Expression Of Forkhead Box C2 Research Articles

Forkhead box C2 is associated with insulin resistance in gestational diabetes mellitus

Objective This study aimed to investigate serum levels of adiponectin, and the mRNA expression of forkhead box C2 (FOXC2) and glucose transporter-4 (GLUT4) in visceral adipose tissue obtained from patients with gestational diabetes mellitus (GDM) and healthy pregnant women. Methods Venous blood samples were obtained from 60 pregnant women with gestational normal glucose tolerance (GNGT) and 21 patients with GDM. Visceral adipose tissues were obtained from 11 women with GDM and 30 with GNGT. Serum adiponectin levels were detected by enzyme-linked immunosorbent assay, and FOXC2 and GLUT4 mRNA expression were detected by quantitative polymerase chain reaction. Results Serum adiponectin concentrations were lower in the women with GDM than in the controls (p < .05). FOXC2 and GLUT4 mRNA expression were decreased in visceral adipose tissue of GDM women than in the controls (p < .05). Correlation analyses showed that FOXC2 tended to have a positive correlation with GLUT4 in GDM patients' visceral adipose tissue (p =.0564). Conclusion Our results revealed that decreased adiponectin, FOXC2, and GLUT4 expression were associated with increased risk of GDM and the regulation mechanism of GLUT4 mediated by FOXC2 would be the focus of further studies.

FOXC2 Alleviates Myocardial Ischemia-Reperfusion Injury in Rats through Regulating Nrf2/HO-1 Signaling Pathway.

Objective Myocardial ischemia-reperfusion injury (MIRI) is the leading cause of death in patients with cardiovascular disease. The purpose of this study is to investigate the effect and mechanism of forkhead box C2 (FOXC2) on MIRI in rats. Methods We made ischemia-reperfusion (I/R) models for rats by performing I/R surgery. After 3 hours, 3 days, and 7 days of reperfusion, we detected the structure and function of rat myocardium by 2, 3, 5-triphenyl tetrazolium chloride staining, echocardiography, lactate dehydrogenase kit, and haematoxylin-eosin staining. The change of FOXC2 expression in myocardial tissue was also detected. Then, we increased the expression of FOXC2 in rats by adenovirus transfection to clarify the effect of FOXC2 on changes of oxidative stress and inflammation of rat myocardium. In addition, we detected the effect of FOXC2 overexpression plasmid on the function of H9c2 cells in vitro. The expression changes of Nrf2/HO-1 in myocardial cells were also detected to clarify the mechanism of action of FOXC2. Results The expression of FOXC2 in I/R rats was significantly lower than that in the sham group. After overexpressing FOXC2 in I/R rats, we found that the expression of SOD1/2 of rat myocardium and inflammatory factors in the serum were significantly reduced. Overexpression of FOXC2 also increased the viability and antioxidant capacity of H9c2 cells. In addition, FOXC2 was found to increase the activity of the Nrf2/HO-1 signaling pathway in myocardial cells, and the inhibition of Nrf2/HO-1 signaling pathway attenuated the protective effect of FOXC2 on myocardial cells. Conclusions MIRI in rats was accompanied by low expression of FOXC2 in myocardial tissue. Overexpression of FOXC2 reduces the level of inflammation and oxidative stress in myocardial tissue by promoting the Nrf2/HO-1 signaling pathway, thereby alleviating MIRI.

Imbalance between Expression of FOXC2 and Its lncRNA in Lymphedema-Distichiasis Caused by Frameshift Mutations.

Forkhead-box C2 (FOXC2) is a transcription factor involved in lymphatic system development. FOXC2 mutations cause Lymphedema-distichiasis syndrome (LD). Recently, a natural antisense was identified, called lncRNA FOXC2-AS1, which increases FOXC2 mRNA stability. No studies have evaluated FOXC2 and FOXC2-AS1 blood expression in LD and healthy subjects. Here, we show that FOXC2 and FOXC-AS1 expression levels were similar in both controls and patients, and a significantly higher amount of both RNAs was observed in females. A positive correlation between FOXC2 and FOXC2-AS1 expression was found in both controls and patients, excluding those with frameshift mutations. In these patients, the FOXC2-AS1/FOXC2 ratio was about 1:1, while it was higher in controls and patients carrying other types of mutations. The overexpression or silencing of FOXC2-AS1 determined a significant increase or reduction in FOXC2 wild-type and frameshift mutant proteins, respectively. Moreover, confocal and bioinformatic analysis revealed that these variations caused the formation of nuclear proteins aggregates also involving DNA. In conclusion, patients with frameshift mutations presented lower values of the FOXC2-AS1/FOXC2 ratio, due to a decrease in FOXC2-AS1 expression. The imbalance between FOXC2 mRNA and its lncRNA could represent a molecular mechanism to reduce the amount of FOXC2 misfolded proteins, protecting cells from damage.

