The microRNA-183/96/182 cluster inhibits lung cancer progression and metastasis by inducing an interleukin-2-mediated antitumor CD8+ cytotoxic T-cell response.

One of the mechanisms by which cancer cells acquire hyperinvasive and migratory properties with progressive loss of epithelial markers is the epithelial-to-mesenchymal transition (EMT). We have previously reported that in different cancer types, including nonsmall cell lung cancer (NSCLC), the microRNA-183/96/182 cluster (m96cl) is highly repressed in cells that have undergone EMT. In the present study, we used a novel conditional m96cl mouse to establish that loss of m96cl accelerated the growth of Kras mutant autochthonous lung adenocarcinomas. In contrast, ectopic expression of the m96cl in NSCLC cells results in a robust suppression of migration and invasion in vitro, and tumor growth and metastasis in vivo. Detailed immune profiling of the tumors revealed a significant enrichment of activated CD8+ cytotoxic T lymphocytes (CD8+ CTLs) in m96cl-expressing tumors, and m96cl-mediated suppression of tumor growth and metastasis was CD8+ CTL-dependent. Using coculture assays with naïve immune cells, we show that m96cl expression drives paracrine stimulation of CD8+ CTL proliferation and function. Using tumor microenvironment-associated gene expression profiling, we identified that m96cl elevates the interleukin-2 (IL2) signaling pathway and results in increased IL2-mediated paracrine stimulation of CD8+ CTLs. Furthermore, we identified that the m96cl modulates the expression of IL2 in cancer cells by regulating the expression of transcriptional repressors Foxf2 and Zeb1, and thereby alters the levels of secreted IL2 in the tumor microenvironment. Last, we show that in vivo depletion of IL2 abrogates m96cl-mediated activation of CD8+ CTLs and results in loss of metastatic suppression. Therefore, we have identified a novel mechanistic role of the m96cl in the suppression of lung cancer growth and metastasis by inducing an IL2-mediated systemic CD8+ CTL immune response.

Stroke-associated intergenic variants modulate a human FOXF2 transcriptional enhancer.

SNPs associated with human stroke risk have been identified in the intergenic region between Forkhead family transcription factors FOXF2 and FOXQ1, but we lack a mechanism for the association. FoxF2 is expressed in vascular mural pericytes and is important for maintaining pericyte number and stabilizing small vessels in zebrafish. The stroke-associated SNPs are located in a previously unknown transcriptional enhancer for FOXF2, functional in human cells and zebrafish. We identify critical enhancer regions for FOXF2 gene expression, including binding sites occupied by transcription factors ETS1, RBPJ, and CTCF. rs74564934, a stroke-associated SNP adjacent to the ETS1 binding site, decreases enhancer function, as does mutation of RPBJ sites. rs74564934 is significantly associated with the increased risk of any stroke, ischemic stroke, small vessel stroke, and elevated white matter hyperintensity burden in humans. Foxf2 has a conserved function cross-species and is expressed in vascular mural pericytes of the vessel wall. Thus, stroke-associated SNPs modulate enhancer activity and expression of a regulator of vascular stabilization, FOXF2, thereby modulating stroke risk.

FOXF2 oppositely regulates stemness in luminal and basal-like breast cancer cells through the Wnt/beta-catenin pathway.

The stemness of cancer cells contributes to tumorigenesis, the heterogeneity of malignancies, cancer metastasis, and therapeutic resistance. However, the roles and regulatory mechanisms maintaining stemness among breast cancer subtypes remain elusive. Our previous studies have demonstrated that ectopic expression and dynamic alteration of the mesenchymal transcription factor forkhead box F2 (FOXF2) differentially regulates breast cancer progression and metastasis organotropism in a cell subtype-specific manner. Here, we reveal the underlying mechanism by which FOXF2 enhances stemness in luminal breast cancer cells but suppresses that in basal-like breast cancer (BLBC) cells. We show that luminal breast cancer and BLBC cells with FOXF2-regulated stemness exhibit partial mesenchymal stem cell properties that toward osteogenic differentiation and myogenic differentiation, respectively. Furthermore, we show that FOXF2 activates the Wnt signaling pathway in luminal breast cancer cells but represses this pathway in BLBC cells by recruiting nuclear receptor coactivator 3 (NCoA3) and nuclear receptor corepressor 1 (NCoR1) to the promoters of Wnt family member 2B (WNT2B) and frizzled class receptor 1 (FZD1) genes to activate and repress their transcription, respectively. We propose that targeting the Wnt signaling pathway is a promising strategy for the treatment of breast cancers with dysregulated expression of FOXF2.

