LIM Homeobox Gene Research Articles

Evo-devo applied to sleep research: an approach whose time has come.

Sleep occurs in all animals but its amount, form, and timing vary considerably between species and between individuals. Currently, little is known about the basis for these differences, in part, because we lack a complete understanding of the brain circuitry controlling sleep-wake states and markers for the cell types which can identify similar circuits across phylogeny. Here, I explain the utility of an "Evo-devo" approach for comparative studies of sleep regulation and function as well as for sleep medicine. This approach focuses on the regulation of evolutionary ancient transcription factors which act as master controllers of cell-type specification. Studying these developmental transcription factor cascades can identify novel cell clusters which control sleep and wakefulness, reveal the mechanisms which control differences in sleep timing, amount, and expression, and identify the timepoint in evolution when different sleep-wake control neurons appeared. Spatial transcriptomic studies, which identify cell clusters based on transcription factor expression, will greatly aid this approach. Conserved developmental pathways regulate sleep in mice, Drosophila, and C. elegans. Members of the LIM Homeobox (Lhx) gene family control the specification of sleep and circadian neurons in the forebrain and hypothalamus. Increased Lhx9 activity may account for increased orexin/hypocretin neurons and reduced sleep in Mexican cavefish. Other transcription factor families specify sleep-wake circuits in the brainstem, hypothalamus, and basal forebrain. The expression of transcription factors allows the generation of specific cell types for transplantation approaches. Furthermore, mutations in developmental transcription factors are linked to variation in sleep duration in humans, risk for restless legs syndrome, and sleep-disordered breathing. This paper is part of the "Genetic and other molecular underpinnings of sleep, sleep disorders, and circadian rhythms including translational approaches" collection.

LIM-HD transcription factors control axial patterning and specify distinct neuronal and intestinal cell identities in planarians.

Adult planarians can regenerate the gut, eyes and even a functional brain. Proper identity and patterning of the newly formed structures require signals that guide and commit their adult stem cells. During embryogenesis, LIM-homeodomain (LIM-HD) transcription factors act in a combinatorial 'LIM code' to control cell fate determination and differentiation. However, our understanding about the role these genes play during regeneration and homeostasis is limited. Here, we report the full repertoire of LIM-HD genes in Schmidtea mediterranea. We found that lim homeobox (lhx) genes appear expressed in complementary patterns along the cephalic ganglia and digestive system of the planarian, with some of them being co-expressed in the same cell types. We have identified that Smed-islet1, -lhx1/5-1, -lhx2/9-3, -lhx6/8, -lmx1a/b-2 and -lmx1a/b-3 are essential to pattern and size the planarian brain as well as for correct regeneration of specific subpopulations of dopaminergic, serotonergic, GABAergic and cholinergic neurons, while Smed-lhx1/5.2 and -lhx2/9.2 are required for the proper expression of intestinal cell type markers, specifically the goblet subtype. LIM-HD are also involved in controlling axonal pathfinding (lhx6/8), axial patterning (islet1, lhx1/5-1, lmx1a/b-3), head/body proportions (islet2) and stem cell proliferation (lhx3/4, lhx2/9-3, lmx1a/b-2, lmx1a/b-3). Altogether, our results suggest that planarians might present a combinatorial LIM code that controls axial patterning and axonal growing and specifies distinct neuronal and intestinal cell identities.

LHX2 Is a Potential Biomarker and Associated with Immune Infiltration in Breast Cancer.

Worldwide, breast cancer is the most common malignancy. LHX2, a member of the LIM homeobox gene family and a transcription factor, plays a crucial role in numerous tumors, but the function of LHX2 in breast cancer progression remains unknown. In this study, we show that LHX2 is upregulated in breast cancer tissues and positively correlated with breast cancer progression. Meanwhile, the clinical characteristics of breast cancer and LHX2 expression showed a strong correlation. GSEA showed that a high LHX2 expression may activate the T-cell activation pathway, PI3K/AKT/mTOR signaling pathway, and apoptosis pathway. Moreover, ssGSEA showed that Th1 cells and Th2 cells had a positive correlation with LHX2 expression in breast cancer. Experiments showed that LHX2 promotes the proliferation, colony formation, migration, and invasion of breast cancer cells. Immunohistochemistry and immunofluorescence assays helped to analyze LHX2-associated immune infiltration in breast cancer. A Western blot assay proved that LHX2 activated the PI3K/AKT/mTOR pathway and the apoptosis pathway. A TUNEL assay confirmed that LHX2 inhibited apoptosis. Taken together, LHX2 plays a vital role in breast cancer's progression and prognosis and could be an immune infiltration biomarker for breast cancer, and LHX2 activates the PI3K/AKT/mTOR pathway and apoptosis pathway in breast cancer.

