LIM Homeobox Research Articles

Long noncoding RNA SNHG3 promotes glioma tumorigenesis by sponging miR-485-5p to upregulate LMX1B expression.

LIM homeobox transcription factor 1-beta (LMX1B) has recently been found to be highly expressed in advanced gliomas and is associated with poor survival. However, the regulatory molecular mechanism of LMX1B expression in gliomas remains unclear. In this study, bioinformatics analysis showed that miR-485-5p may be the potential upstream regulator of LMX1B, and long noncoding RNA (lncRNA) small nucleolar RNA host gene 3 (SNHG3) may function as a competitive endogenous RNA to sponge miR-485-5p. In addition, the expression of SNHG3 and LMX1B in advanced glioma tissues was significantly upregulated, while the expression of miR-485-5p was significantly downregulated. SNHG3 overexpression reduced the expression of miR-485-5p; increased the expression of LMX1B; and promoted the proliferation, migration, and invasion of glioma cells. In contrast, miR-485-5p overexpression reduced the expression of LMX1B and inhibited cell proliferation, migration, and invasion. The luciferase reporter assay and RNA immunoprecipitation assay further confirmed the interaction between SNHG3 and miR-485-5p and between miR-485-5p and LMX1B. In addition, subcutaneous and orthotropic xenograft models confirmed that lncRNA SNHG3 silencing or miR-485-5p overexpression significantly reduced the growth of glioma xenografts and prolonged survival time. These results indicate that lncRNA SNHG3 can regulate the expression of LMX1B by sponging miR-485-5p, thereby promoting the proliferation, migration, and invasion of glioma cells. This study provides the first evidence that the SNHG3/miR-485-5p/LMX1B axis is involved in glioma tumorigenesis and highlights the potential of SNHG3 and miR-485-5p as therapeutic targets for glioma.

Effect of LHX2 gene methylation level and its function on radiotherapy of cervical cancer.

BackgroundCervical cancer is the most common malignancy of the female reproductive system, for which radiotherapy is one of the main treatments. Gene methylation in cells is an important factor in tumorigenesis, and radiotherapy can change DNA methylation in cells. At the same time, combined with the clinical effect of radiotherapy, key genes of LIM homeobox 2 (LHX2) significantly related to cervical cancer. The LHX2 are LIM-homeobox genes that play important roles in signal transduction, cell differentiation, tissue-specific differentiation, and body formation.MethodsIn this study, bisulfite genomic sequencing (BSP-Seq) technology was used to analyze the methylation level of LHX2 in patients with cervical cancer before and after radiotherapy. In addition, combined with the clinical effect of radiotherapy, the function of LHX2 in siHA and C33A cells were analyzed with the help of overexpression, small interfering RNA (siRNA), cell invasion, and migration ability. The expression level of the migration- and apoptosis-related genes which were affected by LHX2 were tested with quantitative real time polymerase chain reaction (qRT-PCR).ResultsCombined with clinical treatment, methylation level difference, and correlation enrichment analysis, it was found that LHX2 genes were closely related to the occurrence and development of cervical cancer. After 5-aza-2’-deoxycytidine (5-Aza-dC) and radiotherapy, the methylation of LHX2 genes in siHA and C33A squamous cell carcinoma cells was decreased, and the messenger RNA (mRNA) and protein expression levels were relatively increased; meanwhile, the LHX2 could accelerate the ability for cell invasion and migration and inhibited the apoptosis of the cell after treatment with radiotherapy.ConclusionsThe methylation and expression levels of LHX2 genes are closely related to cervical cancer. The methylation level of LHX2 was reduced after radiation therapy. The LHX2 gene has a positive effect on cervical cancer through acceleration of the cell invasion and migration ability and inhibition of cell apoptosis after radiotherapy treatment.

