FOXC1 Expression Research Articles

FOXC2 is a prognostic biomarker and contributes to the growth and invasion of human hepatocellular carcinoma

BackgroundForkhead box C2 (FOXC2) is a crucial factor involving in various cancers. However, its functions in hepatocellular carcinoma (HCC) is unknown. Here, we explored the role of FOXC2 in the progression of HCC and its potential mechanisms.MethodsFOXC2 expression in HCC tissue and cells were detected by immunohistochemistry or western blot and real-time PCR. CCK8, wound healing and transwell assay were used to measure cell growth and invasion. Tumor formation experiment was carried out to assess the tumorigenicity of HCC cells. Regulation of FOXC2 on Ang-2 was validated by luciferase assay and complementary experiments.ResultsIncreased FOXC2 expression was found to be associated positively with more aggressive clinicopathologic features. HCC patients with higher FOXC2 expression had significantly shorter overall survival. FOXC2 expression was indentified as an independent risk factor for resectable HCC. Increased FOXC2 expression accelerated the migration and invasion of HCC cells, accompanied by enhanced Ang-2 expression. Likewise, FOXC2 knockdown yielded opposite results. Moreover, FOXC2 stimulated the activation of the Ang-2 promoter. Suppression of Ang-2 expression hindered the FOXC2-mediated EMT processs, cell migration and invasion of HCC.ConclusionsFOXC2 is a novel prognostic predictor for HCC and may facilitate the growth and invasion through Ang-2.

Expression analysis of FOXC1 &FOXCUT genes in patients with gastric cancer

BackgroundThe Forkhead box (FOX) gene family encodes a group of regulatory proteins which plays an important role in cancer development. The FOXC1 gene is from the Forkhead family, upstream of the lncRNA FOXCUT gene can play a regulatory role. The present study was performed to analyze the expression of FOXC1 and FOXCUT genes in gastric cancer (GC) patients. Materials and MethodsThe study was conducted on 39 patients. Total RNA was extracted from tumoral and adjacent tissue samples and was used for expression analysis via two step reverse transcriptase quantitative real-time PCR method. ResultsThe relative expression of FOXCUT and FOXC1 genes was significantly higher in tumor tissue in comparison with its adjacent tissue (p = 0.0059, p = 0.0162 respectively). We also evaluated the correlation between gene expression levels and patients' clinical manifestations which illustrated that stage of disease and patients' gender are significantly correlated with FOXC1 and FOXCUT genes expression, respectively. ConclusionsThe evidences of present study suggest that up-regulation of FOXC1 and FOXCUT genes might be a biomarker for GC diagnosis.

CD97 Is Decreased in Preeclamptic Placentas and Promotes Human Trophoblast Invasion Through PI3K/Akt/mTOR Signaling Pathway

Preeclampsia (PE) is a pregnancy disorder leading to the morbidity and mortality. Despite the development of the understanding of etiology, the only effective treatment of PE is the delivery of the placenta. An improved mastery on the regulation of trophoblast invasion could be meaningful to alleviate the disease burden of PE. Relative expression of CD97 in PE and normal placental tissues was evaluated by quantitative real-time polymerase chain reaction, immunohistology, and Western blot. The CD97 siRNA and expression vector was transfected to cultured human trophoblast HTR-8/SVneo, and the cell invasion as well as the protein expression in PI3K/Akt/mTOR signaling pathway were evaluated. Expression of CD97 is significantly downregulated in PE placental tissues compared to normal controls. The Si-CD97 inhibits HTR-8/SVneo trophoblast cells invasion, as well as the activation of PI3K/Akt/mTOR signaling pathway. In accordance, overexpression of CD97 promotes trophoblast cell invasion. In addition, CD97 regulates FOXC2 expression and showed similar effects on PI3K/Akt/mTOR signaling pathway as specific FOXC2 inhibitor. In short, this study demonstrated the downregulation of CD97 expression in preeclamptic placentas. Further mechanism investigation revealed that CD97 promoted trophoblast invasion by targeting FOXC2 via PI3K/Akt/mTOR signaling pathway, laying the foundation for the development of PE intervention strategy by targeting CD97 in placentation and pathogenesis of PE.

Overexpression of Foxc1 regenerates crushed rat facial nerves by promoting Schwann cells migration via the Wnt/β-catenin signaling pathway.

