Analysis of the Drosophila Ajuba LIM protein defines functions for distinct LIM domains.

The Ajuba LIM protein Jub mediates regulation of Hippo signaling by cytoskeletal tension through interaction with the kinase Warts and participates in feedback regulation of junctional tension through regulation of the cytohesin Steppke. To investigate how Jub interacts with and regulates its distinct partners, we investigated the ability of Jub proteins missing different combinations of its three LIM domains to rescue jub phenotypes and to interact with α-catenin, Warts and Steppke. Multiple regions of Jub contribute to its ability to bind α-catenin and to localize to adherens junctions in Drosophila wing imaginal discs. Co-immunoprecipitation experiments in cultured cells identified a specific requirement for LIM2 for binding to Warts. However, in vivo, both LIM1 and LIM2, but not LIM3, were required for regulation of wing growth, Yorkie activity, and Warts localization. Conversely, LIM2 and LIM3, but not LIM1, were required for regulation of cell shape and Steppke localization in vivo, and for maximal Steppke binding in co-immunoprecipitation experiments. These observations identify distinct functions for the different LIM domains of Jub.

CSRP3, p.Arg122*, is responsible for hypertrophic cardiomyopathy in a Chinese family.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is a hereditary disease manifested by a thickened ventricular wall. Cysteine and glycine-rich protein 3 (CSRP3), the gene encoding muscle LIM protein, is important for initiating hypertrophic gene expression. The mutation of CSRP3 causes dilated cardiomyopathy or HCM. METHODS: In the present study, we enrolled a Chinese family with HCM across three generations. Whole-exome sequencing (WES) was performed in the proband to detect the candidate genes of the family. Sanger sequencing was performed for mutational analysis and confirmation of cosegregation. RESULTS: Through histopathological and imaging examinations, an obvious left ventricular hypertrophy was found in the proband. After WES data filtering, bioinformatic prediction and co-segregation analysis, a nonsense mutation (NM_003476.5:c.364C>T; NP_003467.1:p.Arg122*) of CSRP3 was identified in this family. This variant was predicted to be disease-causing and resulted in a truncated protein. CONCLUSIONS: This is the first HCM family case of CSRP3 (p.Arg122*) variation in Asia. The finding here not only contributes to the genetic diagnosis and counseling of the family, but also provides a new case with detailed phenotypes that may be caused by the CSRP3 variant.

MicroRNA-139-5p Alleviates High Glucose-Triggered Human Retinal Pigment Epithelial Cell Injury by Targeting LIM-Only Factor 4.

Diabetic retinopathy (DR) is a type of diabetes complication, which can result in loss of vision in adults worldwide. Increasing evidence has revealed that microRNAs (miRs) can regulate DR progression. Thus, the present study was aimed at assessing the possible mechanism of miR-139-5p in high glucose- (HG-) incubated retinal pigment epithelial (ARPE-19) cells. The present results demonstrated that miR-139-5p expression was notably reduced in the serum samples of patients with DR, as well as in ARPE-19 cells treated with HG in a time-dependent manner. Moreover, miR-139-5p was markedly overexpressed by transfection of miR-139-5p mimics into ARPE-19 cells. Overexpression of miR-139-5p markedly induced cell viability and repressed HG-triggered apoptosis. Furthermore, overexpression of miR-139-5p relived HG-enhanced oxidative stress injury. It was found that HG induced malondialdehyde levels but decreased superoxide dismutase and glutathione peroxidase activities in ARPE-19 cells. In addition, overexpression of miR-139-5p could markedly decrease intracellular stress. The results demonstrated that overexpression of miR-139-5p effectively repressed HG-activated inflammation, as indicated by the upregulation of inflammation cytokines, including TNF-α, IL-6, and Cox-2, in ARPE-19 cells. Subsequently, it was identified that LIM-only factor 4 (LMO4) could act as a downstream target for miR-139-5p. LMO4 expression was significantly increased in patients with DR and HG-treated ARPE-19 cells. Mechanistically, knockdown of LMO4 reversed the biological role of miR-139-5p in proliferation, apoptosis, oxidative stress, and release of inflammation factors in vitro. Collectively, these results suggested that miR-139-5p significantly decreased ARPE-19 cell injury caused by HG by inducing proliferation and suppressing cell apoptosis, oxidant stress, and inflammation by modulating LMO4.