EMBR-09. EXAMINING THE ROLE OF THE DEVELOPMENTALLY ENCODED TRANSCRIPTION FACTOR, LHX9, IN GROUP 3 MEDULLOBLASTOMA

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Despite major advances in our understanding of the biology of MB, novel treatments remain urgently needed. Using a chemical-genomics driven drug repositioning strategy, we identified the cardiac glycoside family of compounds as potential treatments for Group 3 MB. We subsequently demonstrated that single-agent treatment with digoxin prolongs survival in a patient-derived xenograft model (PDOX) of Group 3 MB to a degree comparable to radiation therapy, a mainstay in the treatment of MB. Finally, we examined the mechanism of digoxin-mediated cell killing using RNA-seq. This work identified LHX9, a member of the LIM homeobox family of transcription factors, as the gene most significantly down-regulated following treatment (Huang and Injac et al, Sci Trans Medicine, 2018). Homologs of LHX9 play key roles in cerebellar development via spatially and temporally restricted expression and LHX9 has been proposed as a core transcription factor (TF) in the regulatory circuitry of Group 3 tumors. Loss of function of other core TFs has been shown to impact MB growth. The role of LHX9 in MB, however, has not been previously experimentally evaluated. We now report that knockdown of LHX9 in MB-derived cell lines results in marked growth inhibition. RNA-seq analysis of LHX9-depleted cells showed changes which included alterations in extracellular matrix-receptor interactions and TGFb signaling. These findings raise the possibility that loss of LHX9 plays a major role in digoxin-mediated cell killing and that LHX9 represents a key dependency required for the growth of Group 3 MB. Clinical targeting of core TFs would represent a novel approach to targeting this devastating disease.

Limited correlation between tumor markers and minimal residual disease detected by seven neuroblastoma-associated mRNAs in high-risk neuroblastoma patients.

Vanillylmandelic acid (VMA), homovanillic acid (HVA), neuron-specific enolase (NSE) and lactate dehydrogenase (LDH) are classical tumor markers and are used as standard clinical evaluations for patients with neuroblastoma (NB). Minimal residual disease (MRD) can be monitored by quantifying several sets of NB-associated mRNAs in the bone marrow (BM) and peripheral blood (PB) of patients with NB. Although MRD in BM and PB has been revealed to be a strong prognostic factor that is independent of standard clinical evaluations, its interrelation with tumor markers remains uncharacterized. The present study determined the levels of tumor markers (VMA, HVA, NSE and LDH) and MRD (BM-MRD and PB-MRD) in 133 pairs of concurrently collected BM, PB and urine samples from 19 patients with high-risk NB. The patients were evaluated during the entire course of treatment, which included 10 diagnoses, 32 treatments, 36 post-treatment, 9 relapses and 46 post-relapse sample pairs. The level of BM-MRD and PB-MRD was determined by quantifying 7 NB-mRNAs (collapsin response mediator protein 1, dopamine beta-hydroxylase, dopa decarboxylase, growth-associated protein 43, ISL LIM homeobox 1, pairedlike homeobox 2b and tyrosine hydroxylase) using droplet digital PCR. In overall sample pairs, tumor markers (VMA, HVA, NSE and LDH) demonstrated weak but significant correlations (P<0.011) with BM-MRD and PB-MRD. In subgroups according to each patient evaluation, the degree of correlation between tumor markers and MRD became stronger in patients with adrenal gland tumors, BM metastasis at diagnosis and relapse/regrowth compared with overall sample pairs. In contrast, tumor markers demonstrated variable correlations with MRD in subgroups according to each sample evaluation (BM infiltration at sampling, collection time point and disease status). The results suggested that tumor markers may demonstrate limited correlation with MRD in patients with high-risk NB.

Downregulation of USP18 reduces tumor-infiltrating activated dendritic cells in extranodal diffuse large B cell lymphoma patients.

Extranodal diffuse large B cell lymphoma (EN DLBCL) often leads to poor outcomes, while the underlying mechanism remains unclear. As immune imbalance plays an important role in lymphoma pathogenesis, we hypothesized that immune genes might be involved in the development of EN DLBCL. Ninety-three differentially expressed immune genes (DEIGs) were identified from 1168 differentially expressed genes (DEGs) between tumor tissues of lymph node DLBCL (LN DLBCL) and EN DLBCL patients in TCGA database. Nine prognostic immune genes were further identified from DEIGs by univariate Cox regression analysis. A multivariate predictive model was established based on these prognostic immune genes. Patients were divided into high- and low-risk groups according to the median model-based risk score. Kaplan-Meier survival curves showed that patients in the high-risk group had a shorter survival time than those in the low-risk group (P < 0.001). Ubiquitin-specific peptidase 18 (USP18) was further recognized as the key immune gene in EN DLBCL on the basis of coexpression of differentially expressed transcription factors (DETFs) and prognostic immune genes. USP18 exhibited low expression in EN DLBCL, which was regulated by LIM homeobox 2 (LHX2) (R = 0.497, P < 0.001, positive). The potential pathway downstream of USP18 was the MAPK pathway, identified by gene set variation analysis (GSVA), gene set enrichment analysis (GSEA) and Pearson correlation analysis (R = 0.294, P < 0.05, positive). The “ssGSEA” algorithm and Pearson correlation analysis identified that activated dendritic cells (aDCs) were the cell type mostly associated with USP18 (R = 0.694, P < 0.001, positive), indicating that USP18 participated in DC-modulating immune responses. The correlations among key biomarkers were supported by multiomics database validation. Indeed, the USP18 protein was confirmed to be expressed at lower levels in tumor tissues in patients with EN DLBCL than in those with LN DLBCL by immunohistochemistry. In short, our study illustrated that the downregulation of USP18 was associated with reduced aDC number in the tumor tissues of EN DLBCL patients, indicating that targeting USP18 might serve as a promising therapy.