LHX2 in germ cells control tubular organization in the developing mouse testis

In the gonads of mammalian XY embryos, the organization of cords is the hallmark of testis development. This organization is thought to be controlled by interactions of the Sertoli cells, endothelial and interstitial cells with little or no role of germ cells. Challenging this notion, herein we show that the germ cells play an active role in the organization of the testicular tubules. We observed that the LIM-homeobox gene, Lhx2 is expressed in the germ cells of the developing testis between E12.5-E15.5. In Lhx2 knockout-fetal testis there was altered expression of several genes not just in germ cells but also in the supporting (Sertoli) cells, endothelial cells, and interstitial cells. Further, loss of Lhx2 led to disrupted endothelial cell migration and expansion of interstitial cells in the XY gonads. The cords in the developing testis of Lhx2 knockout embryos are disorganized with a disrupted basement membrane. Together, our results show an important role of Lhx2 in testicular development and imply the involvement of germ cells in the tubular organization of the differentiating testis. The preprint version of this manuscript is available at https://doi.org/10.1101/2022.12.29.522214

Identification of prognostic indicators, diagnostic markers, and possible therapeutic targets among LIM homeobox transcription factors in breast cancer.

Breast cancer (BRCA) is the most common malignant tumor among women worldwide. Despite advances in treatment, many patients still die from a lack of effective diagnostic and prognostic markers and powerful therapeutic targets. LIM homeobox genes (LHXs) play vital roles in regulating the development of various organisms. However, there are limited reports regarding their roles in the diagnosis, prognosis, and treatment of BRCA. UALCAN, Kaplan-Meier plotter, cBioPortal, GeneMANIA, STRING, DAVID 6.8, TRRUST v2, LinkedOmics, and TIMER were utilized to analyze differential expression, prognostic value, genetic alteration, neighbor gene network, transcription factor targets, kinase targets, and immune cell infiltration of LHXs in BRCA patients. LHX gene expression patterns are clear in BRCA and its different subtypes. Further analyses indicated that this altered expression is possibly affected by genetic and/or epigenetic changes. The prognostic and diagnostic values of certain LHXs are unique to different BRCA subtypes. LHXs are mainly involved in the regulation of differentiation and development, and their neighbor genes are primarily involved in cancer-related pathways. Moreover, most LHXs are closely correlated with immune cell infiltration. Furthermore, LHXs may exert their functions by regulating a series of transcription factor and kinasetargets. LHXs are unique diagnostic and prognostic markers and participate in cancer through different signaling pathways and/or regulatory mechanisms in BRCA. This study provides potential applications of LHXs for the diagnosis, prognosis, and treatment of BRCA and its different subtypes.

Lhx2 in germ cells suppresses endothelial cell migration in the developing ovary

LIM-homeobox genes play multiple roles in developmental processes, but their roles in gonad development are not completely understood. Herein, we report that Lhx2, Ils2, Lmx1a, and Lmx1b are expressed in a sexually dimorphic manner in mouse, rat, and human gonads during sex determination. Amongst these, Lhx2 has female biased expression in the developing gonads of species with environmental and genetic modes of sex determination. Single-cell RNAseq analysis revealed that Lhx2 is exclusively expressed in the germ cells of the developing mouse ovaries. To elucidate the roles of Lhx2 in the germ cells, we analyzed the phenotypes of Lhx2 knockout XX gonads. While the gonads developed appropriately in Lhx2 knockout mice and the somatic cells were correctly specified in the developing ovaries, transcriptome analysis revealed enrichment of genes in the angiogenesis pathway. There was an elevated expression of several pro-angiogenic factors in the Lhx2 knockout ovaries. The elevated expression of pro-angiogenic factors was associated with an increase in numbers of endothelial cells in the Lhx2−/− ovaries at E13.5. Gonad recombination assays revealed that the increased numbers of endothelial cells in the XX gonads in absence of Lhx2 was due to ectopic migration of endothelial cells in a cell non-autonomous manner. We also found that, there was increased expression of several endothelial cell-enriched male-biased genes in Lhx2 knockout ovaries. Also, in absence of Lhx2, the migrated endothelial cells formed an angiogenic network similar to that of the wild type testis, although the coelomic blood vessel did not form. Together, our results suggest that Lhx2 in the germ cells is required to suppress vascularization in the developing ovary. These results suggest a need to explore the roles of germ cells in the control of vascularization in developing gonads. Preprint version of the article is available on BioRxiv at https://doi.org/10.1101/2022.03.07.483280.