Facial paralysis can result in severe implications for patients. A good prognosis depends on the degree of nerve regeneration. Schwann cells (SCs) play an important role in facial nerve development and regeneration through migration. Forkhead box C1 (Foxc1), a member of the forkhead transcription factor family, is implicated in cell migration. However, the role of Foxc1 in the progression after facial nerve crush remains unknown. Our aim was to evaluate the effect of Foxc1 overexpression on SC migration and recovery of facial nerves after crush injury. The rat facial nerve crush injury model was established through the use of unilateral surgery. The results showed that the expression of Foxc1 was increased in the surgery group compared to that of the control group. SCs were isolated from the sciatic nerves and cultured. Foxc1, delivered by an adeno‐associated virus in vivo, or adenovirus in vitro, both induced overexpression of Foxc1, and increased the expression of CXCL12 and β‐catenin. After the transfection of Foxc1, the migration of SC was increased both in vitro and in vivo, was reduced by the inhibition of CXCL12 or β‐catenin. The facial nerve function and the nerve axon remyelination of the rats transfected with Foxc1 were significantly improved after nerve crush injury. Overall, the results demonstrated that overexpression of Foxc1 promoted SC migration by regulating CXCL12 via the Wnt/β‐catenin pathway, thus contributing to improved facial nerve function after crush injury.

The Transcription Factor Foxc1 Promotes Osteogenesis by Directly Regulating Runx2 in Response of Intermittent Parathyroid Hormone (1-34) Treatment.

Parathyroid hormone (PTH) is crucial for bone remodeling. Intermittent PTH (1–34) administration stimulates osteogenesis and promotes bone formation; however, the possible targets and underlying mechanisms still remain unclear. In this study, functional links between PTH and Foxc1, a transcription factor reported to be predominant in skeletal development and formation, were indicated. We determined the impacts of Foxc1 on in vitro osteogenic differentiation and in vivo bone regeneration under intermittent PTH induction, and further explored its possible targets. We found that the expression level of Foxc1 was upregulated during osteogenic induction by intermittent PTH treatment, and the elevated expression of Foxc1 induced by PTH was inhibited by PTH1R silencing, while rescued by intermittent PTH supplement. By gain- and loss-of-function strategies targeting Foxc1 in MC3T3-E1 cells, we demonstrated that Foxc1 could promote in vitro osteogenic differentiation by intermittent PTH induction. Moreover, immunofluorescence analysis indicated the nuclear co-localization of Foxc1 with Runx2. Luciferase-reporter and chromatin immunoprecipitation analysis further confirmed that Foxc1 could bind to the P1 promoter region of Runx2 directly, which plays an indispensable part in osteogenic differentiation and bone mineralization. Meanwhile, we also revealed that Foxc1 could promote bone regeneration induced by intermittent PTH treatment in vivo. Taken together, this study revealed the role and mechanism of Foxc1 on in vitro osteogenic differentiation and in vivo bone regeneration in response of intermittent PTH treatment.

Conditional inactivation of Foxc1 and Foxc2 in neural crest cells leads to cardiac abnormalities

Cardiac neural crest cells (cNCCs) are required for normal heart development. cNCCs are a multipotent and migratory cell lineage that differentiates into multiple cell types. cNCCs migrate into the developing heart to contribute to the septation of the cardiac outflow tract (OFT). Foxc1 and Foxc2 are closely related members of the FOX (Forkhead box) transcription factor family and are expressed in cNCC during heart development. However, the precise role of Foxc1 and Foxc2 in cNCCs has yet to be fully described. We found that compound NCC-specific Foxc1;Foxc2 mutant embryos exhibited persistent truncus arteriosus (PTA), ventricular septal defects (VSDs), and thinning of the ventricular myocardium. Loss of Foxc1/c2 expression in cNCCs resulted in abnormal patterns of cNCC migration into the OFT without the formation of the aorticopulmonary septum. Further, loss of Foxc1 expression in cNCCs resulted in normal OFT development but abnormal ventricular septal formation. In contrast, loss of Foxc2 expression in NCCs led to no obvious cardiac abnormalities. Together, we provide evidence that Foxc1 and Foxc2 in cNCCs are cooperatively required for proper cNCC migration, the formation of the OFT septation, and the development of the ventricles. Our data also suggests that Foxc1 expression may play a larger role in ventricular development compared to Foxc2.