ETS Binding Sites are Critical for Activity of the LHX2‐Associated Sub‐AER Regulatory Module 1 (LASARM1)

Fibroblast growth factors (FGFs) secreted from the apical ectodermal ridge (AER) coordinate proximal-distal patterning, while sonic hedgehog (SHH) from the zone of polarizing activity (ZPA) directs anterior-posterior growth of the limb. These signaling centers maintain each other's expression in a reciprocal feedback loop; however, the molecular intermediates involved are only partially characterized. LIM homeobox 2 (LHX2) has been identified as an intermediate regulator downstream of FGF in the reciprocal loop. We have identified a cis-regulatory module (CRM) located upstream of the LHX2 promoter that is active in the sub-AER LHX2 expression domain. Overexpression of FGF in Hamburger-Hamilton stage 24 (HH24) chicken limb buds, resulted in increased expression of several FGF-associated transcription factors, including E26 transformation-specific proteins (ETS). Members of the ETS family have been known to regulate the expression of SHH via an enhancer known as the Zone of Polarizing Activity Regulatory Sequence (ZRS). We wondered whether the regulation of LHX2 was mediated through this LHX2-associated sub-AER Regulatory Module 1 (LASARM1) via ETS transcription factors. In silico analysis of LASARM1 revealed three ETS binding sites within LASARM1 and hypothesized that mutation of all three binding sites would disrupt FGF-mediated enhancer activity. To determine whether these ETS transcription factor binding sites contribute to LASARM1 activity, we performed site directed mutagenesis on all three ETS binding sites (triple∆ETS) within a LASARM1-ptk-eGFP reporter construct, followed by targeted regional electroporation (TREP) into the limb buds of HH24 chick embryos. Transfection efficiency was determined by co-transfection with a β-actin promoter-driven RFP construct. After 24 hours of incubation, LASARM1 activity was determined by fluorescence microscopy. We found that mutation of all three ETS binding sites within LASARM1 resulted in fluorescence that was undetectable, suggesting reduced LASARM1 activity. Our findings suggest that FGF signaling utilizes ETS transcription factors to regulate LHX2 expression through the LASARM1 enhancer. Further work is needed to confirm this connection. Investigations regarding the relationship between FGF and LHX2 will provide important insights into the mechanisms underlying the FGF-SHH regulatory loop.

Smyd1 Orchestrates Early Heart Development Through Positive and Negative Gene Regulation.

SET and MYND domain-containing protein 1 (Smyd1) is a striated muscle-specific histone methyltransferase. Our previous work demonstrated that deletion of Smyd1 in either cardiomyocytes or the outflow tract (OFT) resulted in embryonic lethality at E9.5, with cardiac structural defects such as truncation of the OFT and right ventricle and impaired expansion and proliferation of the second heart field (SHF). The cardiac phenotype was accompanied by the downregulation of ISL LIM Homeobox 1 (Isl1) and upregulation of atrial natriuretic factor (ANF). However, the mechanisms of Smyd1 regulating Isl1 and ANF during embryonic heart development remain to be elucidated. Here, we employed various biochemical and molecular biological approaches including chromatin immunoprecipitation polymerase chain reaction (ChIP-PCR), pGL3 fluorescence reporter system, and co-immunoprecipitation (CoIP) and found that Smyd1 interacted with absent small homeotic-2-like protein (ASH2L) and activated the promoter of Isl1 by trimethylating H3K4. We also found that Smyd1 associated with HDAC to repress ANF expression using trichostatin A (TSA), a deacetylase inhibitor. In conclusion, Smyd1 participates in early heart development by upregulating the expression of Isl1 and downregulating the expression of ANF.

LIM Homeobox9 knockdown by morpholino does not affect zebrafish retinal development.