LIM Homeobox-2 Suppresses Hallmarks of Adult and Pediatric Liver Cancers by Inactivating MAPK/ERK and Wnt/Beta-Catenin Pathways

Introduction: Hepatocellular carcinoma and hepatoblastoma are two liver cancers characterized by gene deregulations, chromosomal rearrangements, and mutations in Wnt/beta-catenin (Wnt) pathway-related genes. LHX2, a transcriptional factor member of the LIM homeobox gene family, has important functions in embryogenesis and liver development. LHX2 is oncogenic in many solid tumors and leukemia, but its role in liver cancer is unknown. Methods: We analyzed the expression of LHX2 in hepatocellular carcinoma and hepatoblastoma samples using various transcriptomic datasets and biological samples. The role of LHX2 was studied using lentiviral transduction, in vitro cell-based assays (growth, migration, senescence, and apoptosis), molecular approaches (phosphokinase arrays and RNA-seq), bioinformatics, and two in vivo models in chicken and Xenopus embryos. Results: We found a strong connection between LHX2 downregulation and Wnt activation in these two liver cancers. In hepatoblastoma, LHX2 downregulation correlated with multiple poor outcome parameters including higher patient age, intermediate- and high-risk tumors, and low patient survival. Forced expression of LHX2 reduced the proliferation, migration, and survival of liver cancer cells in vitro through the inactivation of MAPK/ERK and Wnt signals. In vivo, LHX2 impeded the development of tumors in chick embryos and repressed the Wnt pathway in Xenopus embryos. RNA-sequencing data and bioinformatic analyses confirmed the deregulation of many biological functions and molecular processes associated with cell migration, cell survival, and liver carcinogenesis in LHX2-expressing hepatoma cells. At a mechanistic level, LHX2 mediated the disassembling of beta-catenin/T-cell factor 4 complex and induced expression of multiple inhibitors of Wnt (e.g., TLE/Groucho) and MAPK/ERK (e.g., DUSPs) pathways. Conclusion: Collectively, our findings demonstrate a tumor suppressive function of LHX2 in adult and pediatric liver cancers.

The combination of hair follicle-specific marker LHX2 and co-expressed marker can distinguish between sweat gland placodes and hair placodes in rat.

Eccrine sweat gland (ESG) and hair follicle (HF) are different skin appendages but share many common development characteristics. Although the morphology of adult ESG and HF is obviously different, it is difficult to distinguish ESG placodes from HFs placodes morphologically. To study the fate determination of ESG and HF, specific antigen markers for ESG placodes and HF placodes must be found first to distinguish them. In the study, we detected the expression of commonly used keratins 4, 5, 7-10, 12, 14, 15, 17-20, 27 and 73, and the reported ESG and HF specific markers, P-cadherin, Lymphoid enhancer factor 1 (LEF1), LIM Homeobox gene 2(LHX2), Na+/K+-ATPase (NKA) and Na+-K+-2Cl- cotransporter 1 (NKCC1) in ESG and HF placodes by single-immunofluorescence staining and double-immunofluorescence staining. To further verify the results of immunofluorescence staining, Western blot (WB) was used to detect the differential antigen and some co-expressed antigens of ESG and HF placodes. The results showed that both ESG and HF placodes expressed K4/5/14/1517/18, P-cadherin and LEF1, neither expressed K7/8/9/10/12/19/20/27/73, NKA or NKCC1. HF placodes specifically expressed LHX2. Combination of LHX2 and co-expressed antigen K14, can distinguish ESG placodes from HF placodes. We conclude that LHX2 is a specific marker for HF placodes, and ESG placodes and HF placodes can be distinguished by double immunofluorescence staining of the specific marker LHX2 and the co-expressed markers, such as K4, K5, K14, K15, K17, K18, P-cadherin and LEF1.