Timed Induction of Gli1 (P3 To P30) Was Expressed In Mouse Trabecular Meshwork

PurposeThe objective of this project is to determine if Gli1 positive cells contribute to the anterior eye structures including the trabecular meshwork and Schlemm’s canal using an inducible Gli1‐CreERT2; tdTomatoflox (Gli1‐tdTomato) mouse model. Gli1 is downstream of the Sonic hedgehog (Shh) signaling pathway. This pathway is mediated by the Gli transcription factors (Gli1, Gli2, and Gli3) that deferentially activate and repress the expression of specific eye development genes. In addition, Pitx2, FOXC1, and FOXC2 are critical transcription factors for anterior eye development. A second objective is to establish if FOXC1, FOXC2, and Pitx2 are co‐expressed in the Gli1 positive cells.MethodsGli1‐tdTomato expressing mice were induced at different stages with tamoxifen. Eyes were examined that were induced on various embryonic and post‐natal days, P3, P7, and P30 and sacrificed several days to weeks later. Mouse tissues were fixed and prepared for frozen or paraffin sectioning. Tissue antigen retrieval was done using citric acid buffer. Indirect immunohistochemistry for FOXC1, FOXC2, and Pitx2 was completed. Some samples were counterstained with phalloidin. Sections were analyzed with confocal microscopy for spatial distribution of the Gli1+ labeled cells and FOXC1, FOXC2, and Pitx2.ResultsIn Gli1‐tdTomato mice induced on P3, P7, and P30, the periocular mesoderm, trabecular meshwork including Schlemm’s canal, eyelids and optic nerve were positive for Gli1 several days to 3 weeks later. In contrast, the cornea, lens and sclera did not have positive cells for Gli1. Expression levels for FOXC1, FOXC2, and Pitx2 were generally higher in mice harvested at younger ages. After postnatal day 30, the expression decreased with little to no expression of FOXC1 and FOXC2 while Pitx2 expression remained relatively constant in anterior angle tissues.ConclusionThis inducible Gli1‐tdTomato mouse model may be an excellent system for determining the role of Gli1 positive cells in trabecular meshwork and Schlemm’s canal development. The elegance of this model is it can be activated at specific developmental stages and Gli1+ cell derivatives can be tracked and other detection probes (phalloidin, nuclear stains, Immunohistochemistry etc), can be incorporated to determine cell characteristics or over‐all tissue structure. Our results provide evidence that Gli1+ cells are contributing to the development of the anterior angle tissues including trabecular meshwork and Schlemm’s canal.Support or Funding InformationBiomedical Sciences Department Seed Grant (K. Svoboda), NIH K08DE025090 and R21 DE027928 (H. Zhao).

MP84-16 FOXC2 OVEREXPRESSION IN ENDOTHELIAL PROGENITOR CELLS ENHANCES RE-ENDOTHELIALIZATION FOLLOWING CAVERNOUS ARTERIAL INJURY

You have accessJournal of UrologySexual Function/Dysfunction: Basic Research & Pathophysiology (MP84)1 Apr 2020MP84-16 FOXC2 OVEREXPRESSION IN ENDOTHELIAL PROGENITOR CELLS ENHANCES RE-ENDOTHELIALIZATION FOLLOWING CAVERNOUS ARTERIAL INJURY Yanping Huang*, Mujun Lu, Wei Liu, Yidong Liu, and Wei Xue Yanping Huang*Yanping Huang* More articles by this author , Mujun LuMujun Lu More articles by this author , Wei LiuWei Liu More articles by this author , Yidong LiuYidong Liu More articles by this author , and Wei XueWei Xue More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000976.016AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Corpora cavernosa vascular endothelium injury is an important cause of erectile dysfunction (ED). Targeted repair of penile vascular endothelium is a vital way for ED treatment. Endothelial progenitor cells (EPCs) can facilitate endothelial repair through direct differentiation into endothelial cells and/or via the paracrine mechanisms. Foxc2 plays an important role in the combinatorial regulation of endothelial gene expression and vasculogenesis. The aim is to investigate that if Foxc2 can promote EPCs’ homing and differentiation, which repair the cavernous vascular endothelium injury eventually. METHODS: Rat cavernous vascular injury was established using a flexible guide wire. The rat subjected to vascular injury were randomly assigned into experimental groups. EPCs were isolated and cultured from wild type rat. EPCs were transfected with Foxc2 expression vector (Foxc2-EPCs) or with empty control vector (Ctrl-EPCs). The rat subjected to cavernous vascular injury received PBS, Ctrl-EPCs or Foxc2-EPCs by tail vein injection. Fluorescent microscopy was used to examine the homing capacity of EPCs to the injury sites. For assessment of re-endothelialization animals were perfused with Evans blue dye at 7 days after EPCs delivery. RESULTS: At 3 days after EPCs delivery, cells were strictly restricted to the injury sites. The number of recruited cells was significantly higher in the rat transfused with Foxc2-EPCs compared with Ctrl-EPCs (about 3-fold of Ctrl-EPCs). At 7 days after EPCs delivery, Foxc2-EPCs delivery significantly increased the degree of re-endothelialization relative to Ctrl-EPCs (84.3±1.2%vs. 50.1±1.1%, P< 0.05). At 28 days, the inhibitory effect of Foxc2-EPCs delivery was lower than Ctrl-EPCs (0.32±0.01vs. 0.78士0.05, P< 0.05). CONCLUSIONS: Foxc2 overexpression may increase EPCs homing and recruitment to the sites of cavernous vascular injury, and thereby enhance the therapeutic benefit of EPCs for facilitating re-endothelialization. Source of Funding: This study was supported by the National Natural Science Foundation of China (NSFC: 81771577). © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e1270-e1271 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yanping Huang* More articles by this author Mujun Lu More articles by this author Wei Liu More articles by this author Yidong Liu More articles by this author Wei Xue More articles by this author Expand All Advertisement PDF downloadLoading ...