A 10-gene prognostic signature points to LIMCH1 and HLA-DQB1 as important players in aggressive cervical cancer disease.

BACKGROUND: Advanced cervical cancer carries a particularly poor prognosis, and few treatment options exist. Identification of effective molecular markers is vital to improve the individualisation of treatment. We investigated transcriptional data from cervical carcinomas related to patient survival and recurrence to identify potential molecular drivers for aggressive disease. METHODS: Primary tumour RNA-sequencing profiles from 20 patients with recurrence and 53 patients with cured disease were compared. Protein levels and prognostic impact for selected markers were identified by immunohistochemistry in a population-based patient cohort. RESULTS: Comparison of tumours relative to recurrence status revealed 121 differentially expressed genes. From this gene set, a 10-gene signature with high prognostic significance (p = 0.001) was identified and validated in an independent patient cohort (p = 0.004). Protein levels of two signature genes, HLA-DQB1 (n = 389) and LIMCH1 (LIM and calponin homology domain 1) (n = 410), were independent predictors of survival (hazard ratio 2.50, p = 0.007 for HLA-DQB1 and 3.19, p = 0.007 for LIMCH1) when adjusting for established prognostic markers. HLA-DQB1 protein expression associated with programmed death ligand 1 positivity (p < 0.001). In gene set enrichment analyses, HLA-DQB1high tumours associated with immune activation and response to interferon-γ (IFN-γ). CONCLUSIONS: This study revealed a 10-gene signature with high prognostic power in cervical cancer. HLA-DQB1 and LIMCH1 are potential biomarkers guiding cervical cancer treatment.

T-ALL can evolve to oncogene independence.

The majority of cases of T-cell acute lymphoblastic leukemia (T-ALL) contain chromosomal abnormalities that drive overexpression of oncogenic transcription factors. However, whether these initiating oncogenes are required for leukemia maintenance is poorly understood. To address this, we developed a tetracycline-regulated mouse model of T-ALL driven by the oncogenic transcription factor Lmo2. This revealed that whilst thymus-resident pre-Leukemic Stem Cells (pre-LSCs) required continuous Lmo2 expression, the majority of leukemias relapsed despite Lmo2 withdrawal. Relapse was associated with a mature phenotype and frequent mutation or loss of tumor suppressor genes including Ikzf1 (Ikaros), with targeted deletion Ikzf1 being sufficient to transform Lmo2-dependent leukemias to Lmo2-independence. Moreover, we found that the related transcription factor TAL1 was dispensable in several human T-ALL cell lines that contain SIL-TAL1 chromosomal deletions driving its overexpression, indicating that evolution to oncogene independence can also occur in human T-ALL. Together these results indicate an evolution of oncogene addiction in murine and human T-ALL and show that loss of Ikaros is a mechanism that can promote self-renewal of T-ALL lymphoblasts in the absence of an initiating oncogenic transcription factor.

FHL1 promotes glioblastoma aggressiveness through regulating EGFR expression.

The four-and-a-half LIM domain protein 1 (FHL1) plays a key role in multiple cancers. Here, we characterized its role in glioblastoma (GBM), the most common and incurable form of brain cancer. Overexpression of FHL1 promotes growth, migration, and invasion of GBM cells in vivo and in vitro. In contrast, FHL1 silencing by RNAi exhibits the opposite effects. FHL1 interacts with the transcription factor SP1 to upregulate epidermal growth factor receptor (EGFR) expression and activate the downstream signaling cascades, including Src, Akt, Erk1/2, and Stat3, leading to GBM malignancy. FHL1 is highly expressed and positively correlated with EGFR levels in human GBM, particularly those of the classical subtype. Our results suggest that the FHL1-SP1-EGFR axis plays a tumor-promoting role, and highlight the translational potential of inhibiting FHL1 for GBM treatment.

LIM and SH3 protein 1 localizes to the leading edge of protruding lamellipodia and regulates axon development.