ABSTRACTLIM homeobox 9 (Lhx9) is a member of the LIM homeodomain transcription factor family, which expresses and functions in various vertebrate tissues, such as the gonads and pineal gland. Previous studies on lhx9 in zebrafish have mainly focused on the brain. However, little is known about the expression pattern of lhx9 during embryogenesis. Here, we detected lhx9 expression in zebrafish embryos using whole-mount in situ hybridization and found lhx9 expressed in heart, pectoral fin, and retina during their development in zebrafish. We then detailed the expression of lhx9 in retinal development. To further investigate the function of Lhx9 in retinogenesis, we performed morpholino (MO) knockdown experiments and found that upon lhx9 knockdown by MO, larvae presented normal eye development, retinal neural development, differentiation, proliferation, apoptosis, and responses to light stimulus. We not only elaborated the expression pattern of lhx9 in zebrafish embryogenesis, but we also demonstrated that lhx9 knockdown by morpholino does not affect the zebrafish retinal development, and our study provides data for further understanding of the role of Lhx9 in zebrafish retinal development.

Genetic background modifies vulnerability to glaucoma-related phenotypes in Lmx1b mutant mice.

ABSTRACTVariants in the LIM homeobox transcription factor 1-beta (LMX1B) gene predispose individuals to elevated intraocular pressure (IOP), a key risk factor for glaucoma. However, the effect of LMX1B mutations varies widely between individuals. To better understand the mechanisms underlying LMX1B-related phenotypes and individual differences, we backcrossed the Lmx1bV265D (also known as Lmx1bIcst) allele onto the C57BL/6J (B6), 129/Sj (129), C3A/BLiA-Pde6b+/J (C3H) and DBA/2J-Gpnmb+ (D2-G) mouse strain backgrounds. Strain background had a significant effect on the onset and severity of ocular phenotypes in Lmx1bV265D/+ mutant mice. Mice of the B6 background were the most susceptible to developing abnormal IOP distribution, severe anterior segment developmental anomalies (including malformed eccentric pupils, iridocorneal strands and corneal abnormalities) and glaucomatous nerve damage. By contrast, Lmx1bV265D mice of the 129 background were the most resistant to developing anterior segment abnormalities, had less severe IOP elevation than B6 mutants at young ages and showed no detectable nerve damage. To identify genetic modifiers of susceptibility to Lmx1bV265D-induced glaucoma-associated phenotypes, we performed a mapping cross between mice of the B6 (susceptible) and 129 (resistant) backgrounds. We identified a modifier locus on Chromosome 18, with the 129 allele(s) substantially lessening severity of ocular phenotypes, as confirmed by congenic analysis. By demonstrating a clear effect of genetic background in modulating Lmx1b-induced phenotypes, providing a panel of strains with different phenotypic severities and identifying a modifier locus, this study lays a foundation for better understanding the roles of LMX1B in glaucoma with the goal of developing new treatments.

MiR-142-5p promotes cervical cancer progression by targeting LMX1A through Wnt/β-catenin pathway.

BackgroundPrevious work has shown that miR-142-5p in cervical cancer tissues increased significantly compared with adjacent normal tissues. However, the function and the mechanism of miR-142-5p in cervical cancer have not been reported.MethodsQuantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to determine the gene expression levels. MTT, flow cytometry, and transwell assays were performed to explore the functions of miR-142-5p in HeLa cells. The potential target gene of miR-142-5p was investigated via luciferase reporter assays. The protein expression levels were analyzed by Western blotting.ResultsWe found that miR-142-5p expression was elevated but LIM homeobox transcription factor 1 alpha (LMX1A) was decreased in cervical cancer tissues and cells. Overexpression of miR-142-5p or knockdown of LMX1A inhibited cell apoptosis, promoted cell proliferation, migration, invasion abilities, and activated the Wnt/β-catenin pathway. However, knockdown of miR-142-5p or overexpression of LMX1A showed opposite results. LMX1A was identified as a direct target of miR-142-5p by luciferase reporter assays. Finally, rescue experiments demonstrated that LMX1A overexpression attenuated the carcinogenic effect of miR-142-5p mimic on HeLa cells.ConclusionsThese findings suggested that miR-142-5p might be a cervical cancer oncogene and could serve as a potential therapeutic target for the treatment of cervical cancer.