Foxc2 Alleviates Ox-LDL-Induced Lipid Accumulation, Inflammation, and Apoptosis of Macrophage via Regulating the Expression of Angptl2.

The present study aimed to investigate the role of Forkhead box protein C2 (Foxc2) in oxidized low-density lipoprotein (ox-LDL)-induced macrophages and identify the potential mechanisms. RAW264.7 cells, the murine macrophage cell line, were stimulated by ox-LDL, and cell proliferation was examined. The levels of inflammation- and oxidative stress-related markers were detected using kits after induction with ox-LDL. Subsequently, the expression of Foxc2 was measured using Western blotting. After transfection with Foxc2 pcDNA3.1, intracellular lipid droplets were examined using oil red O staining. The levels of total cholesterol (TC), free cholesterol (FC), inflammatory cytokines, and oxidative stress markers were determined. Moreover, apoptosis of RAW264.7 cells was detected using flow cytometry, and apoptosis-related proteins were measured using Western blotting. Angiopoietin-like protein 2 (Angptl2) was predicted as a target gene of Foxc2. Therefore, the expression of Angptl2 was examined after Foxc2 overexpression in ox-LDL-induced RAW264.7 cells. Then, the changes of intracellular lipid droplets, TC, FC, inflammatory cytokines, oxidative stress factors, and cell apoptosis were detected after Angptl2 overexpression or co-transfection with Foxc2 and Angptl2 pcDNA3.1. The results revealed that ox-LDL induction inhibited proliferation of RAW264.7 cells and promoted the release of inflammatory factors. Importantly, the expression of Foxc2 was obviously decreased after stimulation by ox-LDL. Foxc2 overexpression suppressed lipid accumulation, TC, FC levels, inflammation, oxidative stress, and apoptosis induced by ox-LDL, whereas these inhibitory effects were relieved after co-transfection with Angptl2 pcDNA3.1. These findings demonstrated that Foxc2 can alleviate ox-LDL-induced lipid accumulation, inflammation, and apoptosis of macrophage via regulating the expression of Angptl2.

Subdural haematomas drain into the extracranial lymphatic system through the meningeal lymphatic vessels

Subdural haematomas (SDHs) are characterized by rapidly or gradually accumulated haematomas between the arachnoid and dura mater. The mechanism of haematoma clearance has not been clearly elucidated until now. The meningeal lymphatic vessel (mLV) drainage pathway is a novel system that takes part in the clearance of waste products in the central nervous system (CNS). This study aimed to explore the roles of the mLV drainage pathway in SDH clearance and its impacting factors. We injected FITC-500D, A488-fibrinogen and autologous blood into the subdural space of mice/rats and found that these substances drained into deep cervical lymph nodes (dCLNs). FITC-500D was also observed in the lymphatic vessels (LYVE+) of the meninges and the dCLNs in mice. The SDH clearance rate in SDH rats that received deep cervical lymph vessel (dCLV) ligation surgery was significantly lower than that in the control group, as evaluated by haemoglobin quantification and MRI scanning. The drainage rate of mLVs was significantly slower after the SDH model was established, and the expression of lymphangiogenesis-related proteins, including LYVE1, FOXC2 and VEGF-C, in meninges was downregulated. In summary, our findings proved that SDH was absorbed through the mLV drainage pathway and that haematomas could inhibit the function of mLVs.

Identification of a novel microRNA-141-3p/Forkhead box C1/β-catenin axis associated with rheumatoid arthritis synovial fibroblast function in vivo and in vitro.