The actin cytoskeleton drives cell motility and is essential for neuronal development and function. LIM and SH3 protein 1 (LASP1) is a unique actin-binding protein that is expressed in a wide range of cells including neurons, but its roles in cellular motility and neuronal development are not well understood. We report that LASP1 is expressed in rat hippocampus early in development, and this expression is maintained through adulthood. High-resolution imaging reveals that LASP1 is selectively concentrated at the leading edge of lamellipodia in migrating cells and axonal growth cones. This local enrichment of LASP1 is dynamically associated with the protrusive activity of lamellipodia, depends on the barbed ends of actin filaments, and requires both the LIM domain and the nebulin repeats of LASP1. Knockdown of LASP1 in cultured rat hippocampal neurons results in a substantial reduction in axonal outgrowth and arborization. Finally, loss of the Drosophila homologue Lasp from a subset of commissural neurons in the developing ventral nerve cord produces defasciculated axon bundles that do not reach their targets. Together, our data support a novel role for LASP1 in actin-based lamellipodial protrusion and establish LASP1 as a positive regulator of both in vitro and in vivo axon development.

Evolutionarily diverse LIM domain-containing proteins bind stressed actin filaments through a conserved mechanism.

The actin cytoskeleton assembles into diverse load-bearing networks, including stress fibers (SFs), muscle sarcomeres, and the cytokinetic ring to both generate and sense mechanical forces. The LIM (Lin11, Isl- 1, and Mec-3) domain family is functionally diverse, but most members can associate with the actin cytoskeleton with apparent force sensitivity. Zyxin rapidly localizes via its LIM domains to failing SFs in cells, known as strain sites, to initiate SF repair and maintain mechanical homeostasis. The mechanism by which these LIM domains associate with stress fiber strain sites (SFSS) is not known. Additionally, it is unknown how widespread strain sensing is within the LIM protein family. We identify that the LIM domain-containing region of 18 proteins from the Zyxin, Paxillin, Tes, and Enigma proteins accumulate to SFSS. Moreover, the LIM domain region from the fission yeast protein paxillin like 1 (Pxl1) also localizes to SFSS in mammalian cells, suggesting that the strain sensing mechanism is ancient and highly conserved. We then used sequence and domain analysis to demonstrate that tandem LIM domains contribute additively, for SFSS localization. Employing in vitro reconstitution, we show that the LIM domain-containing region from mammalian zyxin and fission yeast Pxl1 binds to mechanically stressed F-actin networks but does not associate with relaxed actin filaments. We propose that tandem LIM domains recognize an F-actin conformation that is rare in the relaxed state but is enriched in the presence of mechanical stress.

Paxillin family of focal adhesion adaptor proteins and regulation of cancer cell invasion.

The paxillin family of proteins, including paxillin, Hic-5, and leupaxin, are focal adhesion adaptor/scaffolding proteins which localize to cell-matrix adhesions and are important in cell adhesion and migration of both normal and cancer cells. Historically, the role of these proteins in regulating the actin cytoskeleton through focal adhesion-mediated signaling has been well documented. However, studies in recent years have revealed additional functions in modulating the microtubule and intermediate filament cytoskeletons to affect diverse processes including cell polarization, vesicle trafficking and mechanosignaling. Expression of paxillin family proteins in stromal cells is also important in regulating tumor cell migration and invasion through non-cell autonomous effects on the extracellular matrix. Both paxillin and Hic-5 can also influence gene expression through a variety of mechanisms, while their own expression is frequently dysregulated in various cancers. Accordingly, these proteins may serve as valuable targets for novel diagnostic and treatment approaches in cancer.

Melatonin alters neuronal architecture and increases cysteine-rich protein 1 signaling in the male mouse hippocampus.

