Expression Of FHL2 Research Articles

FHL2 expression by cancer-associated fibroblasts promotes metastasis and angiogenesis in lung adenocarcinoma.

Cancer-associated fibroblasts (CAFs) contribute to the progression of lung cancer. Four and a half LIM domain protein-2 (FHL2) is a component of focal adhesion structures. We analyzed the function of FHL2 expressed by CAFs in lung adenocarcinoma. Expression of FHL2 in fibroblast subtypes was investigated using database of single-cell RNA-sequencing of lung cancer tissue. The role of FHL2 in the proliferation and migration of CAFs was assessed. The effects of FHL2 knockout on the migration and invasion of human lung adenocarcinoma cells and tube formation of endothelial cells induced by CAF-conditioned medium (CM) were evaluated. The effect of FHL2 knockout in CAFs on metastasis was determined using a murine orthotopic lung cancer model. The prognostic significance of stromal FHL2 was assessed by immunohistochemistry in human adenocarcinoma specimens. FHL2 is highly expressed in myofibroblasts in cancer tissue. TGF-β1 upregulated FHL2 expression in CAFs and FHL2 knockdown attenuated CAF proliferation. FHL2 knockout reduced CAF induced migration of A110L and H23 human lung adenocarcinoma cell lines, and the induction of tube formation of endothelial cells. FHL2 knockout reduced CAF-induced metastasis of lung adenocarcinomas in an orthotopic model in vivo. The concentration of Osteopontin (OPN) in CM from CAF was downregulated by FHL2 knockout. siRNA silencing and antibody blocking of OPN reduced the pro-migratory effect of CM from CAF on lung cancer cells. In resected lung adenocarcinoma specimens, positive stromal FHL2 expression was significantly associated with higher microvascular density and worse prognosis. In conclusion, FHL2 expression by CAFs enhances the progression of lung adenocarcinoma by promoting angiogenesis and metastasis.

Long non-coding RNA Small Nucleolar RNA Host Gene 4 ameliorates cigarette smoke-induced proliferation, apoptosis, inflammation, and airway remodeling in alveolar epithelial cells through the modulation of the mitogen-activated protein kinase signaling pathway via the microRNA-409-3p/Four and a Half LIM Domains 1 axis

The long non-coding RNA (lncRNA) Small Nucleolar RNA Host Gene 4 (SNHG4) has been demonstrated to be significantly downregulated in various inflammatory conditions, yet its role in chronic obstructive pulmonary disease (COPD) remains elusive. This study aims to elucidate the biological function of SNHG4 in COPD and to unveil its potential molecular targets. Our findings reveal that both SNHG4 and Four and a Half LIM Domains 1 (FHL1) were markedly downregulated in COPD, whereas microRNA-409-3p (miR-409-3p) was upregulated. Importantly, SNHG4 exhibited a negative correlation with inflammatory markers in patients with COPD, but a positive correlation with forced expiratory volume in 1s percentage (FEV1%). SNHG4 distinguished COPD patients from non-smokers with high sensitivity, specificity, and accuracy. Overexpression of SNHG4 ameliorated cigarette smoke extract (CSE)-mediated inflammation, apoptosis, oxidative stress, and airway remodeling in 16HBE bronchial epithelial cells. These beneficial effects of SNHG4 overexpression were reversed by the overexpression of miR-409-3p or the silencing of FHL1. Mechanistically, SNHG4 competitively bound to miR-409-3p, mediating the expression of FHL1, and consequently improving inflammation, apoptosis, oxidative stress, and airway remodeling in 16HBE cells. Additionally, SNHG4 regulated the miR-409-3p/FHL1 axis to inhibit the activation of the mitogen-activated protein kinase (MAPK) pathway induced by CSE. In a murine model of COPD, knockdown of SNHG4 exacerbated CSE-induced pulmonary inflammation, apoptosis, and oxidative stress. In summary, our data affirm that SNHG4 mitigates pulmonary inflammation, apoptosis, and oxidative damage mediated by COPD through the regulation of the miR-409-3p/FHL1 axis.Graphical