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.

CircFMN2 Sponges miR-1238 to Promote the Expression of LIM-Homeobox Gene 2 in Prostate Cancer Cells

Circular RNAs (circRNAs) regulate gene expression in different malignancies. However, the molecular mechanisms that link circRNAs with the tumorigenesis of prostate cancer (PCa) are not well understood. In the present study, we attempted to provide a novel basis for targeted therapy for PCa from the aspect of circRNA-microRNA (miRNA)-mRNA interaction. We investigated the expression of circRNAs in 5 paired PCa tissues and adjacent non-tumor tissues by microarray analysis. We focused on hsa_circ_0005100, which is located on chromosome 1 and derived from FMN2, and thus we named it circFMN2. The qRT-PCR was used to detect circFMN2 and target miRNA expression in PCa tissues and cell lines. Biological functional experiments were performed to detect the effects of circFMN2 on the biological behavior of PCa cells in vivo and in vitro. Bioinformatic analysis was utilized to predict potential miRNA target sites on circFMN2. High expression of circFMN2 was associated with PCa progression. Function assays revealed that knockdown of circFMN2 significantly reduced PCa cell growth in vitro and in vivo. Finally, we found that circFMN2 acts as a competing endogenous RNA (ceRNA) for miR-1238 to regulate LIM-homeobox gene 2 (LHX2) expression. circFMN2 regulates the miR-1238/LHX2 axis to promote PCa progression.

PDGFA in Cashmere Goat: A Motivation for the Hair Follicle Stem Cells to Activate.

Simple SummaryCashmere goats are the most important goat breed due to the high yield and fineness of the fibers that they produce. Cashmere fiber is a luxury product since it is soft, light and warm. The development of this fiber depends on the hair follicle (HF) cyclical activity, which is characterized by the succession of growth and regressive phases. In the transition between telogen and anagen phases, many growth factors work to activate the HF stem cells and to allow the growth of a new cashmere fiber. As several factors involved in the stem cell activation, Platelet-Derived Growth Factor A (PDGFA), Bone Morphogenetic Protein 2 (BMP2) and Lim-Homeobox gene 2 (LHX2) were analyzed in this work to evaluate their activity during the cashmere HF cycle. These molecules were studied using different approaches and finally, PDGFA and BMP2 appeared to have higher levels of expression during the cycle activation phase with respect to the LHX2, which suggests that they play a main role in the development of a new cashmere fiber. The obtained data will improve the knowledge of the HF cycle in the cashmere goat and they could be a useful tool for improving cashmere fiber production.The cashmere hair follicle (HF) perpetually goes through cycles of growth, involution and rest. The photoperiod is the main factor in the control of seasonal coat change in cashmere goats while stem cells play a crucial role in the HF growth. Several factors, including Platelet-Derived Growth Factor A (PDGFA), Bone Morphogenetic Protein 2 (BMP2) and Lim-Homeobox gene 2 (LHX2) are implicated in HF morphogenesis and cycle. In this work, the mentioned molecules were investigated to evaluate their role in follicular cycle activation. The study was performed on skin samples collected at different periods of HF cycle and the molecular expression of PDGFA, BMP2 and LHX2 was evaluated by Real-Time PCR (qPCR) at each time point. Since PDGFA showed the most variation, the goat PDGFA gene was sequenced and the protein localization was investigated by immunohistochemistry together with PDGF receptor α (PDGFRα). PDGFA immunostaining was observed in the basal layer of the HF outer root sheath and the immunoreaction appeared stronger in the regressive HFs compared to those in the anagen phase according to qPCR analysis. PDGFRα was observed in the HF epithelium, proving the effect of PDGFA on the follicular structure. The data obtained suggest that PDGFA and BMP2 are both implicated in HF cycle in goat. In particular, PDGFA secreted by the HF is involved in the anagen activation.

Smad ubiquitylation regulatory factor 1 promotes LIM-homeobox gene 9 degradation and represses testosterone production in Leydig cells.