LIM Homeobox 4 (lhx4) regulates retinal neural differentiation and visual function in zebrafish

LIM homeobox 4 (LHX4) is expressed in the photoreceptors (PRs) of the outer nuclear layer (ONL) and bipolar cells (BCs) of the inner nuclear layer (INL) in mouse and chicken retina. It regulates the subtype-specific development of rod BCs and cone BCs in the mouse retina. However, no report has been published on its expression and function in the zebrafish retina. In this study, we assessed the expression of Lhx4 using in situ hybridization (ISH) technique and explored its role in zebrafish (Danio rerio) retinal development via morpholino (MO) technology. We found that the expression of lhx4 in the zebrafish retina begins 48 h post-fertilization (hpf) and is continuously expressed in the ONL and INL. A zebrafish model constructed with lhx4 knockdown in the eyes through vivo-MO revealed that: lhx4 knockdown inhibits the differentiation of Parvalbumin+ amacrine cells (ACs) and Rhodopsin+ rod photoreceptors (RPs), enhances the expression of visual system homeobox 2 (vsx2); and damages the responses of zebrafish to light stimulus, without affecting the differentiation of OFF-BCs and rod BCs, and apoptosis in the retina. These findings reveal that lhx4 regulates neural differentiation in the retina and visual function during zebrafish embryonic development.

Association of ISL1 polymorphisms and eosinophilic levels among otitis media patients.

BackgroundOtitis media (OM) is a middle ear inflammatory complex disorder involving genetic and environmental factors. It onsets during childhood and often recurs and perplexes in genetically susceptible patients. Previously, murine models had shown the association of ISL LIM homeobox 1 (ISL1) gene with otitis media with effusion.AimTo investigate the association of ISL1 genetic variants with otitis media.Subjects and methodsA total of 285 cases and 277 controls were recruited for the study. The entire coding region of ISL1 gene was genotyped using Sanger sequencing or single‐strand conformation polymorphism methods. Genotype, haplotype, in silico analysis, and linkage disequilibrium analysis were performed.ResultsThe variants rs2303751 (c.504A>G) and rs121913540 (c.513G>A) were associated with OM, and the OR (95%CI) was 0.74 (0.57–0.95) and 0.43 (0.20–0.91), respectively. Besides, the rs2303751 AA genotype was associated with elevated eosinophil numbers in OM when compared to controls. The 5 SNP haplotype analysis of SNPs c.‐492A>G, c.504A>G, c.513G>A, c.576C>T, and c.*651A>T revealed A‐A‐G‐C‐A to be a risk haplotype in females whereas the 3 SNP haplotype analysis of SNPs c.504A>G, c.513G>A, and c.567C>T suggested G‐A‐C as protective and A‐G‐C to be a risk haplotype for otitis media.ConclusionOurs is the first report which shows a significant association of ISL1 variants (rs2303751 and rs121913540) with hearing‐related disorder like otitis media in humans. These results implicate the possible role of ISL1 gene in the etiopathology of otitis media. The replication of the study in other ethnic populations may strengthen our findings.

Identification of Candidate Genes for Osteoporosis via Integrated Bioinformatics Analysis

Osteoporosis is a common bone disease; however, its pathophysiology is yet unclear. This study aimed to investigate candidate genes in osteoporosis and its pathomechanism. Microarray datasets Genomic Spatial Event-7429, 13850, 56815, and 7158, comprising data obtained from blood samples of osteoporosis patients and healthy controls, were downloaded from the Gene Expression Omnibus database. Differentially expressed genes were identified via intersection of the four datasets, using Affy and Limma software packages. Functional and pathway enrichment analysis of differentially expressed genes were conducted using the database for annotation, visualization, and integrated discovery database. Thereafter, protein-protein interactions between the products of differentially expressed genes and key modules were analyzed using the search tool for the retrieval of interacting genes/proteins database and Cytoscape software. Furthermore, a transcriptional regulatory network was established with the differentially expressed genes, using the Web-based gene set analysis toolkit database. In total, 702 differentially expressed genes were filtered from the intersection of the four datasets, which were primarily enriched in functions and pathways associated with glutamate secretion, Adenosine triphosphate binding, extracellular region, and phosphatidylinositol 3-kinase-protein kinase B signaling pathway. Furthermore, 361 nodes and 846 edges were present in the protein-protein interaction network. Two significant modules were obtained, each containing 22 key genes. Transcriptional factor-target gene regulatory network analysis revealed two vital transcription factors, Forkhead Box O4 and LIM Homeobox 3. Genes including endothelin receptor type A, XC chemokine receptor 1, 5-hydroxytryptamine receptor 2a, phosphatidylinositol-4,5-bisphosphate phosphodiesterase beta-4, glutamate metabotropic receptor 5, kiss-1 metastasis suppressor, 1-phosphatidylinositol-4,5-bisphosphate phosphodiesterase beta-2, cholinergic receptor muscarinic 3, g protein subunit gamma 4, neurotensin receptor 1, neuromedin U, neuromedin B, breast cancer gene 1 associated RING domain 1, exonuclease 1, replication factor C subunit 2, restriction site associated DNA 52 homolog DNA repair protein, DNA topoisomerase III alpha, Hemolytic uremic syndrome 1 checkpoint clamp component, timeless circadian regulator, breast cancer gene 1 DNA repair associated, tumor protein p53 and claspin are potential key genes for osteoporosis and may help elucidate the underlying pathomechanism, and transcription factors Forkhead Box O4 and LIM Homeobox 3 are potential therapeutic targets for osteoporosis.