Rationale: Rheumatoid arthritis (RA) is a prototype of inflammatory arthritis in which synovial fibroblasts (SFs) play key roles in cartilage and bone destruction through tumor-like proliferation, migration, invasion and inflammation. This study aimed to research forkhead box protein C1 (FoxC1) and microRNA (miR)-141-3p, which modulate pathological changes in the synovial membrane, to find possible strategies for treating RA.Methods: FoxC1, β-catenin and miR-141-3p gene expression in synovial tissues and SFs was quantified by real-time PCR; FoxC1 and β-catenin protein levels were evaluated by immunohistochemistry, immunofluorescence, and Western blotting. We transiently transfected human SFs with FoxC1 and β-catenin overexpression and silencing vectors and assessed proliferation, migration, invasion and inflammation by cell function and enzyme-linked immunosorbent assays. We also assessed downstream signaling activation using immunofluorescence, real-time PCR and Western blotting. Double luciferase, coimmunoprecipitation and chromatin immunoprecipitation assays were used to verify miR-141-3p, FoxC1 and β-catenin gene and protein combinations. Finally, the therapeutic effects of FoxC1 silencing and miR-141-3p overexpression were evaluated in type II collagen-induced arthritis (CIA) rats.Results: We found that FoxC1 expression was significantly upregulated in synovium and SFs in both RA patients and rats with collagen-induced arthritis (CIA). FoxC1 overexpression increased β-catenin messenger RNA (mRNA) and protein levels and upregulated cyclin D1, c-Myc, fibronectin and matrix metalloproteinase 3 (MMP3) mRNA and protein expression in RA SFs (RASFs). In contrast, FoxC1 knockdown reduced β-catenin mRNA and protein levels as well as cyclin D1, c-Myc, and fibronectin mRNA and protein levels in RASFs. Furthermore, altering FoxC1 expression did not significantly change GSK3β and pGSK3β levels. FoxC1 overexpression promoted proliferation, migration, invasion and proinflammatory cytokine (interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α)) production and reduced anti-inflammatory cytokine (IL-10) levels in RASFs. FoxC1 bound to the β-catenin promoter, and β-catenin mediated the FoxC1-induced pathological changes. We also observed downregulated microRNA (miR)-141-3p expression in SFs from both RA patients and CIA rats and further found that miR-141-3p bound to the FoxC1 3′UTR and suppressed FoxC1 expression. Intra-ankle miR-141-3p agomir or FoxC1-specific siRNA injection hindered CIA development in rats.Conclusions: FoxC1 and miR-141-3p participate in RA pathogenesis by mediating inflammation and SF proliferation, migration, and invasion and thus could be novel targets for RA therapy as a nonimmunosuppressive approach.

Forkhead box C1 promotes the pathology of osteoarthritis by upregulating β-catenin in synovial fibroblasts.

Osteoarthritis (OA) is a joint disease characterized by articular cartilage degeneration, and no effective treatment is available. The OA classification has shifted from a cartilage-only disease to a whole-joint disease, and the synovial membrane plays an important role. Therefore, studies are needed to identify additional genes that regulate the pathologicalchanges in the synovial membrane to develop a promising therapeutic strategy for OA. Here, we validated that the expression of forkhead box protein C1 (FoxC1) and β-catenin was upregulated in OA synovial membranes and synovial fibroblasts (SFs). Gain- and loss-of-function studies revealed that FoxC1 overexpression promoted, whilst silencing inhibited OA synovial fibroblast (OASF) proliferation and pro-inflammatory cytokine [interleukin 6 (IL-6), interleukin 8 (IL-8) and tumour necrosis factor-α (TNF-α)] production. FoxC1 overexpression increased β-catenin mRNA, total and nuclear protein expression in OASFs and upregulated a disintegrin and metalloproteinase with thrombospondin motif 5 (ADAMTS-5), fibronectin, matrix metalloproteinase 3 (MMP3) and matrix metalloproteinase 13 (MMP13) mRNA and total protein expression in OASFs. Conversely, FoxC1 knockdown reduced β-catenin mRNA, total and nuclear protein expression in OASFs and reduced ADAMTS-5, fibronectin, MMP3 and MMP13 mRNA and total protein expression in OASFs. β-catenin mediates FoxC1-induced pathologicalchanges (proliferation, catabolic regulation and inflammation) in OASFs. MicroRNA-200a-3p (miR-200a-3p) binds to the 3'-UTR of FoxC1 and mediates FoxC1 expression. Intra-articular FoxC1-specific siRNA transfection hindered OA development in mice. Therefore, our results demonstrate the key role FoxC1 plays invivo and invitro in OA synovial pathology, possibly identifying a potential novel therapeutic target for OA.

