudostratified layer of cells found within the mammalian airway, and serves as a barrier between the environment and the rest of the body. Its function is crucial for proper respiration and innate immunity. At homeostasis the SAE has very low cellular turnover, yet has the capacity to regenerate itself after injury or infection at an astonishingly fast rate. This is due primarily to the population of resident stem cells found within the SAE called basal cells. Though much work has been done to identify the molecular mechanisms that govern basal cell proliferation, migration, and differentiation, many questions still remain.The transcription factor Lymphoid Enhancer Binding Factor 1 (Lef‐1) is a Wnt effector protein expressed in stem cell populations in various epithelial tissues, including the developing lung. Our lab has shown that overexpression of Lef‐1 in primary airway stem cell cultures leads to increased proliferation and migration. Additionally, Lef‐1 is required for the development of airway submucosal glands (SMGs), epithelial structures that are continuous with the SAE. However, it is not known if Lef‐1 plays a role in basal cell function. Therefore, we sought to determine the effects of Lef‐1 deletion in basal cells. Though its expression in basal cells is almost undetectable on a population‐wide scale, deletion of Lef‐1 from primary mouse basal cells severely decreases their ability to proliferate in culture, and skews them more toward a secretory club cell fate. Lef‐1 deletion leads to a loss in DNA damage response (DDR) gene expression, including Tp73, Rad51, and Chek1. Lef‐1 knockout basal cells tend to stall in the cell cycle at the G1/S transition. Together these studies indicate an important role for Lef‐1 in basal cell replication and proliferation.Support or Funding InformationUniversity of Iowa Molecular Medicine ProgramUniversity of Iowa Department of Anatomy &amp; Cell Biology Funding: 2R01 DK047967

LncRNA LEF1-AS1 silencing diminishes EZH2 expression to delay hepatocellular carcinoma development by impairing CEBPB-interaction with CDCA7

ABSTRACT Hepatocellular carcinoma (HCC) is recognized for its high mortality rate worldwide. Based on intensive studies, long non-coding RNA (lncRNA) expression exerts significant effects on tumor suppression. Herein, we investigated the molecular mechanism of lymphoid enhancer-binding factor-1 antisense RNA 1 (LEF1-AS1) in HCC cells. Microarray-based gene expression analysis was adopted to predict and verify the differentially expressed genes in HCC, which predicted cell division cycle-associated 7 (CDCA7) and LEF1-AS1 to be highly expressed in HCC. The expression of LEF1-AS1, CDCA7, CCAAT/enhancer-binding protein beta (CEBPB) and enhancer of zeste homolog 2 (EZH2) was determined by means of reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. LncMap was used to predict the lncRNA-transcription factor-gene interaction in HCC. ChIP, RIP assay and dual luciferase reporter gene assay were employed to verify the relationship between the transcription factor and gene. Silencing of LEF1-AS1 could downregulate CDCA7 expression through CEBPB. Overexpression of LEF1-AS1, EZH2 and CDCA7 promoted proliferation and invasion in HCC cells. LEF1-AS1 promoted CDCA7 expression to further upregulate EZH2. Tumor formation in nude mice was assessed to verify the experimental results. Silencing of LEF1-AS1 inhibited the growth of tumors in vivo. Collectively, silencing LEF1-AS1 inhibited the proliferation and invasion of HCC cells by down-regulating EZH2 through the CEBPB-CDCA7 signaling pathway, which provides scientific evidence for the treatment of HCC. Abbreviations: HCC: Hepatocellular carcinoma; lncRNA: long non-coding RNA; LEF1-AS1: lymphoid enhancer-binding factor-1 antisense RNA 1; EZH2: enhancer of zeste homolog 2; CDCA7: cell division cycle-associated 7; GEO: Gene Expression Omnibus; NC: negative control; oe: overexpressed; RT-qPCR: reverse transcription quantitative polymerase chain reaction; PBS: phosphate buffered saline; HRP: horseradish peroxidase; OD: optical density; RIP: Radioimmunoprecipitation; ChIP: Chromatin immunoprecipitation; WT: wild type.

Deletion of hypoxia-responsive microRNA-210 results in a sex-specific decrease in nephron number.

Low nephron number results in an increased risk of developing hypertension and chronic kidney disease. Intrauterine growth restriction is associated with a nephron deficit in humans, and is commonly caused by placental insufficiency, which results in fetal hypoxia. The underlying mechanisms by which hypoxia impacts kidney development are poorly understood. microRNA-210 is the most consistently induced microRNA in hypoxia and is known to promote cell survival in a hypoxic environment. In this study, the role of microRNA-210 in kidney development was evaluated using a global microRNA-210 knockout mouse. A male-specific 35% nephron deficit in microRNA-210 knockout mice was observed. Wnt/β-catenin signaling, a pathway crucial for nephron differentiation, was misregulated in male kidneys with increased expression of the canonical Wnt target lymphoid enhancer binding factor 1. This coincided with increased expression of caspase-8-associated protein 2, a known microRNA-210 target and apoptosis signal transducer. Together, these data are consistent with a sex-specific requirement for microRNA-210 in kidney development.

Long non-coding RNA LEF1-AS1 is involved in the progression of retinoblastoma through regulating the Wnt/β-catenin pathway.