The expression of AGGF1, FOXC2, and E-cadherin in esophageal carcinoma and their clinical significance.

Angiogenic factor with G-patch and FHA domain 1 (AGGF1) is a newly initiator of angiogenesis. Forkhead box C2 (FOXC2) that is a member of the winged spiral transcription factor family plays an important role in epithelial-mesenchymal transition (EMT). Epithelial-cadherin (E-cad) that is an adhesion molecule is also involved in EMT. The purpose of this study is to investigate the expression of AGGF1, FOXC2, and E-cad in esophageal squamous cell carcinoma (ESCC) and their clinical significance.Immunohistochemistry was performed to investigate the expression of AGGF1, FOXC2, and E-cad in 170 ESCC specimens and corresponding normal esophageal mucosa tissues. Follow-up data was also collected.The positive rates of AGGF1 and FOXC2 expression were significantly higher in ESCC group when compared with the control group; the positive rate of E-cad expression was significantly lower in ESCC group when compared with the control group. Positive rates of AGGF1, FOXC2, and E-cad expression were significantly associated with grades of differentiation, tumor grades, lymph node metastasis stages, as well as tumor-node-metastasis stages. Kaplan–Meier analysis demonstrated that positive expression of AGGF1 or FOXC2 for ESCC patients had significantly unfavorably overall survival time when compared with patients with negative expression of AGGF1 or FOXC2; and positive expression of E-cad for ESCC patients had significantly longer overall survival time when compared with patients with negative expression of E-cad. Multivariate analysis indicated that AGGF1, FOXC2, and E-cad expression and tumor-node-metastasis stages were postoperative independent prognostic factors for ESCC patients.AGGF1, FOXC2, and E-cad may be considered promising biomarkers of ESCC patients’ prognosis.

MiR-23b inhibits proliferation and migration of triple negative breast cancer cells

Objective To investigate the effect and mechanism of miR-23b on the proliferation and migration of triple negative breast cancer (TNBC)cells. Methods Real time polymerase chain reaction (PCR) was used to detect the expression of miR-23b in triple negative breast cancer tissues. MDA-MB-231/miR-23b, BT549/miR-23b cell lines are constructed. Proliferation assay, scaling healing experiment and Transwell migration assay were used to detect the effect of miR-23b on the proliferation and migration of triple negative breast cancer cells. Dual-luciferase reporter gene assay was employed to examine the interactions between miR-23b and forkhead box C2 (FOXC2). Real time PCR and Western blot were performed to detect the effect of miR-23b on the expression of FOXC2. Results The expression level of miR-23b in triple negative breast cancer tissues was significantly less than that in adjacent normal tissues. miR-23b could reduced the proliferation and migration of triple negative breast cancer cells. Dual-luciferase assay confirmed that miR-23b could regulate the expression and activity of FOXC2. The expression of FOXC2 in mRNA and protein level was inhibited by miR-23b. Conclusions miR-23b can inhibit the expression of FOXC2 and affect the proliferation and migration of triple negative breast cancer cells. Key words: MicroRNAs; Breast neoplasms; Cell proliferation; Cell migration assays; In vitro

The Forkhead Transcription Factor FOXC2 Is Required for Maintaining Murine Spermatogonial Stem Cells.

Continuous spermatogenesis from puberty to old age in males relies on spermatogonial stem cells (SSCs) that possess the property of self-renewal and differentiation. The delicate balance between self-renewal and differentiation is of great importance. In mice, SSCs exist as a subpopulation of undifferentiated spermatogonia. SSCs are controlled by intrinsic molecular pathways that can be activated by extrinsic signals. Our results here first show that the expression of forkhead box C2 (FOXC2) is restricted to GFRα1-positive spermatogonia in the testis. Whole-mount immunofluorescence results reveal that FOXC2 is expressed predominately in As and Apr spermatogonia. Reduction of Foxc2 gene expression by shRNA lentivirus treatment significantly impairs the maintenance of SSCs in vitro. Furthermore, knock-down of Foxc2 decreases SSC colonization to only 10.42% compared to the control by transplantation. Reverse transcription and real-time quantitative PCR gene analyses following knock-down of Foxc2 indicate that Foxc2 may act as a suppressor for SSC differentiation. Extrinsic stimuli treatments show that glial cell line-derived neurotrophic factor and retinoic acid act in opposite ways to regulate FOXC2 expression and subsequent SSC property. These results suggest that FOXC2 is a critical intrinsic regulator of SSC self-renewal and differentiation.