Primary cilia deficiency in neural crest cells models anterior segment dysgenesis in mouse.

Defects affecting tissues of the anterior segment (AS) of the eye lead to a group of highly debilitating disorders called Anterior Segment Dysgenesis (ASD). Despite the identification of some causative genes, the pathogenesis of ASD remains unclear. Interestingly, several ciliopathies display conditions of the AS. Using conditional targeting of Ift88 with Wnt1-Cre, we show that primary cilia of neural crest cells (NCC), precursors of most AS structures, are indispensable for normal AS development and their ablation leads to ASD conditions including abnormal corneal dimensions, defective iridocorneal angle, reduced anterior chamber volume and corneal neovascularization. Mechanistically, NCC cilia ablation abolishes hedgehog (Hh) signaling in the periocular mesenchyme (POM) canonically activated by choroid-secreted Indian Hh, reduces proliferation of POM cells surrounding the retinal pigment epithelium and decreases the expression of Foxc1 and Pitx2, two transcription factors identified as major ASD causative genes. Thus, we uncovered a signaling axis linking cilia and ASD.

Coactivation of Endogenous Wnt10b and Foxc2 by CRISPR Activation Enhances BMSC Osteogenesis and Promotes Calvarial Bone Regeneration.

CRISPR activation (CRISPRa) is a burgeoning technology for programmable gene activation, but its potential for tissue regeneration has yet to be fully explored. Bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into osteogenic or adipogenic pathways, which are governed by the Wnt (Wingless-related integration site) signaling cascade. To promote BMSC differentiation toward osteogenesis and improve calvarial bone healing by BMSCs, we harnessed a highly efficient hybrid baculovirus vector for gene delivery and exploited a synergistic activation mediator (SAM)-based CRISPRa system to activate Wnt10b (that triggers the canonical Wnt pathway) and forkhead c2 (Foxc2) (that elicits the noncanonical Wnt pathway) in BMSCs. We constructed a Bac-CRISPRa vector to deliver the SAM-based CRISPRa system into rat BMSCs. We showed that Bac-CRISPRa enabled CRISPRa delivery and potently activated endogenous Wnt10b and Foxc2 expression in BMSCs for >14days. Activation of Wnt10b or Foxc2 alone was sufficient to promote osteogenesis and repress adipogenesis invitro. Furthermore, the robust and prolonged coactivation of both Wnt10b and Foxc2 additively enhanced osteogenic differentiation while inhibiting adipogenic differentiation of BMSCs. The CRISPRa-engineered BMSCs with activated Wnt10b and Foxc2 remarkably improved the calvarial bone healing after implantation into the critical-sized calvarial defects in rats. These data implicate the potentials of CRISPRa technology for bone tissue regeneration.

FOXC2-AS1 regulates phenotypic transition, proliferation and migration of human great saphenous vein smooth muscle cells

ObjectivesIn varicose veins, vascular smooth muscle cells (VSMCs) often shows phenotypic transition and abnormal proliferation and migration. Evidence suggests the FOXC2–Notch pathway may be involved in the pathogenesis of varicose veins. Here, this study aimed to explore the role of long non-coding RNA FOXC2-AS1 (FOXC2 antisense RNA 1) in phenotypic transition, proliferation, and migration of varicose vein-derived VSMCs and to explore whether the FOXC2-Notch pathway was involved in this process.MethodsThe effect of FOXC2-AS1 on the proliferation and migration of human great saphenous vein smooth muscle cells (SV-SMCs) was analyzed using MTT assay and Transwell migration assay, respectively. The levels of contractile marker SM22α and synthetic marker osteopontin were measured by immunohistochemistry and Western blot to assess the phenotypic transition.ResultsThe human varicose veins showed thickened intima, media and adventitia layers, increased synthetic VSMCs, as well as upregulated FOXC2-AS1 and FOXC2 expression. In vitro assays showed that FOXC2-AS1 overexpression promoted phenotypic transition, proliferation, and migration of SV-SMCs. However, the effect of FOXC2-AS1 overexpression could be abrogated by both FOXC2 silencing and the Notch signaling inhibitor FLI-06. Furthermore, FOXC2-AS1 overexpression activated the Notch pathway by upregulating FOXC2.ConclusionFOXC2-AS1 overexpression promotes phenotypic transition, proliferation, and migration of SV-SMCs, at least partially, by activating the FOXC2-Notch pathway.