Fox proteins are modular competency factors for facial cartilage and tooth specification.

Facial form depends on the precise positioning of cartilage, bone, and tooth fields in the embryonic pharyngeal arches. How complex signaling information is integrated to specify these cell types remains a mystery. We find that modular expression of Forkhead domain transcription factors (Fox proteins) in the zebrafish face arises through integration of Hh, Fgf, Bmp, Edn1 and Jagged-Notch pathways. Whereas loss of C-class Fox proteins results in reduced upper facial cartilages, loss of F-class Fox proteins results in distal jaw truncations and absent midline cartilages and teeth. We show that Fox proteins are required for Sox9a to promote chondrogenic gene expression. Fox proteins are sufficient in neural crest-derived cells for cartilage development, and neural crest-specific misexpression of Fox proteins expands the cartilage domain but inhibits bone. These results support a modular role for Fox proteins in establishing the competency of progenitors to form cartilage and teeth in the face.

Foxf2 and Smad6 co-regulation of collagen 5A2 transcription is involved in the pathogenesis of intrauterine adhesion.

The replacement of normal endometrial epithelium by fibrotic tissue is the pathological feature of intrauterine adhesion (IUA), which is caused by trauma to the basal layer of the endometrium. COL5A2 is a molecular subtype of collagen V that regulates collagen production in fibrotic tissue. Here, we investigated the roles of Foxf2 and Smad6 in regulating the transcription of COL5A2 and their involvement in the pathogenesis of IUA. Small interference-mediated Foxf2 (si-Foxf2) silencing and pcDNA3.1-mediated Smad6 (pcDNA3.1-Smad6) up-regulation were performed in a TGF-ß1-induced human endometrial stromal cell line (HESC) fibrosis model. Assessment of collagen expression by Western blotting, immunofluorescence and qRT-PCR showed that COL5A2, COL1A1 and FN were significantly down-regulated in response to si-Foxf2 and pcDNA3.1-Smad6. Transfection of lentivirus vector-Foxf2 (LV-Foxf2) and pcDNA3.1-Smad6 into HESCs and qRT-PCR showed that Foxf2 promoted COL5A2 expression and Smad6 inhibited Foxf2-induced COL5A2 expression. Co-immunoprecipitation, chromatin immunoprecipitation and dual-luciferase reporter assays to detect the interaction between Foxf2 and Smad6 and their role in COL5A2 transcription showed that Foxf2 interacted with Smad6 and bond the same promoter region of COL5A2. In a rat IUA model, injection of ADV2-Foxf2-1810 and ADV4-Smad6 into the uterine wall showed that Foxf2 down-regulation and Smad6 up-regulation decreased fibrosis and the expression of COL5A2 and COL1A1, as detected by haematoxylin/eosin, Masson trichrome staining and immunohistochemistry. Taken together, these results suggested that Foxf2 interacted with Smad6 and co-regulated COL5A2 transcription in the pathogenesis of IUA, whereas they played opposite roles in fibrosis.

Familial Absent Uvula With Velopharyngeal Incompetence-A New Syndrome?

We present a family with a previously undescribed abnormality of the palate and oropharynx which involved the absence of the uvula and the anterior pillar of the fauces, rudimentary posterior pillar of the fauces, and hypernasality. Eight family members over 4 generations are affected in a pattern consistent with autosomal dominant inheritance. A causal role for the FOXF2 gene has been identified and previously reported. We describe the management of the proband, which involved attempting to lengthen the palate and to retroposition the abnormally anteriorly directed velar musculature, along with speech therapy.

Foxf2 plays a dual role during transforming growth factor beta-induced epithelial to mesenchymal transition by promoting apoptosis yet enabling cell junction dissolution and migration.