Prognostic and Clinicopathological Value of the Expression of FOXC2 &amp;amp; YKL-40 in Carcinoma of the Breast, an Immunohistochemical Study

Background: Breast cancer is considered the commonest and the most fatal female cancer worldwide. There is to an urgent need for discovering recent therapies to identify patient prognosis and improve treatment strategies. Fork-head Box C2 (FOXC2) is a transcription factor which is a key regulator of cancer stem cells (CSC) properties and epithelial mesenchymal transition (EMT) e.g. cancer initiation, metastatic capacity, and resistance to chemotherapy. FOXC2 roles in CSCs properties and EMT regulation in breast cancer needs detailed studies. YKL-40 is known as chitinase-3-like-1 belongs to a family of mammalian proteins that have an amino acid sequence which is similar to the 18-glycosyl hydrolase bacterial chitinases group. Recent studies have found aberrant YKL-40 elevated expression in cancer of various organs, so it may be used as a recent prognostic biomarker for patients with breast cancer. Former researchers have assessed the expression of FOXC2 & YKL-40 separately in cancer patients and relations to prognosis of patients; however, no studies assessed them together in breast cancer patients and the previous results were inconclusive. Accordingly, our study aimed at evaluation of immunohistochemical expressions of FOXC2 & YKL-40 in carcinoma of the breast in a trial to clarify the relation among their expressions, clinicopathological parameters and recurrence of the disease after successive therapy and patients’ prognosis. Methods: we have evaluated expressions of FOXC2 & YKL-40 in sections from 50 paraffin blocks of carcinoma of the breast using immunohistochemistry. We followed up our patients for 3 years for assessment of recurrence of the disease after successive therapy and survival rates. We analyzed the relationship between their combined expression clinicopathological and prognostic parameters. Results: high FOXC2 expression was associated with older age of the patient (p = 0.002), negative ER (p = 0.009), & PR (p = 0.008), positive HER2neu (p = 0.02), aggressive molecular subtype, higher grade of the tumor (p = 0.03), high incidence of distant metastasis (p = 0.011), high incidence of lymph node metastasis, higher KI labeling index, advanced stage, (p expression was positively correlated with older age of the patient (p = 0.002), negative ER (p = 0.03), & PR (p = 0.04), positive HER2neu (p = 0.02), higher grade of the tumor (p = 0.003), high incidence of distant metastasis (p = 0.04), higher KI labeling index, aggressive molecular subtype, advanced stage, high incidence of lymph node metastasis (p Conclusion: Higher expression levels of FOXC2 & YKL-40 are markers of poor prognosis in breast cancer.

Effects of forkhead box C2 on carcinogenesis and lymphatic metastasis in endometrial carcinoma.

We investigated the distribution of endometrial lymphatic vessels and expression of forkhead box C2 (FOXC2) in normal endometrium during menstrual cycle and in endometrial adenocarcinoma. Full-thickness uterine samples and endometrial adenocarcinoma samples were collected for immunohistochemical analysis using D2-40 and FOXC2 mouse monoclonal antibodies. The lymphatic vessel density (LVD) of the endometrium was significantly reduced compared with the myometrium during the cycle. Intra-tumoral LVD was significantly decreased in both stages of endometrioid adenocarcinoma compared with normal endometrium and myometrium. Intra-tumoral LVD significantly decreased from stage IA to stage IIIC. Peri-tumoral LVD for stage IA and stage IIIC tumors was significantly increased compared with normal endometrial LVD, but decreased compared with normal myometrial LVD. Stage IIIC showed increased peri-tumoral LVD when compared with stage IA. The positive rate of FOXC2 was 73.3% in proliferative endometrium and 80% in secretory endometrium. Secretory endometrium showed significantly increased FOXC2 expression compared with proliferative endometrium. Endometrioid adenocarcinoma showed significantly increased FOXC2 expression compared with normal endometrium, both in the epithelium and stroma. FOXC2 expression in the stroma significantly increased when pelvic and/or para-aotic lymph nodes were involved. FOXC2 was immunolocalized in low-risk endometrial carcinoma in endometrioid adenocarcinoma, but not in normal endometrium. Endometrial lymphatic vessels were located in normal endometrium and myometrium across the menstrual cycle and in intra-and peri-endometrioid adenocarcinoma, and increased in endometrial adenocarcinoma. Peri-tumoral lymphatics were associated with increased lymphatic metastasis. FOXC2 may be associated with the genesis of endometrial carcinoma and lymphangiogensis in endometrial adenocarcinoma in intra- and peri-tumoral lymphatics.