FHL2 is a novel target in pulmonary arterial hypertension

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): - Longfonds Nederland Background/ Objectives Pulmonary arterial hypertension (PAH) is a fatal disorder characterized by excessive proliferation of endothelial (EC) and smooth muscle cells, and impaired BMP signaling in small lung arteries. The LIM-only protein FHL2 is expressed in the vessel wall in ECs and smooth muscle cells and plays a crucial role in vessel wall homeostasis. However, the molecular mechanisms how FHL2 exerts its effects and its role in pulmonary artery EC function remain unclear. Here, we investigated the effect of FHL2 on EC proliferation and unraveled the underlying mechanisms involved. Methods/Results We found that FHL2 is highly expressed in lungs of PAH rats compared to control rats as evidenced by qPCR and western blot analysis. Human microvascular ECs (MVECs) derived from lungs of PAH patients displayed enhanced expression of FHL2. Ectopic expression of FHL2 in MVECs significantly increased mRNA levels of BMPR2 and Id1. To substantiate these results, luciferase assays were performed using BMP response element reporter construct and found that FHL2 increases Id1 expression. Furthermore, overexpression of FHL2 decreased TNF-α induced expression of pro-inflammatory cytokines. Finally, ectopic expression of FHL2 reduced proliferation of ECs. Conclusion Our data indicate that FHL2 is a novel negative regulator of MVEC proliferation through enhancing BMP signaling and inhibiting inflammation. Modulation of FHL2 may potentially represent a novel therapeutic strategy for the treatment of PAH.

FHL2 in arterial medial calcification in chronic kidney disease.

Arterial medial calcification (AMC) is a common complication in individuals with chronic kidney disease (CKD), which can lead to cardiovascular morbidity and mortality. The progression of AMC is controlled by a key transcription factor called runt-related transcription factor 2 (RUNX2), which induces vascular smooth muscle cells (VSMCs) transdifferentiation into a osteogenic phenotype. However, RUNX2 has not been targeted for therapy due to its essential role in bone development. The objective of our study was to discover a RUNX2 coactivator that is highly expressed in arterial VSMCs as a potential therapy for AMC. We employed transcriptomic analysis of human data and an animal reporter system to pinpoint FHL2 as a potential target. Subsequently, we investigated the mRNA and protein expression patterns of FHL2 in the aortas of both human and animal subjects with CKD. To examine the role of FHL2 in the RUNX2 transcription machinery, we conducted coimmunoprecipitation (Co-IP) and chromatin immunoprecipitation (ChIP) experiments. Next, we manipulated FHL2 expression in cultured VSMCs to examine its impact on high phosphate-induced transdifferentiation. Finally, we employed FHL2 null mice to confirm the role of FHL2 in the development of AMC in vivo. Among all the potential RUNX2 cofactor, FHL2 displays selective expression within the cardiovascular system. In the context of CKD subjects, FHL2 undergoes upregulation and translocation from the cytosol to the nucleus of arterial VSMCs. Once in the nucleus, FHL2 interacts structurally and functionally with RUNX2, acting as a coactivator of RUNX2. Notably, the inhibition of FHL2 expression averts transdifferentiation of VSMCs into an osteogenic phenotype and mitigates aortic calcification in uremic animals, without causing any detrimental effects on the skeletal system. These observations provide evidence that FHL2 is a promising target for treating arterial calcification in patients with CKD.

Four and a half LIM domains 2 (FHL2) attenuates tumorigenesis of gastrointestinal stromal tumors (GISTs) by negatively regulating KIT signaling.

Gastrointestinal stromal tumors (GISTs) are predominately induced by KIT mutants. In this study, we found that four and a half LIM domains 2 (FHL2) was highly expressed in GISTs and KIT signaling dramatically increased FHL2 transcription while FHL2 inhibited KIT transcription. In addition, our results showed that FHL2 associated with KIT and increased the ubiquitination of both wild-type KIT and primary KIT mutants in GISTs, leading to decreased expression and activation of KIT although primary KIT mutants were less inhibited by FHL2 than wild-type KIT. In the animal experiments, loss of FHL2 expression in mice carrying germline KIT/V558A mutation which can develop GISTs resulted in increased tumor growth, but increased sensitivity of GISTs to imatinib treatment which is used as the first-line targeted therapy of GISTs, suggesting that FHL2 plays a role in the response of GISTs to KIT inhibitor. Unlike wild-type KIT and primary KIT mutants, we further found that FHL2 didn't alter the expression and activation of drug-resistant secondary KIT mutants. Taken together, our results indicated that FHL2 acts as the negative feedback of KIT signaling in GISTs while primary KIT mutants are less sensitive and secondary KIT mutants are resistant to the inhibition of FHL2.