Neuronal plasticity describes changes in structure, function, and connections of neurons. The hippocampus, in particular, has been shown to exhibit considerable plasticity regarding both physiological and morphological functions. Melatonin, a hormone released by the pineal gland, promotes cell survival and dendrite maturation of neurons in the newborn brain and protects against neurological disorders. In this study, we investigated the effect of exogenous melatonin on neuronal architecture and its possible mechanism in the hippocampus of adult male C57BL/6 mice. Melatonin treatment significantly increased the total length and complexity of dendrites in the apical and basal cornu ammonis (CA) 1 and in the dentate gyrus in mouse hippocampi. Spine density in CA1 apical dendrites was increased, but no significant differences in other subregions were observed. In primary cultured hippocampal neurons, the length and arborization of neurites were significantly augmented by melatonin treatment. Additionally, western blot and immunohistochemical analyses in both in vivo and in vitro systems revealed significant increases in the level of cysteine-rich protein 1 (crp-1) protein, which is known to be involved in dendritic branching in mouse hippocampal neurons after melatonin treatment. Our results suggest that exogenous melatonin leads to significant alterations of neuronal micromorphometry in the adult hippocampus, possibly via crp-1 signaling.

LIM-Nebulette Reinforces Podocyte Structural Integrity by Linking Actin and Vimentin Filaments.

BACKGROUND: Maintenance of the intricate interdigitating morphology of podocytes is crucial for glomerular filtration. One of the key aspects of specialized podocyte morphology is the segregation and organization of distinct cytoskeletal filaments into different subcellular components, for which the exact mechanisms remain poorly understood. METHODS: Cells from rats, mice, and humans were used to describe the cytoskeletal configuration underlying podocyte structure. Screening the time-dependent proteomic changes in the rat puromycin aminonucleoside-induced nephropathy model correlated the actin-binding protein LIM-nebulette strongly with glomerular function. Single-cell RNA sequencing and immunogold labeling were used to determine Nebl expression specificity in podocytes. Automated high-content imaging, super-resolution microscopy, atomic force microscopy (AFM), live-cell imaging of calcium, and measurement of motility and adhesion dynamics characterized the physiologic role of LIM-nebulette in podocytes. RESULTS: Nebl knockout mice have increased susceptibility to adriamycin-induced nephropathy and display morphologic, cytoskeletal, and focal adhesion abnormalities with altered calcium dynamics, motility, and Rho GTPase activity. LIM-nebulette expression is decreased in diabetic nephropathy and FSGS patients at both the transcript and protein level. In mice, rats, and humans, LIM-nebulette expression is localized to primary, secondary, and tertiary processes of podocytes, where it colocalizes with focal adhesions as well as with vimentin fibers. LIM-nebulette shRNA knockdown in immortalized human podocytes leads to dysregulation of vimentin filament organization and reduced cellular elasticity as measured by AFM indentation. CONCLUSIONS: LIM-nebulette is a multifunctional cytoskeletal protein that is critical in the maintenance of podocyte structural integrity through active reorganization of focal adhesions, the actin cytoskeleton, and intermediate filaments.

Ldb1 is required for Lmo2 oncogene-induced thymocyte self-renewal and T-cell acute lymphoblastic leukemia.

Prolonged or enhanced expression of the proto-oncogene Lmo2 is associated with a severe form of T-cell acute lymphoblastic leukemia (T-ALL), designated early T-cell precursor ALL, which is characterized by the aberrant self-renewal and subsequent oncogenic transformation of immature thymocytes. It has been suggested that Lmo2 exerts these effects by functioning as component of a multi-subunit transcription complex that includes the ubiquitous adapter Ldb1 along with b-HLH and/or GATA family transcription factors; however, direct experimental evidence for this mechanism is lacking. In this study, we investigated the importance of Ldb1 for Lmo2-induced T-ALL by conditional deletion of Ldb1 in thymocytes in an Lmo2 transgenic mouse model of T-ALL. Our results identify a critical requirement for Ldb1 in Lmo2-induced thymocyte self-renewal and thymocyte radiation resistance and for the transition of preleukemic thymocytes to overt T-ALL. Moreover, Ldb1 was also required for acquisition of the aberrant preleukemic ETP gene expression signature in immature Lmo2 transgenic thymocytes. Co-binding of Ldb1 and Lmo2 was detected at the promoters of key upregulated T-ALL driver genes (Hhex, Lyl1, and Nfe2) in preleukemic Lmo2 transgenic thymocytes, and binding of both Ldb1 and Lmo2 at these sites was reduced following Cre-mediated deletion of Ldb1. Together, these results identify a key role for Ldb1, a nonproto-oncogene, in T-ALL and support a model in which Lmo2-induced T-ALL results from failure to downregulate Ldb1/Lmo2-nucleated transcription complexes which normally function to enforce self-renewal in bone marrow hematopoietic progenitors.