The role of pineal microRNA-325 in regulating circadian rhythms after neonatal hypoxic-ischemic brain damage.

Circadian rhythm disorder is a common, but often neglected, consequence of neonatal hypoxic-ischemic brain damage (HIBD). However, the underlying molecular mechanisms remain largely unknown. We previously showed that, in a rat model of HIBD, up-regulation of microRNA-325 (miR-325) in the pineal gland is responsible for the suppression of Aanat, a key enzyme involved in melatonin synthesis and circadian rhythm regulation. To better understand the mechanism by which miR-325 affects circadian rhythms in neonates with HIBD, we compared clinical samples from neonates with HIBD and samples from healthy neonates recruited from the First Affiliated Hospital of Soochow University (Dushuhu Branch) in 2019. We found that circulating miR-325 levels correlated positively with the severity of sleep and circadian rhythm disorders in neonates with HIBD. Furthermore, a luciferase reporter gene assay revealed that LIM homeobox 3 (LHX3) is a novel downstream target of miR-325. In addition, in miR-325 knock-down mice, the transcription factor LHX3 exhibited an miR-325-dependent circadian pattern of expression in the pineal gland. We established a neonatal mouse model of HIBD by performing double-layer ligation of the left common carotid artery and exposing the pups to a low-oxygen environment for 2 hours. Lhx3 mRNA expression was significantly down-regulated in these mice and partially rescued in miR-325 knockout mice subjected to the same conditions. Finally, we showed that improvement in circadian rhythm-related behaviors in animals with HIBD was dependent on both miR-325 and LHX3. Taken together, our findings suggest that the miR-325-LHX3 axis is responsible for regulating circadian rhythms and provide novel insights into the identification of potential therapeutic targets for circadian rhythm disorders in patients with neonatal HIBD. The clinical trial was approved by Institutional Review Board of Children's Hospital of Soochow University (approval No. 2015028) on July 20, 2015. Animal experiments were approved by Animal Care and Use Committee, School of Medicine, Soochow University, China (approval No. XD-2016-1) on January 15, 2016.

MBRS-17. EXAMINING THE ROLE OF LHX9 IN GROUP 3 MEDULLOBLASTOMA

Medulloblastoma (MB) is the most common malignant brain tumor of childhood. Despite major advances in our understanding of the biology of MB, novel treatments remain urgently needed. Using a chemical-genomics driven drug repositioning strategy, we identified the cardiac glycoside family of compounds as potential treatments for Group 3 MB. We subsequently demonstrated that single-agent treatment with digoxin prolongs survival in a patient-derived xenograft model (PDOX) of Group 3 MB to a degree comparable to radiation therapy, a mainstay in the treatment of MB. Finally, we examined the mechanism of digoxin-mediated cell killing using RNA-seq. This work identified LHX9, a member of the LIM homeobox family of transcription factors, as the gene most significantly down-regulated following treatment (Huang and Injac et al, Sci Trans Medicine, 2018). Homologs of LHX9 play key roles in cerebellar development via spatially and temporally restricted expression and LHX9 has been proposed as a core transcription factor (TF) in the regulatory circuitry of Group 3 tumors. Loss of function of other core TFs has been shown to impact MB growth. The role of LHX9 in MB, however, has not been previously experimentally evaluated. We now report that knockdown of LHX9 in MB-derived cell lines results in marked growth inhibition raising the possibility that loss of LHX9 plays a major role in digoxin-mediated cell killing and that LHX9 represents a key dependency required for the growth of Group 3 MB. Clinical targeting of core TFs would represent a novel approach to targeting this devastating disease.

Identification of the potential roles of ring finger protein 8 in TP53-mutant breast cancer.