MiR-3682-3p promotes esophageal cancer progression by targeting FHL1 and activating the Wnt/β-catenin signaling pathway

BackgroundEsophageal cancer (EC) is highly ranked among all cancers in terms of its incidence and mortality rates. MicroRNAs (miRNAs) are considered to play key regulatory parts in EC. Multiple research studies have indicated the involvement of miR-3682-3p and four and a half LIM domain protein 1 (FHL1) in the achievement of tumors. The aim of this research was to clarify the significance of these genes and their possible molecular mechanism in EC. MethodsData from a database and the tissue microarray were made to analyze the expression and clinical significance of miR-3682-3p or FHL1 in EC. Reverse transcription quantitative PCR and Western blotting were used to detect the expression levels of miR-3682-3p and FHL1 in EC cells. CCK8, EdU, wound healing, Transwell, flow cytometry, and Western blotting assays were performed to ascertain the biological roles of miR-3682-3p and FHL1 in EC cells. To confirm the impact of miR-3682-3p in vivo, a subcutaneous tumor model was created in nude mice. The direct interaction between miR-3682-3p and FHL1 was demonstrated through a luciferase assay, and the western blotting technique was employed to assess the levels of crucial proteins within the Wnt/β-catenin pathway. ResultsThe noticeable increase in the expression of miR-3682-3p and the decrease in the expression of FHL1 were observed, which correlated with a negative impact on the patients' overall survival. Upregulation of miR-3682-3p expression promoted the growth and metastasis of EC, while overexpression of FHL1 partially reversed these effects. Finally, miR-3682-3p motivates the Wnt/β-catenin signal transduction by directly targeting FHL1. ConclusionMiR-3682-3p along the FHL1 axis activated the Wnt/β-catenin signaling pathway and thus promoted EC malignancy.

Hsa-miR-92b-3p Targeting FHL2 to Enhance Radiosensitivity of Nasopharyngeal Carcinoma.

Radiotherapy resistance is a major cause of treatment failure and leads to poor prognosis in nasopharyngeal carcinoma (NPC). Evidences indicate that microRNA (miRNAs) are closely associated with radiotherapy for NPC. In this study, we found that the expression level of miR-92b-3p was significantly higher in radiotherapy-sensitive NPC patients than in radiotherapy-resistant patients. High expression of miR-92b-3p was associated with good prognosis in patients with NPC, and high expression of FHL2 was associated with poor prognosis in patients with NPC. It was predicted that miR-92b-3p could directly target and bind FHL2. Overexpression of miR-92b-3p significantly inhibited FHL2 expression at the mRNA as well as protein levels, while inhibition of miR-92b-3p expression significantly upregulated FHL2 expression. Overexpression of miR-92b-3p significantly reduced proliferation and colony formation in NPC cells. Inhibition of miR-92b-3p attenuated the sensitivity of nasopharyngeal carcinoma to radiotherapy, while simultaneous inhibition of miR-92b-3p and FHL2 increased the sensitivity of NPC to radiotherapy. Our findings highlighted that miR-92b-3p is closely associated with radiotherapy sensitivity and prognosis in NPC patients and may improve the sensitivity of NPC to radiotherapy by targeting FHL2.

Tabersonine Enhances Olaparib Sensitivity through FHL1-Mediated Epithelial-Mesenchymal Transition in an Ovarian Tumor.

Ovarian cancer (OVC) is one of the most aggressive gynecological malignancies worldwide. Although olaparib treatment has shown favorable outcomes against the treatment of OVC, its effectiveness remains limited in some OVC patients. Investigating new strategies to improve the therapeutic efficacy of olaparib against OVC is imperative. Our study identified tabersonine, a natural indole alkaloid, for its potential to increase the chemosensitivity of olaparib in OVC. The combined treatment of olaparib and tabersonine synergistically inhibited cell proliferation in OVC cells and suppressed tumor growth in A2780 xenografts. The combined treatment effectively suppressed epithelial-mesenchymal transition (EMT) by altering the expression of E-cadherin, N-cadherin, and vimentin and induced DNA damage responses. Integrating quantitative proteomics, FHL1 was identified as a potential regulator to modulate EMT after tabersonine treatment. Increased expression of FHL1 was induced by tabersonine treatment, while downregulation of FHL1 reversed the inhibitory effects of tabersonine on OVC cells by mediating EMT. In vivo findings further reflected that the combined treatment of tabersonine and olaparib significantly inhibited tumor growth and OVC metastasis through upregulation of FHL1. Our findings reveal the role of tabersonine in improving the sensitivity of olaparib in OVC through FHL1-mediated EMT, suggesting that tabersonine holds promise for future application in OVC treatment.