Testosterone is essential for spermatogenesis and the maintenance of secondary sexual characteristics in males. An important transcription factor, LIM-homeobox gene 9 (Lhx9) is indispensable for testis development and testosterone production; however, post-translational modifications of Lhx9 are largely unknown. Here, for the first time to our knowledge, we demonstrate that the level of Lhx9 protein increases in human chorionic gonadotropin-exposed Leydig cells and can be polyubiquitylated. We found that Smad ubiquitylation regulatory factor 1 (Smurf1), an E3 ubiquitin ligase, targets Lhx9 for ubiquitin-mediated proteasome degradation, thereby negatively modulating its function. Increasing Smurf1 decreases the level of Lhx9 and inhibits the Lhx9 transactivation capacity of steroidogenic factor 1 [nuclear receptor subfamily 5, group A, member 1 (NR5A1)]. In contrast, the depletion of Smurf1 leads to increased expression of Lhx9 protein and enhances testosterone biosynthesis-related gene transcripts [NR5A1, steroidogenic acute regulatory protein, CYP17A1, hydroxy-δ-5-steroid dehydrogenase, hydroxy-δ-5-steroid dehydrogenase isomerase 6, and hydroxysteroid (17-β) dehydrogenase 3] and testosterone production in Leydig cells. Furthermore, we found that Smurf1 knockout mice exhibit higher levels of Lhx9 protein and steroidogenesis, which leads to increased serum testosterone concentration. These findings reveal that Smurf1 promotes Lhx9 ubiquitylation and is involved in testosterone production in Leydig cells directly. Our results provide new insights into the molecular events that play a role in the homeostasis of testosterone levels and may provide a new target for testosterone regulation.-Hu, F., Zhu, Q., Sun, B., Cui, C., Li, C., Zhang, L. Smad ubiquitylation regulatory factor 1 promotes LIM-homeobox gene 9 degradation and represses testosterone production in Leydig cells.

Sexually Dimorphic unc-6/Netrin Expression Controls Sex-Specific Maintenance of Synaptic Connectivity

Nervous systems display intriguing patterns of sexual dimorphisms across the animal kingdom, but the mechanisms that generate such dimorphisms remain poorly characterized. In the nematode Caenorhabditis elegans, a number of neurons present in both sexes are synaptically connected to one another in a sexually dimorphic manner as a result of sex-specific synaptic pruning and maintenance [1-3]. We define here a mechanism for the male-specific maintenance of the synaptic connections of the phasmid sensory neuron PHB and its male-specific target, the sex-shared AVG interneuron. We show that the C.elegans Netrin ortholog UNC-6, signaling through its cognate receptor UNC-40/DCC and the CED-5/DOCK180 guanine nucleotide exchange factor, is both required and sufficient for male-specific synaptic maintenance. The dimorphism of unc-6 activity is brought about by sex-specific regulation of unc-6 transcription. Although unc-6 is transcribed in the AVG neuron of males and hermaphrodites during juvenile stages, unc-6 expression is downregulated in AVG in hermaphrodites during sexual maturation but is maintained during sexual maturation of males. unc-6 downregulation in hermaphrodites is conferred by the master regulator of hermaphrodite sexual identity, the Gli/CI homolog TRA-1, which antagonizes the non-sex-specific function of the LIM homeobox gene lin-11, a terminal selector and activator of unc-6 in AVG. Preventing the downregulation of unc-6 in AVG of hermaphrodites through ectopic expression of unc-6 in transgenic animals results in the maintenance of the PHB>AVG synapses in hermaphrodites. Taken together, intersectional transcriptional regulationofunc-6/Netrin is required and sufficient to cellautonomously pattern sexually dimorphic synapses.

Regulation and/or Repression of Cholinergic Differentiation of Murine Embryonic Stem Cells Using RNAi Directed Against Transcription Factor L3/Lhx8.

Techniques for controlling the expression of a specific gene in embryonic stem cells are effective and important for clarifying the functions of the gene. Regarding differentiation of cells into nervous system components, these techniques would play key roles in elucidating, not only the differentiation mechanisms of neuronal and glial cells but also how neuronal phenotypes are determined. In this chapter, we describe a RNA interference method for suppressing cholinergic differentiation in murine embryonic stem cells by knockdown of expression of the transcription factor L3/Lhx8, a Lim homeobox gene family protein. This method will greatly facilitate functional analyses of the factors involved in neuronal differentiation and regeneration and will contribute to cell transplantation studies.

Diversity of human and mouse homeobox gene expression in development and adult tissues.