Prickle isoforms determine handedness of helical morphogenesis.

Subcellular asymmetry directed by the planar cell polarity (PCP) signaling pathway orients numerous morphogenetic events in both invertebrates and vertebrates. Here, we describe a morphogenetic movement in which the intertwined socket and shaft cells of the Drosophila anterior wing margin mechanosensory bristles undergo PCP-directed apical rotation, inducing twisting that results in a helical structure of defined chirality. We show that the Frizzled/Vang PCP signaling module coordinates polarity among and between bristles and surrounding cells to direct this rotation. Furthermore, we show that dynamic interplay between two isoforms of the Prickle protein determines right- or left-handed bristle morphogenesis. We provide evidence that, Frizzled/Vang signaling couples to the Fat/Dachsous PCP directional signal in opposite directions depending on whether Pkpk or Pksple predominates. Dynamic interplay between Pk isoforms is likely to be an important determinant of PCP outcomes in diverse contexts. Similar mechanisms may orient other lateralizing morphogenetic processes.

Our right and left hands are mirror images of each other and cannot be precisely superimposed. This property, known as chirality, is vital for many tissues and organs to form correctly in humans and other animals. For example, fruit flies have hair-like sensory organs on the edges of their wings known as bristles. One of the cells in each bristle forms a shaft that generally tilts away from the main body of the fly and is anchored in place by another cell known as the socket.A signaling pathway known as PCP signaling controls the directions in which many chiral tissues and organs in animals form. The pathway contains two signaling modules: the global module collects "directional" information about the orientation of the body and sends it to the core module, which interprets this information to control how the tissue or organ grows.Fruit flies have two different versions of one of the core module components ­ known as Prickle and Spiny legs ­ that are thought to alter the direction the core module responds to the information it receives. Mutant flies known as pk^pk mutants are unable to make Prickle and their wing bristles tilt in the opposite direction compared to those in normal flies, but it was not clear exactly why this happens.To address this question, Cho et al. studied PCP signaling in the wings of normal and pk^pk mutant flies. The experiments showed that Prickle directed the bristles on the right wing of a normal fly to grow in left-handed corkscrew-like patterns in which the emerging shaft and socket of each bristle twisted around each other. As a result, the bristles tilted away from the bodies of the flies. In the pk^pk mutants, however, Spiny legs substituted for Prickle, causing the equivalent bristles to grow in a right-handed corkscrew pattern and tilt towards the body.The findings of Cho et al. show that PCP signaling controls the direction fly bristles grow by selectively using Prickle and Spiny legs. In the future, this work may also aid efforts to develop effective screening and treatments for birth defects that result from the failure of chiral tissues and organs to form properly.

The Nebulin Family LIM and SH3 Proteins Regulate Postsynaptic Development and Function.

Neuronal dendrites have specialized actin-rich structures called dendritic spines that receive and integrate most excitatory synaptic inputs. The stabilization of dendrites and spines during neuronal maturation is essential for proper neural circuit formation. Changes in dendritic morphology and stability are largely mediated by regulation of the actin cytoskeleton; however, the underlying mechanisms remain to be fully elucidated. Here, we present evidence that the nebulin family members LASP1 and LASP2 play an important role in the postsynaptic development of rat hippocampal neurons from both sexes. We find that both LASP1 and LASP2 are enriched in dendritic spines, and their knockdown impairs spine development and synapse formation. Furthermore, LASP2 exerts a distinct role in dendritic arbor and dendritic spine stabilization. Importantly, the actin-binding N-terminal LIM domain and nebulin repeats of LASP2 are required for spine stability and dendritic arbor complexity. These findings identify LASP1 and LASP2 as novel regulators of neuronal circuitry.SIGNIFICANCE STATEMENT Proper regulation of the actin cytoskeleton is essential for the structural stability of dendrites and dendritic spines. Consequently, the malformation of dendritic structures accompanies numerous neurologic disorders, such as schizophrenia and autism. Nebulin family members are best known for their role in regulating the stabilization and function of actin thin filaments in muscle. The two smallest family members, LASP1 and LASP2, are more structurally diverse and are expressed in a broader array of tissues. While both LASP1 and LASP2 are highly expressed in the brain, little is currently known about their function in the nervous system. In this study, we demonstrate the first evidence that LASP1 and LASP2 are involved in the formation and long-term maintenance of dendrites and dendritic spines.