Low FHL1 expression indicates a good prognosis and drug sensitivity in ovarian cancer.

Chemotherapy resistance is the main reason for the poor prognosis of ovarian cancer (OC). FHL1 is an important tumour regulator, but its relationship with the prognosis, drug resistance, and tumour microenvironment of OC is unknown. Immunohistochemistry was used to determine FHL1 expression in OC. Kaplan‒Meier plotter was used for survival analysis. The value of gene expression in predicting drug resistance was estimated using the area under the curve (AUC). Bivariate correlation was used to determine the coexpression of two genes. Functional cluster and pathway enrichment were used to uncover hidden signalling pathways. The relationship between gene levels and the tumour microenvironment was visualised through the ggstatsplot and pheatmap packages. The mRNA and protein levels of FHL1 were downregulated in 426 and 100 OC tissues, respectively. Low FHL1 expression was correlated with good progression-free survival (PFS), postprogression survival, and overall survival (OS) in 1815 OC patients,and was further confirmed to be associated with good OS by immunohistochemistry in 152 OC tissues. Furthermore, FHL1 was downregulated in drug-sensitive tissues, while its high expression predicted drug resistance (AUC > 0.65). Mechanistically, FHL1 was coexpressed with FLNC, CAV1, PPP1R12B, and FLNA at the mRNA and protein levels in 558 and 174 OC tissues, respectively, and their expression was downregulated in OC. Additionally, very strong coexpression of FHL1 with the four genes was identified in at least 23 different tumours. Low expression of the four genes was associated with good PFS, and the combination of FHL1 with the four genes provided better prognostic power. Meanwhile, the expression of all five genes was strongly and positively associated with the abundance of macrophages. Low FHL1 expression acts as a favourable factor in OC, probably via positive coexpression with FLNC, CAV1, PPP1R12B, and FLNA.

FHL2 Inhibits SARS-CoV-2 Replication by Enhancing IFN-β Expression through Regulating IRF-3.

SARS-CoV-2 triggered the global COVID-19 pandemic, posing a severe threat to public health worldwide. The innate immune response in cells infected by SARS-CoV-2 is primarily orchestrated by type I interferon (IFN), with IFN-β exhibiting a notable inhibitory impact on SARS-CoV-2 replication. FHL2, acting as a docking site, facilitates the assembly of multiprotein complexes and regulates the transcription of diverse genes. However, the association between SARS-CoV-2 and FHL2 remains unclear. In this study, we report for the first time that SARS-CoV-2 infection in Caco2 cells results in the upregulation of FHL2 expression, while the virus's N proteins can enhance FHL2 expression. Notably, the knockdown of FHL2 significantly amplifies SARS-CoV-2 replication in vitro. Conversely, the overexpression of FHL2 leads to a marked reduction in SARS-CoV-2 replication, with the antiviral property of FHL2 being independent of the cell or virus type. Subsequent experiments reveal that FHL2 supports IFN-β transcription by upregulating the expression and phosphorylation of IRF-3, thereby impeding SARS-CoV-2 replication in cells. These findings highlight FHL2 as a potential antiviral target for treating SARS-CoV-2 infections.

Expression and Predictive Significance of FHL1 and SLIT3 in Surgically Resected Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most common type of lung cancer. However, predictive biomarkers for early efficacy and prognosis evaluation in patients with surgically resected LUAD are not completely explained. Differentially expressed genes (DEGs), gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were identified by RNA sequencing (RNA-Seq) between thirteen LUAD tissues and five normal lung tissues. The expression of DEGs was confirmed by qRT-PCR and a validated cohort from GEPIA. Protein-protein interaction (PPI) network of the top 5% DEGs was constructed by STRING and visualized in Cytoscape. Immunofluorescence results were acquired from clinical specimens from LUAD patients. The expression of FHL1 was analyzed by ImageJ. Survival analysis was performed using the GEPIA dataset. Consistent with the RNA-Seq data, validation of DEGs expression by qRT-PCR and GEPIA cohort showed that FHL1 and SLIT3 were down-regulated in LUAD patient tissues compared with non-tumor tissues. Moreover, FHL1 was significantly reduced in LUAD cell lines compared to the bronchial epithelium cell line (P < 0.01). However, SLIT3 was elevated in A549 and H1299 cells (wide type EGFR) (P < 0.05) while decreased in HCC827 and PC9 cells (mutant EGFR) compared to BESA-2B cells (P < 0.01). PPI network revealed the most significant cluster with 10 nodes and 43 edges. Immunofluorescent staining also showed that the expression of FHL1 was lower in LUAD tissues compared with that in normal lung tissues (P < 0.01). The expressions of SLIT3 and FHL1 were positively correlated. Specifically, the higher expression level of SLIT3 and FHL1 independently predicted a better prognosis (P < 0.01 or P < 0.05). Our findings provide two novel candidates, FHL1 and SLIT3, for prognostic evaluation and treatments after surgery.