Breast cancer is one of the malignant tumors with the highest mortality rate. With the development of precise treatment technology for cancer, numerous molecular targets have been identified and applied in the treatment of diseases. The present study investigated the potential role of ring finger protein 8 (RNF8) in TP53-mutant breast cancer and explored its possible mechanisms of action through a combination of bioinformatics techniques and cell biology. The results revealed that significantly different genes were expressed in RNF8-knockout mice sequencing data compared with in the control group in the presence of TP53 mutations. Downregulated genes were significantly enriched in several pathways of cell proliferation and apoptosis regulation, development and transcription regulation, while upregulated genes were mainly enriched in immune response-associated signaling pathways. Therefore, the consensus genes of the major signaling pathways were further analyzed, revealing that among patients with TP53 wild-type breast cancer, the prognosis of patients with low expression levels of fibroblast growth factor receptor 1, LIM homeobox 2 and EPH receptor B2 was improved compared with that of patients with high expression levels, while among patients with TP53-mutant breast cancer, there was no significant difference in survival status. In addition, among patients with TP53-mutant breast cancer, the prognosis of patients with high BR serine/threonine kinase 1 expression was significantly improved compared with that in patients with low expression. Finally, cell biology experiments demonstrated that in TP53-mutant breast cancer cells (HCC1937), inhibition of RNF8 significantly inhibited the proliferation of TP53-mutant HCC1937 cells and promoted their apoptosis. The present findings may enrich the understanding of the role of RNF8 and indicated that RNF8 may be used as a potential molecular target in TP53-mutant breast cancer, which may lead to the development of clinical treatment strategies.

Joint regulation of Wnt and bone morphogenetic protein signaling pathways to promote differentiation of human induced pluripotent stem cells into cardiomyocytes

To explore the role of joint regulation of Wnt and bone morphogenetic protein (BMP) signaling pathways in the differentiation of human induced pluripotent stem cells (hiPSCs) into cardiomyocytes. HiPSCs were cultured and observed under inverted phase contrast microscope. Immunofluorescence staining was used to observe the expressions of hiPSCs pluripotent markers (OCT3/4, NANOG, and TRA-1-60). HiPSCs were passaged which were taken for subsequent experiments within the 35th passage. When the fusion degree of hiPSCs was close to 100%, the CHIR99021 (Wnt pathway activator) was added on the 0th day of differentiation. Different concentrations of IWP4 (inhibitor of Wnt production) were added on the 3rd day of differentiation, and the best concentration of IWP4 was added at different time points. The optimal concentration and the best effective period of IWP4 were obtained by detecting the expression of troponin T (TNNT2) mRNA by real-time fluorescence quantitative PCR. Then, on the basis of adding CHIR99021 and IWP4, different concentrations of BMP-4 were added on the 5th day of differentiation, and the best concentration of BMP-4 was added at different time points. The optimal concentration and best effective period of BMP-4 were obtained by detecting the expression of TNNT2 mRNA. Finally, hiPSCs were divided into three groups: Wnt group, BMP group, and Wnt+BMP group. On the basis of adding CHIR99021 on the 0th day of differentiation, IWP4, BMP-4, and IWP4+BMP-4 were added into Wnt group, BMP group, and Wnt+BMP group respectively according to the screening results. Cells were collected on the 7th and the 15th days of differentiation. The expressions of myocardial precursor cell markers [ISL LIM homeobox 1 (ISL1), NK2 homeobox 5 (NKX2-5)] and cardiomyocyte specific markers [myocyte enhancer factor 2C (MEF2C), myosin light chain 2 (MYL2), MYL7, and TNNT2] were detected by real-time fluorescent quantitative PCR. Cells were collected on the 28th day of differentiation, and the expression of cardiac troponin T (cTnT) was detected by flow cytometry and immunofluorescence staining. The results of cell mophology and immunoflurescence staining showed that the OCT3/4, NANOG, and TRA-1-60 were highly expressed in hiPSCs, which suggested that hiPSCs had characteristics of pluripotency. The optimal concentration of IWP4 was 10.0 μmol/L ( P<0.05) and the best effective period was the 3rd day ( P<0.05) in inducing hiPSCs to differentiate into cardiomyocytes. The optimal concentration of BMP-4 was 20.0 ng/mL ( P<0.05) and the best effective period was the 3rd day ( P<0.05). The relative expressions of ISL1, NKX2-5, MEF2C, MYL2, MYL7, and TNNT2 mRNAs, the positive expression ratio of cTnT detected by flow cytometry, and sarcomere structure detected by immunofluorescence staining of Wnt+BMP group were superior to those of Wnt group ( P<0.05). Joint regulation of Wnt and BMP signaling pathways can improve the differentiation efficiency of hiPSCs into cardiomyocytes.