LIM and cysteine-rich domains 1 (LMCD1) regulates skeletal muscle hypertrophy, calcium handling, and force.

BACKGROUND: Skeletal muscle mass and strength are crucial determinants of health. Muscle mass loss is associated with weakness, fatigue, and insulin resistance. In fact, it is predicted that controlling muscle atrophy can reduce morbidity and mortality associated with diseases such as cancer cachexia and sarcopenia. METHODS: We analyzed gene expression data from muscle of mice or human patients with diverse muscle pathologies and identified LMCD1 as a gene strongly associated with skeletal muscle function. We transiently expressed or silenced LMCD1 in mouse gastrocnemius muscle or in mouse primary muscle cells and determined muscle/cell size, targeted gene expression, kinase activity with kinase arrays, protein immunoblotting, and protein synthesis levels. To evaluate force, calcium handling, and fatigue, we transduced the flexor digitorum brevis muscle with a LMCD1-expressing adenovirus and measured specific force and sarcoplasmic reticulum Ca2+ release in individual fibers. Finally, to explore the relationship between LMCD1 and calcineurin, we ectopically expressed Lmcd1 in the gastrocnemius muscle and treated those mice with cyclosporine A (calcineurin inhibitor). In addition, we used a luciferase reporter construct containing the myoregulin gene promoter to confirm the role of a LMCD1-calcineurin-myoregulin axis in skeletal muscle mass control and calcium handling. RESULTS: Here, we identify LIM and cysteine-rich domains 1 (LMCD1) as a positive regulator of muscle mass, that increases muscle protein synthesis and fiber size. LMCD1 expression in vivo was sufficient to increase specific force with lower requirement for calcium handling and to reduce muscle fatigue. Conversely, silencing LMCD1 expression impairs calcium handling and force, and induces muscle fatigue without overt atrophy. The actions of LMCD1 were dependent on calcineurin, as its inhibition using cyclosporine A reverted the observed hypertrophic phenotype. Finally, we determined that LMCD1 represses the expression of myoregulin, a known negative regulator of muscle performance. Interestingly, we observed that skeletal muscle LMCD1 expression is reduced in patients with skeletal muscle disease. CONCLUSIONS: Our gain- and loss-of-function studies show that LMCD1 controls protein synthesis, muscle fiber size, specific force, Ca2+ handling, and fatigue resistance. This work uncovers a novel role for LMCD1 in the regulation of skeletal muscle mass and function with potential therapeutic implications.

Overexpression Of hsa-miR-664a-3p Is Associated With Cigarette Smoke-Induced Chronic Obstructive Pulmonary Disease Via Targeting FHL1.