Exploring the Functional Basis of Epigenetic Aging in Relation to Body Fat Phenotypes in the Norfolk Island Cohort.

DNA methylation is an epigenetic factor that is modifiable and can change over a lifespan. While many studies have identified methylation sites (CpGs) related to aging, the relationship of these to gene function and age-related disease phenotypes remains unclear. This research explores this question by testing for the conjoint association of age-related CpGs with gene expression and the relation of these to body fat phenotypes. The study included blood-based gene transcripts and intragenic CpG methylation data from Illumina 450 K arrays in 74 healthy adults from the Norfolk Island population. First, a series of regression analyses were performed to detect associations between gene transcript level and intragenic CpGs and their conjoint relationship with age. Second, we explored how these age-related expression CpGs (eCpGs) correlated with obesity-related phenotypes, including body fat percentage, body mass index, and waist-to-hip ratio. We identified 35 age-related eCpGs associated with age. Of these, ten eCpGs were associated with at least one body fat phenotype. Collagen Type XI Alpha 2 Chain (COL11A2), Complement C1s (C1s), and four and a half LIM domains 2 (FHL2) genes were among the most significant genes with multiple eCpGs associated with both age and multiple body fat phenotypes. The COL11A2 gene contributes to the correct assembly of the extracellular matrix in maintaining the healthy structural arrangement of various components, with the C1s gene part of complement systems functioning in inflammation. Moreover, FHL2 expression was upregulated under hypermethylation in both blood and adipose tissue with aging. These results suggest new targets for future studies and require further validation to confirm the specific function of these genes on body fat regulation.

FHL2 regulates microglia M1/M2 polarization after spinal cord injury via PARP14-depended STAT1/6 pathway

Neuronal apoptosis and inflammation exacerbate the secondary injury after spinal cord injury (SCI). Four and a half domains 2 (FHL2) is a multifunctional scaffold protein with tissue- and cell-type specific effects on the regulation of inflammation, but its role in SCI remains unclear. The T10 mouse spinal cord contusion model was established, and the mice were immediately injected with lentiviruses carrying FHL2 shRNA after SCI. The results showed that FHL2 expression was increased following SCI, and then gradually decreased. Moreover, FHL2 depletion aggravated functional impairment, neuronal necrosis, and enlarged lesion cavity areas in the injured spinal cord. FHL2 deficiency facilitated neuronal apoptosis by elevating cleaved caspase 3/9 expression, neuroinflammation by regulating microglia polarization, and bone loss. Indeed, FHL2 deficiency increased the secretion of TNF-α and IL-6, M1 microglia polarization, and the activation of STAT1 pathway but decreased the secretion of IL-10 and IL-4, M2 microglia polarization, and the activation of the STAT6 pathway in the spinal cord. In vitro, FHL2 silencing promoted LPS + IFN-γ-induced microglia M1 polarization through activating the STAT1 pathway and alleviated IL-4-induced microglia M2 polarization via inhibiting the STAT6 pathway. FHL2 positively regulated the expression of poly (ADP-ribose) polymerase family member 14 (PARP14) by promoting its transcription. PARP14 overexpression inhibited FHL2 silencing-induced microglia M1 polarization and relieved the inhibitory effect of FHL2 silencing on microglia M2 polarization. Collectively, the study suggests that FHL2 reduces the microglia M1/M2 polarization-mediated inflammation via PARP14-dependent STAT1/6 pathway and thereby improves functional recovery after SCI.