Genome-Wide Analysis Identifies an Essential Human TBX3 Pacemaker Enhancer.

The development and function of the pacemaker cardiomyocytes of the sinoatrial node (SAN), the leading pacemaker of the heart, are tightly controlled by a conserved network of transcription factors, including TBX3 (T-box transcription factor 3), ISL1 (ISL LIM homeobox 1), and SHOX2 (short stature homeobox 2). Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined. Identification of the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation. We performed Assay for Transposase-Accessible Chromatin using sequencing on human pluripotent stem cell-derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative REs specific for either human cell type. We validated pacemaker cell-specific elements in the SHOX2 and TBX3 loci. CRISPR-mediated homozygous deletion of the mouse ortholog of a noncoding region with candidate pacemaker-specific REs in the SHOX2 locus resulted in selective loss of Shox2 expression from the developing SAN and embryonic lethality. Putative pacemaker-specific REs were identified up to 1 Mbp upstream of TBX3 in a region close to MED13L harboring variants associated with heart rate recovery after exercise. The orthologous region was deleted in mice, which resulted in selective loss of expression of Tbx3 from the SAN and (cardiac) ganglia and in neonatal lethality. Expression of Tbx3 was maintained in other tissues including the atrioventricular conduction system, lungs, and liver. Heterozygous adult mice showed increased SAN recovery times after pacing. The human REs harboring the associated variants robustly drove expression in the SAN of transgenic mouse embryos. We provided a genome-wide collection of candidate human pacemaker-specific REs, including the loci of SHOX2, TBX3, and ISL1, and identified a link between human genetic variants influencing heart rate recovery after exercise and a variant RE with highly conserved function, driving SAN expression of TBX3.

LIM homeobox 6 (Lhx6)+ neurons in the ventral zona incerta project to the core portion of the lateral supramammillary nucleus in the rat

There was a recent report suggesting that LIM homeobox 6 (Lhx6)+ GABA-releasing neurons of the ventral zona incerta (ZI) promote sleep. We demonstrated in the previous study that Lhx6+ ZI neurons are activated during paradoxical sleep (PS) hypersomnia which was induced by 48-hour PS deprivation, implying their roles in the control of PS like melanin-concentrating hormone (MCH) cells. Since the core portion of the lateral supramammillary nucleus (SUMl) is the major hypothalamic area activating the dentate gyrus as well as other limbic cortices during PS, we examined in the present study whether Lhx6+ ZI cells provide efferent projections to the SUMl, using the retrograde-tracing method. The majority of Lhx6+ neurons projecting to the SUMl occupied the ventral border (or ventral one-third) of the ventral ZI. Based on the quantitative analysis, the mean number of retrogradely-labeled Lhx6+ neurons was comparable to that of retrogradely-labeled MCH cells in the ZI. However, the total (i.e., single- plus double-labeled) number of Lhx6+ cells was approximately three times larger than that of MCH cells in the ZI. Thus, the proportion (about 7.8%) of retrogradely-labeled Lhx6+ neurons over the total Lhx6+ cells was approximately one-third of the percentage (about 20.9%) of retrogradely-labeled MCH neurons over the total MCH cells. On the other hand, a combination of retrogradely-labeled, Lhx6 and MCH cells occupied approximately 43.7% of the total retrogradely-labeled neurons in the ventral ZI. The present observations suggested that Lhx6+ neurons in the ventral ZI might play an important role in the regulation of PS, partly via the neural network involving the SUMl.

Electrically-Driven Cell-Type Specific Neuromodulation in the External Globus Pallidus

The identification of distinct cell-types throughout the basal ganglia has been essential in advancing our understanding of network function and improving neurological therapies. In the globus pallidus externa (GPe), interventions targeted to neuronal subpopulations have profound therapeutic potential, but are challenging to implement in clinical settings. We investigated whether electrical stimulation can be tuned to engage cell-type specific responses in the GPe. Although conventional stimulation was non-specific, variance in the synaptic inputs onto Parvalbumin (PV-GPe) and Lim homeobox 6 (Lhx6-GPe) subpopulations enabled us to segregate the responses of these neurons using brief, high frequency bursts of stimulation. The circuit elements responsible for this synaptic variation were found to be asymmetrical inputs from the STN and collaterals of striatonigral neurons. Using a machine-learning approach, we established stimulus parameters that maximize this cell-type specific neuromodulation. These results establish the feasibility of rapidly transforming knowledge about circuit architecture into translatable therapeutic approaches.