BackgroundHomeobox genes encode a diverse set of transcription factors implicated in a vast range of biological processes including, but not limited to, embryonic cell fate specification and patterning. Although numerous studies report expression of particular sets of homeobox genes, a systematic analysis of the tissue specificity of homeobox genes is lacking.ResultsHere we analyse publicly-available transcriptome data from human and mouse developmental stages, and adult human tissues, to identify groups of homeobox genes with similar expression patterns. We calculate expression profiles for 242 human and 278 mouse homeobox loci across a combination of 59 human and 12 mouse adult tissues, early and late developmental stages. This revealed 20 human homeobox genes with widespread expression, primarily from the TALE, CERS and ZF classes. Most homeobox genes, however, have greater tissue-specificity, allowing us to compile homeobox gene expression lists for neural tissues, immune tissues, reproductive and developmental samples, and for numerous organ systems. In mouse development, we propose four distinct phases of homeobox gene expression: oocyte to zygote; 2-cell; 4-cell to blastocyst; early to mid post-implantation. The final phase change is marked by expression of ANTP class genes. We also use these data to compare expression specificity between evolutionarily-based gene classes, revealing that ANTP, PRD, LIM and POU homeobox gene classes have highest tissue specificity while HNF, TALE, CUT and CERS are most widely expressed.ConclusionsThe homeobox genes comprise a large superclass and their expression patterns are correspondingly diverse, although in a broad sense related to an evolutionarily-based classification. The ubiquitous expression of some genes suggests roles in general cellular processes; in contrast, most human homeobox genes have greater tissue specificity and we compile useful homeobox datasets for particular tissues, organs and developmental stages. The identification of a set of eutherian-specific homeobox genes peaking from human 8-cell to morula stages suggests co-option of new genes to new developmental roles in evolution.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-016-0140-y) contains supplementary material, which is available to authorized users.

MiR-1238 inhibits cell proliferation by targeting LHX2 in non-small cell lung cancer.

In human cancers, dysregulated expression of LIM-homeobox gene 2 (LHX2) and downregulation of miR-1238 has been reported separately. However, the relationship between them remains unclear. We investigated the functional contribution of miR-1238 to the regulation of LHX2 in non-small cell lung cancer (NSCLC). Here, computational algorithms predicted that the 3'-untranslated region (3'-UTR) of LHX2 is a target of miR-1238. Luciferase assays validated that miR-1238 directly bound to 3'-UTR of LHX2. qRT-PCR and western blot analyses further confirmed that overexpression of miR-1238 mimic in NSCLC A549 and LTEP-α-2 cells inhibited endogenous expression of LHX2 mRNA and protein. Moreover, ectopic expression of miR-1238 in NSCLC A549 and LTEP-α-2 cells suppressed cellular viability and proliferation. siRNA-induced knockdown of LHX2 copied the phenotype of miR-1238 overexpression in NSCLC A549 and LTEP-α-2 cells and LHX2 knockdown inhibited cell cycle. In addition, miR-1238 expression was frequently decreased in human NSCLC tissues and reversely correlated with LHX2 expression, which was increased in NSCLC tissues. Collectively, our findings demonstrate that miR-1238 inhibit the proliferation of NSCLC cells at least partly via repression of LHX2, shedding light on the mechanistic interaction of miR-1238 and LHX2 in NSCLC carcinogenesis. Furthermore, our data suggest that expression of miR-1238 could be a promising therapeutic strategy for NSCLC treatment.

Apterous A modulates wing size, bristle formation and patterning in Nilaparvata lugens.

Apterous A (apA), a member of the LIM-homeobox gene family, plays a critical role in the development of wing. The achaete-scute Complex (AS-C) encodes basic helix-loop-helix (bHLH) transcription factors and functions in bristle development. In the present study, we cloned apA (NlapA) and an achaete-scute homologue (NlASH) from N. lugens. Levels of NlapA and NlASH were higher in nymphs than adults, with particularly high expression in the thorax of nymphs. NlapA expressed more highly in nymphs of the macropterous strain (MS) than those of the brachypterous strain (BS) at 2nd and 4th instar. Knockdown of NlapA and NlASH in vivo generated similar phenotypic defects in the wing (loss-of-bristles, twisted or erect wing). Silencing of NlapA in nymphs of MS led to decreased wing size in adults. Moreover, depletion of NlapA suppressed expression of NlDl, Nlsal, Nlser, Nlvg and Nlwg, both in MS and BS, but induced differential responses of Nlubx and Nlnotch expression between MS and BS. Notably, expression of NlASH was regulated by NlapA. These results collectively indicate that NlapA is an upstream modulator of wing size, bristle formation and patterning. Further studies on DNA-protein and protein-protein interactions are required to elucidate NlapA-mediated regulation of wing development.