Downregulation of FHL2 suppressed trophoblast migration, invasion and epithelial–mesenchymal transition in recurrent miscarriage

Research questionIs four and a half LIM domain 2 (FHL2) involved in trophoblast migration, invasion and epithelial–mesenchymal transition (EMT) in recurrent miscarriage? DesignVillus tissue was collected from 24 patients who had experienced recurrent miscarriage and 24 healthy controls. FHL2 mRNA and protein expression in villus specimens were observed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. Small interfering RNA and overexpression plasmid were used to change the FHL2 expression. JAR and HTR8/SVneo cell lines were used to conduct scratch-wound assay and transwell assay to detect trophoblast migration and invasion of FHL2. Downstream molecule expression of mRNA and protein and EMT markers were verified by qRT-PCR and Western blot. ResultsSignificantly lower FHL2 mRNA (P = 0.019) and protein (P = 0.0014) expression was found in trophoblasts from the recurrent miscarriage group compared with healthy controls. FHL2 knockdown repressed migration (P = 0.0046), invasion (P < 0.001) and EMT, as shown by significant differences in mRNA and protein expression of the EMT markers N-cadherin, E-cadherin, Vimentin and Snail (all P < 0.05) of extravillus trophoblasts. FHL2 overexpression enhanced migration (P = 0.025), invasion (P < 0.001) and EMT of extravillus trophoblasts (all EMT markers P < 0.05). The positive upstream factor FHL2 in the extracellular signal-related kinase pathway induced JunD expression, thereby promoting trophoblast migration and invasion via matrix metalloproteinase 2. ConclusionsFHL2 is involved in a regulatory pathway of trophoblast migration, invasion and EMT during early pregnancy, and may have a role in recurrent miscarriage pathogenesis, which can serve as a possible target for novel therapeutic development.

236-LB: FHL2-/- Perivascular Adipose Tissue Improves Vessel Tone and Insulin-Stimulated Vasodilation by Inhibiting ERK1/2

Background: Type 2 diabetes, obesity and aging increase risk of developing cardiovascular diseases. Perivascular adipose tissue (PVAT) is a crucial regulator of vascular function. Four and Half Lim domains 2 (FHL2) is a protein upregulated in aging which targets various proteins with established links to adipose tissue growth, potentially regulating metabolic signaling. Objective: we hypothesized that FHL2 regulates adipose tissue regulation of vascular function. Methods: Femoral arteries were isolated from Wild type (WT) and FHL2-/- mice and vasoreactivity to insulin and acetylcholine was studied with and without perivascular adipose tissue. Results: FHL2-/- vessels with PVAT had increased tone compared to WT vessels with PVAT as well as improved insulin-stimulated vasodilation. Exchange experiments using vessels with PVAT from the opposite genotype showed that FHL2-PVAT enhanced tone and insulin-stimulated vasodilatation compared to WT-PVAT. Gene expression analysis showed downregulation of adiponectin in FHL2-PVAT. Western blot analysis of insulin-stimulated phosphorylation of ERK and Akt in human microvascular endothelial cells (HMECs) stimulated with PVAT-conditioned medium revealed that FHL2-PVAT but not WT-PVAT reduced insulin-stimulated phosphorylation of ERK but not activation of Akt. In line with the effect of FHL2 on insulin-induced vasodilatation and activation of ERK1/2, analysis of publicly available RNA sequencing data of peri-aortic PVAT showed that FHL2 expression in PVAT positively correlates with expression of the endothelial vasoconstrictor peptide endothelin-1 (edn1). Conclusion: FHL2 regulates vascular function through enhancement of smooth muscle tone, regulation of adipokine secretion affecting insulin-stimulated ERK1/2 activation and Edn1 expression in endothelial cells. These finding provide a new mechanism for vascular dysfunction in type 2 diabetes. Disclosure J. Habibe: None. D. van Raalte: Consultant; Boehringer Ingelheim and Eli Lilly Alliance, AstraZeneca, Merck &amp; Co., Inc. Research Support; Boehringer Ingelheim and Eli Lilly Alliance, AstraZeneca, Merck &amp; Co., Inc. M. Nieuwdorp: Advisory Panel; caelus health. C. de Vries: None. E. C. Eringa: None. Funding Amsterdam Cardiovascular Sciences (ACS201703)

Comprehensive pan-cancer analysis identifies FHL2 associated with poor prognosis in lung adenocarcinoma.