The Feasibility to Isolate and Expand Tympanic Membrane Squamous Epithelium Stem Cells From Scarred Perforation Margins.

The scarred rim of chronic tympanic membrane (TM) perforation contains keratinocytes with potential for regeneration while maintaining their morphological and genetic characteristics. The squamous epithelium of the TM has a good regeneration capacity. Successful isolation and expansion of human TM keratinocytes (hTMKR) was reported from a full, en-bloc, healthy TM. Trimmed margins of the TM perforation (harvested during tympanoplasty) underwent enzymatic digestion (collagenase or trypsin) and were seeded either with serum-containing medium (SCM) or keratinocyte serum-free medium (KSFM) and progenitor cell growth medium (PR) (KSFM:PR, 1:1). Gene expression analysis by real-time qRT-PCR was used to compare between human TM cells derived from scarred perforation margins (hTMKR), normal human skin keratinocytes (NhSKR), and human fibroblasts. Twelve patients were included in the study. In 9 of 12 cases (75%) single-cell isolation with fibroblastic or epithelial cell morphology (or both) was achieved. Cells seeded with KSFM:PR yielded epithelial morphology (hTMKR) while SCM culturing resulted in a fibroblastic morphology (hTMFib). Gene expression analysis revealed significant higher expression of VCAN (p = 0.002) and FOXC2 (p = 0.015) at the mRNA levels (normal hTMKR markers) in hTMKR compared to NhSKR. In addition, a comparison of gene expression between hTMKR and hTMFib revealed significantly higher levels of both VCAN (p = 0.045) and SLC6A14 (p = 0.036) among hTMKR. For the first time, we developed a protocol to isolate hTMKR from scarred TM perforation margins. Furthermore, we succeeded in achieving tissue expansion that preserved the characteristic of healthy TM cells. This study bridges "regenerative medicine" approach with clinical and surgical objectives.

Inhibition of lung cancer growth and metastasis by DHA and its metabolite, RvD1, through miR-138-5p/FOXC1 pathway

BackgroundNon small cell lung cancer (NSCLC) is one of the most common cancers in the world. DHA is known to be capable of suppressing NSCLC cell proliferation and metastasis. However, the mechanisms by which DHA exhibits its antitumor effects are unknown. Here we aimed to identify the effects and mechanisms of DHA and its metabolites on lung cancer cell growth and invasion.MethodsAs measures of cell proliferation and invasion ability, the cell viability and transwell assays were used in vitro. Transgenic mfat-1 mice, which convert ω-6 PUFAs to ω-3 PUFAs, were used to detect the effect of endogenous DHA on tumor transplantation. An LC − MS/MS analysis identified the elevation of several eicosanoid metabolites of DHA. By using qPCR miRNA microarray, online prediction software, luciferase reporter assays and Western blot analysis, we further elucidated the mechanisms.ResultsAddition of exogenous DHA inhibited the growth and invasion in NSCLC cells in vitro. Endogenously produced DHA attenuated LLC-derived tumor growth and metastasis in the transgenic mfat-1 mice. Among the elevation of DHA metabolites, resolvin D1 (RvD1) significantly contributed to the inhibition in cell growth and invasion. MiRNA microarray revealed that the level of miR-138-5p was significantly increased after RvD1 treatment. MiR-138-5p mimics decreased cell viability and invasion; while miR-138-5p inhibitor abolished RvD1-mediated suppression of cell viability and invasion. The expression of FOXC1 was significantly reduced upon overexpression of miR-138-5p while luciferase reporter assay showed that FOXC1 was a direct target of miR-138-5p. In vivo, endogenous DHA by the mfat-1 transgene enhanced miR-138-5p expression and decreased FOXC1 expression. Furthermore, overexpression of FOXC1 reversed the inhibition in cell viability and invasion induced by RvD1 treatment.ConclusionsThese data identified the RvD1/miR-138-5p/FOXC1 pathway as a novel mechanism by DHA and its metabolite, RvD1, and the potential of targeting such pathway as a therapeutic strategy in treating NSCLC.

The Epithelial to Mesenchymal Transition Promotes Glutamine Independence by Suppressing GLS2 Expression.