Background: Chronic obstructive pulmonary disease (COPD) is recognized as a chronic lung disease with incomplete reversible airflow limitation, but its pathophysiology was still not clear. This study aimed at investigating regulatory roles of special miRNA-mRNA axis in COPD development. Methods: Differentially expressed miRNAs and downstream mRNAs were screened from the Gene Expression Omnibus (GEO) dataset by using the LIMMA package in R software. Weighted Gene Co-expression Network Analysis (WGCNA) was used to construct a co-expression network for COPD. The correlation of dysregulated miRNA(s) and COPD was analyzed, and miRNAs with significant differences were validated in peripheral blood mononuclear cells (PBMCs) from COPD patients by real-time PCR. Regulatory roles of candidate miRNAs and targeted mRNAs were investigated in vitro study. Results: Thirteen modules of co-expressed miRNAs and mRNAs were constructed from a selected cohort with WGCNA. Turquoise module with 12 differentially expressed miRNAs and 120 mRNAs was significantly correlated with COPD. The expression of hsa-miR-664a-3p, an upregulated miRNA in the module, was increased both in lung tissue and PBMCs from COPD patients, whereas that targeted four and a half LIM domains 1 (FHL1) gene was decreased and positively correlated with forced expiratory volume in 1 sec (FEV1)/forced vital capacity (FVC%) (r = 0.59, p < 0.01). In vitro, luciferase activity assay revealed FHL1 as a target of hsa-miR-664a-3p and it could be directly downregulated by overexpression of hsa-miR-664a-3p. Furthermore, cigarette smoke extract could increase hsa-miR-664a-3p level and decrease FHL1 level in Beas-2B cells. Conclusion: The present study validated significant upregulation of hsa-miR-664a-3p in COPD patients, and its target gene FHL1 was downregulated and positively correlated with FEV1/FVC%; both hsa-miR-664a-3p and FHL1 could be regulated by cigarette smoke extract. Results of bioinformatic analyses and expanded validation suggest that the axis from hsa-miR-664a-3p to FHL1 might play a key role in cigarette smoke-induced COPD, and the exact mechanism should be confirmed in further studies.

PTHR1 May Be Involved in Progression of Osteosarcoma by Regulating miR-124-3p-AR-Tgfb1i1, miR-27a-3p-PPARG-Abca1, and miR-103/590-3p-AXIN2 Axes.

Our previous study has indicated that the parathyroid hormone type 1 receptor (PTHR1) may play important roles in development and progression of osteosarcoma (OS) by regulating Wnt, angiogenesis, and inflammation pathway genes. The goal of this study was to further illuminate the roles of PTHR1 in OS by investigating upstream regulation mechanisms (including microRNA [miRNA] and transcription factors [TFs]) of crucial genes. The microarray dataset GSE46861 was downloaded from the Gene Expression Omnibus database, in which six tumors with short hairpin RNA (shRNA) PTHR1 knockdown (PTHR1.358) and six tumors with shRNA control knockdown (Ren.1309) were collected from mice. Differentially expressed genes (DEGs) between PTHR1.358 and Ren.1309 were identified using the linear models for microarray data (LIMMA) method, and then the miRNA-TF-mRNA regulatory network was constructed using data from corresponding databases, followed by module analysis, to screen crucial regulatory relationships. OS-related human miRNAs were extracted from the curated Osteosarcoma Database. Gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were enriched using the Database for Annotation, Visualization, and Integrated Discovery (DAVID) tool. As a result, the miRNA-TF-mRNA regulatory network, including 1049 nodes (516 miRNA, 25 TFs, and 508 DEGs) and 15942 edges (interaction relationships, such as Pparg-Abca1 and miR-590-3p-AXIN2), was constructed, from which three significant modules were extracted and modules 2 and 3 contained interactions between miRNAs/TFs and DEGs such as miR-103-3p-AXIN2, miR-124-3p-AR-Tgfb1i1, and miR-27a-3p-PPARG-Abca1. miR-27a-3p was a known miRNA associated with OS. Abca1, AR, and miR-124-3p were hub genes in the miRNA-TF-mRNA network. Tgfb1i1 was involved in cell proliferation, Abca1 participated in the cholesterol metabolic process, and AXIN2 was associated with the canonical Wnt signaling pathway. Furthermore, we also confirmed upregulation of miR-590-3p and downregulation of AXIN2 in the mouse OS cell line K7M2-WT transfected with PTHR1 shRNA. In conclusion, PTHR1 may play important roles in progression of OS by activating miR-124-3p-AR-Tgfb1i1, miR-27a-3p-PPARG-Abca1, and miR-103/590-3p-AXIN2 axes.

HCV and flaviviruses hijack cellular mechanisms for nuclear STAT2 degradation: Up-regulation of PDLIM2 suppresses the innate immune response.