The FHL family (four-and-a-half-LIM-only protein family) contains five multifunctional proteins (FHL1-5) that are involved in cell survival, transcriptional regulation, and signal transduction. Among these proteins, FHL2 is one of the most reported members in tumors, which is differentially expressed in numerous tumors. However, no systematic pan-cancer analysis of FHL2 has been performed so far. We obtained The Cancer Genome Atlas (TCGA) expression profiles and clinical data from Xena database and the Tumor Immune Estimation Resource (TIMER) database. Gene expression, prognosis, mRNA modification, and immune infiltration of FHL2 in pan-cancer were analyzed. Functional analysis validated the potential mechanism of FHL2 in lung adenocarcinoma (LUAD). FHL2 is differentially expressed in a wide range of tumors and has prognostic value. Digging into the immune landscape of FHL2, we found that FHL2 is significantly associated with tumor-associated fibroblasts. Furthermore, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) suggested that FHL2 may be involved in epithelial-mesenchymal transition (EMT)-associated pathways such as NF-KB and TGF-β in LUAD. Our comprehensive bioinformatics analysis identified mRNA level expression of FHL2 correlates with prognosis in different cancers. This study may help to more fully explore the role of FHL2 in tumor progression and metastasis.

Transcriptome analysis of the gene expression of M. iliotibialis lateralis affected by dietary methionine restriction.

Introduction: Methionine (Met) is an important amino acid related to the development of skeletal muscle. This study investigated the effects of dietary Met restriction on the gene expression of M. iliotibialis lateralis. Methods: A total of 84 day-old broiler chicks (Zhuanghe Dagu) with a similar initial body weight (207.62 ± 8.54 g) were used in this study. All birds were divided into two groups (CON; L-Met) based on the initial body weight. Each group consisted of six replicates with seven birds per replicate. The experimental period was 63 days (phase 1, days 1-21; phase 2, days 22-63). According to the nutritional requirements of Zhuanghe Dagu chickens, we provided a basal diet (0.39% Met levels during phase 1 and 0.35% Met levels during phase 2, as-fed basis) to the birds in the CON group, while we provided a Met-restricted diet (0.31% Met levels during phase 1 and 0.28% Met levels during phase 2, as-fed basis) to the birds in the L-Met group. The growth performance of broiler chicks and their M. iliotibialis lateralis development parameters were measured on days 21 and 63. Results and Discussion: In this study, dietary Met restriction did not affect the growth performance of broiler chicks but hindered the development of M. iliotibialis lateralis at both sampling timepoints. On the final day, three birds selected from each group (three from CON and three from L-Met) were used to obtain M. iliotibialis lateralis samples from leg muscle for further transcriptome analysis. Transcriptome analysis revealed that dietary Met restriction significantly upregulated 247 differentially expressed genes (DEGs) and downregulated 173 DEGs. Additionally, DEGs were mainly enriched in 10 pathways. Among DEGs, we observed that dietary Met restriction downregulated the expression of CSRP3, KY, FHL1, LMCD1, and MYOZ2 in M. iliotibialis lateralis. Therefore, we considered that dietary Met restriction had negative effects on the development of M. iliotibialis lateralis, and CSRP3, KY, FHL1, LMCD1, and MYOZ2 may serve as potential functional genes involved in this process.

FHL2 deficiency impairs follicular development and fertility by attenuating EGF/EGFR/YAP signaling in ovarian granulosa cells

Female subfertility is an increasing reproductive issue worldwide, which is partially related to abnormal ovarian follicular development. Granulosa cells (GCs), by providing the necessary physical support and microenvironment for follicular development, play critical roles in maintaining female fertility. We previously showed that ectopic expression of four and a half LIM domains 2 (FHL2) promoted ovarian granulosa cell tumor progression. However, its function in follicular development and fertility remains unknown. Here, we confirmed that FHL2 is highly expressed in human and mouse ovaries. FHL2 immunosignals were predominantly expressed in ovarian GCs. A Fhl2 knockout (KO) mouse model was generated to examine its roles in follicular development and fertility. Compared with wildtype, knockout of Fhl2 significantly decreased female litter size and offspring number. Furthermore, Fhl2 deficiency reduced ovarian size and impaired follicular development. RNA-sequencing analysis of GCs isolated from either KO or WT mice revealed that, Fhl2 deletion impaired multiple biological functions and signaling pathways, such as Ovarian Putative Early Atresia Granulosa Cell, ErbB, Hippo/YAP, etc. In vitro studies confirmed that FHL2 silencing suppressed GCs growth and EGF-induced GCs proliferation, while its overexpression promoted GC proliferation and decreased apoptosis. Mechanistic studies indicated that FHL2, via forming complexes with transcriptional factors AP-1 or NF-κB, regulated Egf and Egfr expression, respectively. Besides, FHL2 depletion decreased YAP1 expression, especially the active form of YAP1 (nuclear YAP1) in GCs of growing follicles. EGF, serving as an autocrine/paracrine factor, not only induced FHL2 expression and nuclear accumulation, but also stimulated YAP1 expression and activation. Collectively, our study suggests that FHL2 interacts with EGFR and Hippo/YAP signaling to regulate follicular development and maintain fertility. This study illuminates a novel mechanism for follicular development and a potential therapeutic target to address subfertility.