BACKGROUND: The most life-threatening step during malignant tumor progression is reached when cancer cells leave the primary tumor mass and seed metastasis in distant organs. To infiltrate the surrounding tissue and disseminate throughout the body, single motile tumor cells leave the tumor mass by breaking down cell-cell contacts in a process called epithelial to mesenchymal transition (EMT). An EMT is a complex molecular and cellular program enabling epithelial cells to abandon their differentiated phenotype, including cell-cell adhesion and cell polarity, and to acquire mesenchymal features and invasive properties. METHODS: We employed gene expression profiling and functional experiments to study transcriptional control of transforming growth factor (TGF)ß-induced EMT in normal murine mammary gland epithelial (NMuMG) cells. RESULTS: We identified that expression of the transcription factor forkhead box protein F2 (Foxf2) is upregulated during the EMT process. Although it is not required to gain mesenchymal markers, Foxf2 is essential for the disruption of cell junctions and the downregulation of epithelial markers in NMuMG cells treated with TGFß. Foxf2 is critical for the downregulation of E-cadherin by promoting the expression of the transcriptional repressors of E-cadherin, Zeb1 and Zeb2, while repressing expression of the epithelial maintenance factor Id2 and miRNA 200 family members. Moreover, Foxf2 is required for TGFß-mediated apoptosis during EMT by the transcriptional activation of the proapoptotic BH3-only protein Noxa and by the negative regulation of epidermal growth factor receptor (EGFR)-mediated survival signaling through direct repression of its ligands betacellulin and amphiregulin. The dual function of Foxf2 during EMT is underscored by the finding that high Foxf2 expression correlates with good prognosis in patients with early noninvasive stages of breast cancer, but with poor prognosis in advanced breast cancer. CONCLUSIONS: Our data identify the transcription factor Foxf2 as one of the important regulators of EMT, displaying a dual function in promoting tumor cell apoptosis as well as tumor cell migration.

miR-130b participates in wear particle-induced inflammation and osteolysis via FOXF2/NF-κB pathway.

OBJECTIVE: To reveal other miR-130b-mediated signaling pathway in the involvement of wear particle-induced inflammation and osteolysis. MATERIALS AND METHODS: Particle-induced osteolysis (PIO) mice model was established. Secretion levels of TNF-α, IL-1ß, IL-6, and IL-10 were measured by ELISA. miR-130b and forkhead box F2 (FOXF2) mRNA were detected by qRT-PCR. Protein levels of FOXF2, phosphorylation-p65 (p-p65), and p-IκB were observed by Western blot. Luciferase reporter assay was performed to confirm the regulation of miR-130b on FOXF2. RESULTS: Compared with normal mice, secretion levels of TNF-α, IL-1ß, and IL-6 in PIO mice were significantly up-regulated and IL-10 was significantly down-regulated; miR-130b and p-p65 expressions were up-regulated and FOXF2 expression was down-regulated. In addition, the trends of miR-130b, FOXF2, and p-p65 expressions in Co-Cr-Mo treated Raw264.7 cells were the same as that in PIO mice. After transfection with miR-130b inhibitor, secretion levels of TNF-α, IL-1ß, and IL-6 in Raw264.7 cells were significantly decreased and secretion level of IL-10 was significantly increased. We also proved FOXF2 was a target of miR-130b, and FOXF2 siRNA increased secretion levels of TNF-α, IL-1ß, and IL-6 and decreased secretion level of IL-10. Finally, we found nuclear factor-kappa B (NF-κB) inhibitor BAY 11-7082 further decreased secretion levels of TNF-α, IL-1ß, and IL-6 and increased IL-10 level. CONCLUSION: The role of miR-130b/FOXF2/NF-κB pathway in PIO was firstly revealed, which provided new targets for the treatment of periprosthetic osteolysis.

FOXF2 promoter methylation is associated with prognosis in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma is a commonly malignant tumor of digestive tract with poor prognosis. Previous studies suggested that forkhead box F2 ( FOXF2) could be a candidate gene for assessing and predicting the prognosis of human cancers. However, the relationship between FOXF2 promoter methylation and the prognosis of esophageal squamous cell carcinoma remained unclear. Formalin-fixed, paraffin-embedded esophageal squamous cell carcinoma tissues of 135 esophageal squamous cell carcinoma patients were detected for FOXF2 promoter methylation status by methylation-specific polymerase chain reaction approach. DNA methylation results were evaluated with regard to clinicopathological features and overall survival. Our study confirmed that FOXF2 promoter hypermethylation could independently predict a poorer overall survival of esophageal squamous cell carcinoma patients ( p = 0.002), which was consistent with the data mining results of the data from 82 esophageal squamous cell carcinoma patients in The Cancer Genome Atlas datasets ( p = 0.036). In addition, no correlation was found between FOXF2 promoter methylation and other clinic pathological parameters (age, gender, differentiation, lymph node metastasis, stage, cutting edge, vascular invasion, smoking behavior, and drinking history). In conclusion, FOXF2 methylation might be a useful prognostic biomarker for esophageal squamous cell carcinoma patients.