Host encounters with viruses lead to an innate immune response that must be rapid and broadly targeted but also tightly regulated to avoid the detrimental effects of unregulated interferon expression. Viral stimulation of host negative regulatory mechanisms is an alternate method of suppressing the host innate immune response. We examined three key mediators of the innate immune response: NF-KB, STAT1 and STAT2 during HCV infection in order to investigate the paradoxical induction of an innate immune response by HCV despite a multitude of mechanisms combating the host response. During infection, we find that all three are repressed only in HCV infected cells but not in uninfected bystander cells, both in vivo in chimeric mouse livers and in cultured Huh7.5 cells after IFNα treatment. We show here that HCV and Flaviviruses suppress the innate immune response by upregulation of PDLIM2, independent of the host interferon response. We show PDLIM2 is an E3 ubiquitin ligase that also acts to stimulate nuclear degradation of STAT2. Interferon dependent relocalization of STAT1/2 to the nucleus leads to PDLIM2 ubiquitination of STAT2 but not STAT1 and the proteasome-dependent degradation of STAT2, predominantly within the nucleus. CRISPR/Cas9 knockout of PDLIM2 results in increased levels of STAT2 following IFNα treatment, retention of STAT2 within the nucleus of HCV infected cells after IFNα stimulation, increased interferon response, and increased resistance to infection by several flaviviruses, indicating that PDLIM2 is a global regulator of the interferon response.

[Effect of silencing LASP1 on biological behaviors of oral squamous cell carcinoma cells and IC50 of three anti-tumor drugs].

PURPOSE: This study was designed to investigate the effects of LASP1 on proliferation, metastasis, invasion, and cycle of oral squamous cell carcinoma cells and analyze the changes of IC50 in three antitumor drugs: cisplatin, apatinib and docetaxel. METHODS: The correlation between LASP1 and survival rate and prognosis of patients with head and neck cancer were analyzed on the human protein atlas data. RT-PCR and Western blot were used to detect mRNA and protein expression of LASP1 in oral squamous cell carcinoma cell lines. LASP1 silenced HN30 stable transfectant cell line was constructed by lentivirus. CCK-8 assay was used to detect cell proliferation. Plate colony assay was used to detect cell clone formation ability. Transwell assay was used to detect cell migration and invasion ability. Flow cytometry was used to detect cell cycle changes. Oral squamous cell carcinoma metastases were established in nude mouse, the number of metastatic lung nodules was counted and stained with H-E. CCK-8 method was used to analyze the changes of IC50 in three antitumor drugs: cisplatin, apatinib and docetaxel. Statistical analysis was performed using SPSS 11.0 software package. RESULTS: LASP1 was closely related to the survival rate and prognosis of head and neck cancer. LASP1 promoted proliferation, colony formation, metastasis and invasion of oral squamous cell carcinoma cell line HN30, promoted G2/M phase transition of cell cycle, and significantly reduced the formation of lung metastasis in nude mice after silencing. There was significant correlation with docetaxel IC50 but no significant impact on cisplatin IC50 and aptatinib IC50. CONCLUSIONS: LASP1 enhances cell proliferation, plate cloning, metastasis and invasion, G2/M phase transition of cell cycle, promotes lung metastasis in nude mice and docetaxel resistance of oral squamous cell carcinoma cell line HN30.

LMO7 deficiency reveals the significance of the cuticular plate for hearing function.

Sensory hair cells, the mechanoreceptors of the auditory and vestibular systems, harbor two specialized elaborations of the apical surface, the hair bundle and the cuticular plate. In contrast to the extensively studied mechanosensory hair bundle, the cuticular plate is not as well understood. It is believed to provide a rigid foundation for stereocilia motion, but specifics about its function, especially the significance of its integrity for long-term maintenance of hair cell mechanotransduction, are not known. We discovered that a hair cell protein called LIM only protein 7 (LMO7) is specifically localized in the cuticular plate and the cell junction. Lmo7 KO mice suffer multiple cuticular plate deficiencies, including reduced filamentous actin density and abnormal stereociliar rootlets. In addition to the cuticular plate defects, older Lmo7 KO mice develop abnormalities in inner hair cell stereocilia. Together, these defects affect cochlear tuning and sensitivity and give rise to late-onset progressive hearing loss.