Identifying bioenergetics that facilitate the epithelial to mesenchymal transition (EMT) in breast cancer cells may uncover targets to treat incurable metastatic disease. Metastasis is the number one cause of cancer-related deaths; therefore, it is urgent to identify new treatment strategies to prevent the initiation of metastasis. To characterize the bioenergetics of EMT, we compared metabolic activities and gene expression in cells induced to differentiate into the mesenchymal state with their epithelial counterparts. We found that levels of GLS2, which encodes a glutaminase, are inversely associated with EMT. GLS2 down-regulation was correlated with reduced mitochondrial activity and glutamine independence even in low-glucose conditions. Restoration of GLS2 expression in GLS2-negative breast cancer cells rescued mitochondrial activity, enhanced glutamine utilization, and inhibited stem-cell properties. Additionally, inhibition of expression of the transcription factor FOXC2, a critical regulator of EMT in GLS2-negative cells, restored GLS2 expression and glutamine utilization. Furthermore, in breast cancer patients, high GLS2 expression is associated with improved survival. These findings suggest that epithelial cancer cells rely on glutamine and that cells induced to undergo EMT become glutamine independent. Moreover, the inhibition of EMT leads to a GLS2-directed metabolic shift in mesenchymal cancer cells, which may make these cells susceptible to chemotherapies.

Evidence for miR-548c-5p regulation of FOXC2 transcription through a distal genomic target site in human podocytes.

Podocytes are highly differentiated epithelial cells outlining the glomerular vessels. FOXC2 is a transcription factor essential for inducing podocyte differentiation, development and maturation, and is considered to be the earliest podocyte marker. miRNA prediction analysis revealed a full-length target site for the primate-specific miR-548c-5p at a genomic region > 8kb upstream of FOXC2. We hypothesised that the transcription rates of FOXC2 during podocyte differentiation might be tuned by miR-548c-5p through this target site. Experiments were performed with cultured human podocytes, transfected with luciferase reporter constructs bearing this target site region within an enhancer element of the native plasmid. The results confirmed a seed region-driven targeting potential by the miRNA, with mimics downregulating and inhibitors enhancing luciferase activity. Introducing mutations into the miRNA target seed region abolished the expected response. In cultured podocytes, FOXC2 mRNA and protein levels responded to miR-548c-5p abundance in a coordinated manner before and after induction of differentiation, with high statistical significance. Ago-ChIP experiments revealed occupancy of the miRNA target site by miRNA/RISC in undifferentiated cells and its release when differentiation is initiated, allowing its interaction with the gene's promoter region to amplify FOXC2 expression, as shown by chromosome conformation capture and qRT-PCR. Moreover, the expression pattern of FOXC2 during podocyte differentiation seems to be affected by miR-548c-5p, as removal of either endogenous or mimic miR-548c-5p results in increased FOXC2 protein levels and cells resembling those undergoing differentiation. Collectively, results indicate a well-orchestrated regulatory model of FOXC2 expression by a remote upstream target site for miR-548c-5p.

LncRNA Tug1 involves in the pulmonary vascular remodeling in mice with hypoxic pulmonary hypertension via the microRNA-374c-mediated Foxc1

Hypoxic pulmonary hypertension (HPH) is a serious and potentially devastating disorder of the pulmonary circulation with complicated mechanisms. Long non-coding RNA (lncRNA) has been revealed to participate in HPH development. This study aimed to explore how lncRNA Tug1 affected the pulmonary vascular remodeling in HPH. A mouse model of HPH and a pulmonary artery smooth muscle cell (PASMC) model of HPH (HPH-PASMCs) were established, where the expression of lncRNA Tug1 was determined. Then, the interaction among lncRNA Tug1, miR-374c, and Foxc1 was assessed. Finally, in order to determine the effects of lncRNA Tug1 on PASMC activities and pulmonary vascular remodeling after HPH, the expression of lncRNA Tug1 was silenced in HPH-PASMCs and HPH mice, with the proliferation, apoptosis, and migration of PASMCs as well as blood pressure in mice measured, respectively. The obtained data revealed that lncRNA Tug1 was highly expressed in HPH mice and HPH-PASMCs. In addition, lncRNA Tug1 up-regulated the expression of Foxc1 by binding to miR-374c. Notably, silencing of lncRNA Tug1 inhibited the proliferation and migration, but promoted the apoptosis of PASMCs. Moreover, lncRNA Tug1 silencing was observed to attenuate the pulmonary vascular remodeling in HPH mice through the Foxc1-mediated NOTCH signaling pathway. Taken conjointly, silencing of lncRNA Tug1 down-regulated the Foxc1 expression by binding to miR-374c, thereby inhibiting the proliferation and migration, while promoting apoptosis of PASMCs to impede pulmonary vascular remodeling in HPH via the Notch signaling pathway. This study provided novel therapeutic insights for treating HPH.