Exploring genotype–phenotype relationships of the LHX3 gene on growth traits in beef cattle

The LIM-homeobox gene 3 (LHX3) plays an essential role in pituitary gland and nervous system development. Sequence variants (SVs) in coding and non-coding regions of LHX3 gene have an impact on LHX3 transcription and growth traits in cattle. Previously, we have identified 3 single nucleotide polymorphisms (SNPs: 1–3) in all exons and intron 2 regions of the LHX3 gene in cattle. Here, 7 novel SNPs (SNPs: 4-10) were identified by DNA sequencing and polymerase chain reaction single-stranded conformational polymorphism (PCR-SSCP) methods. In the present study, a total of 10 SNPs were assessed linkage disequilibrium (LD) in 802 cows representing four main cattle breeds from China (Nanyang, Qinchuan, Jiaxian, and Chinese Holstein). The assessment results demonstrated that 17 haplotypes and 18 diplotypes were revealed in these cattle populations. Moreover, association analysis indicated that the genotypes of SNPs 1–6 are associated with the body weight at 6, 12 and 18months of age in Nanyang cattle (P<0.01 or P<0.05), whereas no significant association was found between the 18 diplotypes and growth traits. Our results provide evidence that some SNPs in LHX3 gene may be associated with body weight at certain age, and LHX3 gene may be used as candidate gene for marker-assisted selection (MAS) in beef cattle breeding.

Overexpression of Lhx8 inhibits cell proliferation and induces cell cycle arrest in PC12 cell line.

LIM-homeobox genes play a pivotal function in tissue patterning and differentiation, Lhx8 is a member of LIM-homeobox gene family, and it is selectively expressed in embryonic basal forebrain and is a key factor for the determination of cholinergic cells fate. However, besides cholinergic differentiation, little is known about the potential role of Lhx8 in cell biology. In this study, we transfected Lhx8 complementary DNA (cDNA) into PC12 cell line using lentiviral vectors to acquire the cells which stably expressed high level of Lhx8, and we provide the experimental evidence that overexpression of Lhx8 inhibits cell proliferation and induces cell cycle arrest but not apoptosis in vitro. In conclusion, besides cholinergic differentiation, our results suggest that Lhx8 also plays as a suppressor gene of proliferation in cell biology.

Islet1 and its co-factor Ldb1 are expressed in quiescent cells of mouse intestinal epithelium.

Islet1 belongs to Lim homeobox (Lhx) gene family which encodes transcription factors that have been conserved in evolution. They form complexes with other transcriptional regulators, among them obligatory co-factors encoded by Ldb genes. Isl1 (Islet1), Lhx and Ldb1 genes play a crucial role in organ patterning, cell fate determination and cell differentiation in both embryonic and adult tissues. In this study we analyzed expression pattern of Isl1 and its co-factor Ldb1 in small intestine. We also studied the biological role of Ldb1 in gut endoderm. Quantitative PCR analysis revealed a relatively high level of expression of Lhx1, Isl1, Isl2, Lmx1a, Ldb1 and Ldb2 mRNAs in the gut tissue as compared to the level of less abundant detectable Lmx1b mRNA. Immunohistochemical studies demonstrated a unique pattern of Ldb1 and Islet1 proteins in the crypt compartment. Ldb1 is produced at a low level in majority of crypt cells; but, its abundant expression was demonstrated for some single cells. Islet1 is also expressed in single cells of the crypt. Double staining experiments with Ldb1 and Isl1 antibodies showed that both genes are co-expressed in certain cells of the crypt. Further analysis revealed the Ldb1-expressing cells in the gut are both of endodermal and mesodermal origin. Proliferation studies using antibodies to phospho-histone H3 and Ki-67 antigens, as well as long-term BrdU labeling, showed that cells prominently expressing Ldb1/Islet1 are quiescent but do not belong to any known terminally differentiated cell lineages. They may represent a group of stem-like cells in the crypt. Further experiments by cell lineage tracing should be performed to better characterize this cell population. Functional studies of mice with Ldb1 gene ablated in gut endoderm revealed no specific role of Ldb1 in that tissue.