The lymphoid enhancer binding factor 1 antisense RNA 1 (LEF1-AS1) has been suggested to function as a tumour-associated lncRNA in several types of human cancers, but there is no study to date about the role of LEF1-AS1 in retinoblastoma. In our study, LEF1-AS1 expression was increased in retinoblastoma tissues and cell lines compared with paired adjacent normal tissues and the retinal pigment epithelial cell line, respectively. Meanwhile, we found that patients with retinoblastoma with IIRC D-E or undifferentiated type had notably higher levels of LEF1-AS1 expression than those with IIRC A-C or differentiated type. High LEF1-AS1 expression predicted poor disease-free survival in patients with retinoblastoma. The in vitro assays suggested that silencing of LEF1-AS1 suppressed retinoblastoma cell proliferation, migration, and invasion through regulating the Wnt/β-catenin pathway. In conclusion, LEF1-AS1 functions as an oncogenic lncRNA in retinoblastoma.

Silencing of long noncoding RNA LEF1-AS1 prevents the progression of hepatocellular carcinoma via the crosstalk with microRNA-136-5p/WNK1.

Long noncoding RNAs (lncRNAs) have been recognized as cancer-associated biological molecules, favoring hepatocellular carcinoma (HCC) progression. This study was conducted to elucidate the effects lncRNA lymphoid enhancer-binding Factor 1 antisense RNA (LEF1-AS1) on the pathological development of HCC, along with the crosstalk involving microRNA-136-5p (miR-136-5p) and with-no-K (lysine) kinase 1 (WNK1). The study recruited primary HCC tissues and their corresponding nonneoplastic liver tissues. The gain- and loss-of-function studies were performed in HCC cells HuH-7 and tumor xenografts in nude mice. The dual luciferase reporter gene assay system, RNA pull-down, and radioimmunoprecipitation assays were applied to detect their interactions among lncRNA LEF1-AS1, miR-136-5p, and WNK1. 5-Ethynyl-2'-deoxyuridine staining, scratch test, Transwell assays, and in vitro tube formation assays were conducted to examine HCC cell proliferation, migration, and invasion and HUVEC angiogenesis. HCC tissues and cells contained high lncRNA LEF1-AS1 expression. LncRNA LEF1-AS1 upregulation triggered markedly increased HCC cell proliferation, migration, and invasion and human umbilical vein endothelial cell angiogenesis. In vivo silencing lncRNA LEF1-AS1 resulted in reduced tumor cell vitality and matrix metalloproteinase-9 and the vascular endothelial growth factor expression. Additionally, the role of lncRNA LEF1-AS1 was found to be largely dependent on WNK1. Association of lncRNA LEF1-AS1 with WNK1 blocked the inhibitory effect of miR-136-5p on WNK1, which was confirmed by in vivo experiments. Altogether, our results revealed an important role of lncRNA LEF1-AS1 in regulating the HCC progression by regulating WNK1, providing a potential biomarker for the therapeutic modalities regarding HCC.

LEF1 haploinsufficiency causes ectodermal dysplasia.

Ectodermal dysplasias are a family of genodermatoses commonly associated with variants in the ectodysplasin/NF-κB or the Wnt/β-catenin pathways. Both pathways are involved in signal transduction from ectoderm to mesenchyme during the development of ectoderm-derived structures. Wnt/β-catenin pathway requires the lymphoid enhancer-binding factor 1 (LEF1), a nuclear mediator, to activate target gene expression. In mice, targeted inactivation of the LEF1 gene results in a complete block of development of multiple ectodermal appendages. We report two unrelated patients with 4q25 de novo deletion encompassing LEF1, associated with severe oligodontia of primary and permanent dentition, hypotrichosis and hypohidrosis compatible with hypohidrotic ectodermal dysplasia. Taurodontism and a particular alveolar bone defect were also observed in both patients. So far, no pathogenic variants or variations involving the LEF1 gene have been reported in human. We provide further evidence for LEF1 haploinsufficiency role in ectodermal dysplasia and delineate its clinical phenotype.

Carboxy-Terminal Cementum Protein 1-Derived Peptide 4 (cemp1-p4) Promotes Mineralization through wnt/β-catenin Signaling in Human Oral Mucosa Stem Cells.

Human cementum protein 1 (CEMP1) is known to induce cementoblast and osteoblast differentiation and alkaline phosphatase (ALP) activity in human periodontal ligament-derived cells in vitro and promotes bone regeneration in vivo. CEMP1′s secondary structure analysis shows that it has a random-coiled structure and is considered an Intrinsic Disordered Protein (IDP). CEMP1′s short peptide sequences mimic the biological capabilities of CEMP1. However, the role and mechanisms of CEMP1′s C-terminal-derived synthetic peptide (CEMP1-p4) in the canonical Wnt/β-catenin signaling pathway are yet to be described. Here we report that CEMP1-p4 promotes proliferation and differentiation of Human Oral Mucosa Stem Cells (HOMSCs) by activating the Wnt/β-catenin pathway. CEMP1-p4 stimulation upregulated the expression of β-catenin and glycogen synthase kinase 3 beta (GSK-3B) and activated the transcription factors TCF1/7 and Lymphoid Enhancer binding Factor 1 (LEF1) at the mRNA and protein levels. We found translocation of β-catenin to the nucleus in CEMP1-p4-treated cultures. The peptide also penetrates the cell membrane and aggregates around the cell nucleus. Analysis of CEMP1-p4 secondary structure revealed that it has a random-coiled structure. Its biological activities included the induction to nucleate hydroxyapatite crystals. In CEMP1-p4-treated HOMSCs, ALP activity and calcium deposits increased. Expression of Osterix (OSX), Runt-related transcription factor 2 (RUNX2), Integrin binding sialoproptein (IBSP) and osteocalcin (OCN) were upregulated. Altogether, these data show that CEMP1-p4 plays a direct role in the differentiation of HOMSCs to a “mineralizing-like” phenotype by activating the β-catenin signaling cascade.