HBXIP knockdown inhibits FHL2 to promote cycle arrest and suppress cervical cancer cell proliferation, invasion and migration.

Hepatitis B X-interacting protein (HBXIP) and four and a half LIM domain 2 (FHL2) have been reported to serve as independent biomarkers for cervical cancer. The present study evaluated the effects of HBXIP on cervical cancer in terms of its cellular malignant characteristics. Reverse transcription-quantitative PCR and western blotting were used to assess the mRNA and protein expression levels of HBXIP and FHL2 in the human endocervical epithelial End1/E6E7 cell line and the cervical cancer HeLa, CaSki, C33A and SiHa cell lines. After knocking down HBXIP expression by transfection of small interfering RNAs targeting HBXIP, cell cycle progression was assessed using flow cytometry with PI staining. Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine staining, wound healing and Transwell assays were used to assess cell proliferation, migration and invasion, respectively. Furthermore, co-immunoprecipitation assay was used to evaluate the potential binding relationship between HBXIP and FHL2. Western blotting was used for the analysis of HBXIP and FHL2, cell cycle-associated proteins, including cyclin D1 and cyclin D2, metastasis-associated proteins, including MMP2 and MMP9, and Wnt/β-catenin signaling-associated proteins, including β-catenin and c-Myc. Both HBXIP and FHL2 were found to be highly expressed in cervical cancer cells compared with that in the human endocervical epithelial cell line. HBXIP knockdown suppressed the proliferation, invasion and migration of HeLa cells, but promoted cell cycle arrest at the G0/G1 phase. HBXIP was demonstrated to interact with FHL2, and HBXIP knockdown also inhibited FHL2 mRNA and protein expression. By contrast, FHL2 overexpression reversed the inhibitory effects of HBXIP knockdown on the malignant characteristics of cervical cancer cells. Furthermore, HBXIP knockdown blocked the Wnt/β-catenin signaling pathway in HeLa cells, which was also partially reversed by FHL2 overexpression; the decreased β-catenin and c-Myc expression caused by HBXIP knockdown was increased again after FHL2 was overexpressed. In conclusion, these results suggest that HBXIP knockdown suppressed the malignant characteristics of cervical cancer cells through the downregulation of FHL2 expression, indicating a promising insight into the therapeutic target of cervical cancer.

Preferential production and secretion of the complement regulator factor H-like protein 1 (FHL-1) by human myeloid cells

Factor H is a pivotal complement regulatory protein that is preferentially produced by the liver and circulates in high concentrations in serum. There has been an increasing interest in the extrahepatic production of complement factors, including by cells of the immune system, since this contributes to non-canonical functions of local complement activation and regulation. Here we investigated the production and regulation of factor H and its splice variant factor H-like protein 1 (FHL-1) by human myeloid cells. As validation, we confirmed the predominant presence of intact factor H in serum, despite a strong but comparable mRNA expression of CFH and FHL1 in liver. Comparable levels of CFH and FHL1 were also observed in renal tissue, although a dominant staining for FHL-1 was shown within the proximal tubules. Human in vitro generated pro- and anti-inflammatory macrophages both expressed and produced factor H/FHL-1, but this was strongest in pro-inflammatory macrophages. Production was not affected by LPS activation, but was increased upon stimulation with IFN-γ or CD40L. Importantly, in both macrophage subsets mRNA expression of FHL1 was significantly higher than CFH. Moreover, production of FHL-1 protein could be confirmed using precipitation and immunoblotting of culture supernatants. These data identify macrophages as producers of factor H and FHL-1, thereby potentially contributing to local complement regulation at sites of inflammation.