Correlation of Forkhead Box c2 with subtypes and invasive ability of invasive breast cancer.

Forkhead Box c2 (FOXC2) is a member of forkhead/winged-helix family of transcription factors. The relationship between FOXC2 and invasive breast cancers, including basal-like breast cancer (BLBC, a subtype of breast cancer), remains to be elucidated. In this study, immunohistochemistry was used to detect the expression of FOXC2 in samples from 103 cases of invasive breast cancers and 15 cases of normal mammary glands. The relationship between FOXC2 and clinical parameters of invasive breast cancers such as patient's age, tumor size, lymph node metastasis, tumor grade, the expression of ER, PR, HER-2 and p53, and Ki-67 labeling index (LI) was evaluated. The expression of FOXC2 was detected in parent MCF7 cells, MCF cells transfected with FOXC2 expression vectors and MDA-MB-435 cells by immunohistochemistry and Western blotting. Transwell assay was used to determine the invasive ability of these cells. The results showed that FOXC2 was strongly expressed in basal epithelial cells in normal mammary glands and weakly expressed or even not expressed in glandular epithelial cells. The majority of invasive breast cancers (71.8%, 74/103) had negative or weak expression of FOXC2. However, FOXC2 was strongly expressed in 60.7% of BLBCs. Moreover, FOXC2 was related with tumor grade, p53 expression, ki-67 LI and lymph nodes metastasis. It was expressed in FOXC2-transfected MCF cells and MDA-MB-435 cells but not in parent MCF cells. Transwell assay revealed that MCF cells transfected with FOXC2 expression vectors were more aggressive than the parent MCF cells, suggesting a positive correlation between FOXC2 and the invasion of breast cancer. It was concluded that there is a significant association between FOXC2 and the metastasis of invasive breast cancer. FOXC2 may be used as a new marker for the diagnosis and prognosis prediction of different subtypes of invasive breast cancers.

Expression of Transcription Factor FOXC2 in Cervical Cancer and Effects of Silencing on Cervical Cancer Cell Proliferation

Forkhead box C2 (FOXC2) is a member of the winged helix/forkhead box (Fox) family of transcription factors. It has been suggested to regulate tumor vasculature, growth, invasion and metastasis, although it has not been studied in cervical cancer. Here, we analyzed FOXC2 expression in cervical tissues corresponding to different stages of cervical cancer development and examined its correlation with clinicopathological characteristics. In addition, we examined the effects of targeting FOXC2 on the biological behavior of human cervical cancer cells. The expression of FOXC2 in normal human cervix, CIN I-III and cervical cancer was examined by immunohistochemistry and compared among the three groups and between cervical cancers with different pathological subtypes. Endogenous expression of FOXC2 was transiently knocked down in human Hela and SiHa cervical cells by siRNA, and cell viability and migration were examined by scratch and CCK8 assays, respectively. In normal cervical tissue the frequency of positive staining was 25% (10/40 cases), with a staining intensity (PI) of 0.297 ± 0.520, in CIN was 65% (26/40 cases), with a PI of 3.00 ± 3.29, and in cancer was 91.8% (68/74 cases), with a PI of 5.568 ± 3.449. The frequency was 100% in adenocarcinoma (5/5 cases) and 91.3% in SCCs (63/69 cases). The FOXC2 positive expression rate was 88.5% in patients with cervical SCC stage I and 100% in stage II, showing significant differences compared with normal cervix and CIN. With age, pathologic differentiation degree and tumor size, FOXC2 expression showed no significant variation. On transient transfection of Hela and SiHa cells, FOXC2-siRNA inhibition rates were 76.2% and 75.7%; CCK8 results showed reduced proliferation and relative migration (in Hela cells from 64.5 ± 3.16 to 49.5 ± 9.24 and in SiHa cells from 60.1 ± 3.05 to 44.3 ± 3.98) (P < 0.05). FOXC2 gene expression increases with malignancy, especially with blood vessel hyperplasia and invasion degree. Targeted silencing was associated with reduced cell proliferation as well as invasion potential.

Nuclear localization of GLI1 and elevated expression of FOXC2 in breast cancer is associated with the basal-like phenotype.

Aberrant sonic hedgehog (SHH)/glioma-associated oncogene (GLI) signaling has been shown in the development of many tumors. The aims of the present study are to determine the expression of two SHH signaling molecules, the glioma-associated oncogene homolog 1 (GLI1) and forkhead box C2 (FOXC2), in invasive breast cancers (IBC), to evaluate their association with clinicopathological parameters, and to determine their prognostic significance in breast cancer patients. Expression of GLI1 and FOXC2 were assessed by immunohistochemical analysis of a tissue microarray containing 262 unselected IBC cases. A statistical analysis was performed to assess the correlation of GLI1 and FOXC2 expression with the patients' clinicopathological parameters, postoperative survival rate, and molecular subtypes. Immunoreactivity of GLI1 and FOXC2 was observed in 84% and 75% of all breast cancer tissues, respectively. There was a significant correlation between nuclear FOXC2 and GLI1 expressions in these breast cancers, which was associated with estrogen receptor (ER) negativity. Furthermore, there was a significant association between nuclear expression of GLI1 and FOXC2 and a basal-like breast cancer phenotype. Patients with nuclear GLI1 or FOXC2-expressing tumors had a significantly shorter survival time than those without nuclear FOXC2 or GLI1 expression. Multivariate analysis showed that nuclear GLI1 or FOXC2 expression was an independent factor for predicting the prognosis of basal-like breast cancer. In conclusion, there was a significant correlation between expression of nuclear GLI1 or FOXC2 and human breast cancer. More specifically, elevated levels of these proteins were associated with the basal-like breast cancer phenotype and with a poor rate of disease-free survival. These data suggest that GLI1 and FOXC2 are involved in tumorigenesis and that they may be useful as diagnostic and therapeutic targets for human basal-like breast cancers. Additional studies are warranted to better understand the biological significance of GLI1 and FOXC2, to further refine statistics related to patient prognosis, and to optimize treatment of patients with basal-like breast cancer.

MiR-520h-mediated FOXC2 regulation is critical for inhibition of lung cancer progression by resveratrol

Resveratrol, a phytochemical found in various plants and Chinese herbs, is associated with multiple tumor-suppressing activities, has been tested in clinical trials. However, the molecular mechanisms involved in resveratrol-mediated tumor suppressing activities are not yet completely defined. Here, we showed that treatment with resveratrol inhibited cell mobility through induction of the mesenchymal-epithelial transition (MET) in lung cancer cells. We also found that downregulation of FOXC2 (forkhead box C2) is critical for resveratrol-mediated suppression of tumor metastasis in an in vitro and in vivo models. We also identified a signal cascade, namely, resveratrol-∣miRNA-520h-∣PP2A/C-∣Akt → NF-κB → FOXC2, in which resveratrol inhibited the expression of FOXC2 through regulation of miRNA-520h-mediated signal cascade. This study identified a new miRNA-520h-related signal cascade involved in resveratrol-mediated tumor suppression activity and provide the clinical significances of miR-520h, PP2A/C and FOXC2 in lung cancer patients. Our results indicated a functional link between resveratrol-mediated miRNA-520h regulation and tumor suppressing ability, and provide a new insight into the role of resveratrol-induced molecular and epigenetic regulations in tumor suppression.

FOXC2 controls Ang-2 expression and modulates angiogenesis, vascular patterning, remodeling, and functions in adipose tissue

Adipogenesis is spatiotemporally coupled to angiogenesis throughout adult life, and the interplay between these two processes is communicated by multiple factors. Here we show that in a transgenic mouse model, increased expression of forkhead box C2 (FOXC2) in the adipose tissue affects angiogenesis, vascular patterning, and functions. White and brown adipose tissues contain a considerably high density of microvessels appearing as vascular plexuses, which show redistribution of vascular smooth muscle cells and pericytes. Dysfunction of these primitive vessels is reflected by impairment of skin wound healing. We further provide a mechanistic insight of the vascular phenotype by showing that FOXC2 controls Ang-2 expression by direct activation of its promoter in adipocytes. Remarkably, an Ang-2-specific antagonist almost completely reverses this vascular phenotype. Thus, the FOXC2-Ang-2 signaling system is crucial for controlling adipose vascular function, which is part of an adaptation to increased adipose tissue metabolism.