MicroRNA-182 targets FOXF2 to promote the development of triple-negative breast cancer.

To explore the function of microRNA-182 (miR-182) on MCF7 and MDA-MB-231 cells behaviors, and possible mechanisms of triple-negative breast cancer (TNBC) development. Totally, 30 TNBC patients were enrolled to investigate the correlation between miR-182 expression and TNBC clinical indicators. miR-182 expression in TNBC tissues was measured by qRT-PCR, followed by bioinformatics methods and luciferase reporter assay to investigate whether FOXF2 was a direct target of miR-182. Besides, miR-182 mimics were transfected into MCF7 cells while miR-182 inhibitor into MDA-MB-231 cells, followed by cell proliferation and migration detection. miR-182 expression was significantly correlated with TNBC clinical indicators, such as lymph node metastasis TNM (stage III), intravascular cancer emboli and TNBC recurrence and metastasis. miR-182 expression was significantly higher in TNBC tissues than that in matched normal tissues, and was significantly higher in MDA-MB-231 cells than that in MCF7 cells. miR-182 knockdown inhibited the proliferation and migration of MDA-MB-231 cells while miR-182 overexpression markedly promoted the proliferation and migration of MCF7 cells. Besides, FOXF2 was identified as a direct target of miR-182. Our findings indicate that miR-182 may promote cell proliferation and migration in TNBC possible via down-regulation of FOXF2. miR-182 may serve as a potential target in TNBC treatment.

Forkhead box F2 regulation of platelet-derived growth factor and myocardin/serum response factor signaling is essential for intestinal development.

Alterations in the forkhead box F2 gene expression have been reported in numerous pathologies, and Foxf2(-/-) mice are perinatal lethal with multiple malformations; however, molecular mechanisms pertaining to Foxf2 signaling are severely lacking. In this study, Foxf2 requirements in murine smooth muscle cells were examined using a conditional knock-out approach. We generated novel Foxf2-floxed mice, which we bred to smMHC-Cre-eGFP mice to generate a mouse line with Foxf2 deleted specifically from smooth muscle. These mice exhibited growth retardation due to reduced intestinal length as well as inflammation and remodeling of the small intestine. Colons of Tg(smMHC-Cre-eGFP(+/-));Foxf2(-/-) mice had expansion of the myenteric nerve plexus and increased proliferation of smooth muscle cells leading to thickening of the longitudinal smooth muscle layer. Foxf2 deficiency in colonic smooth muscle was associated with increased expression of Foxf1, PDGFa, PDGFb, PDGF receptor α, and myocardin. FOXF2 bound to promoter regions of these genes indicating direct transcriptional regulation. Foxf2 repressed Foxf1 promoter activity in co-transfection experiments. We also show that knockdown of Foxf2 in colonic smooth muscle cells in vitro and in transgenic mice increased myocardin/serum response factor signaling and increased expression of contractile proteins. Foxf2 attenuated myocardin/serum response factor signaling in smooth muscle cells through direct binding to the N-terminal region of myocardin. Our results indicate that Foxf2 signaling in smooth muscle cells is essential for intestinal development and serum response factor signaling.

Novel evidence of association with nonsyndromic cleft lip with or without cleft palate was shown for single nucleotide polymorphisms in FOXF2 gene in an Asian population.

BACKGROUND: The forkhead box F2 gene (FOXF2) located in chromosome 6p25.3 has been shown to play a crucial role in palatal development in mouse and rat models. To date, no evidence of linkage or association has been reported for this gene in humans with oral clefts. METHODS: Allelic transmission disequilibrium tests were used to robustly assess evidence of linkage and association with nonsyndromic cleft lip with or without cleft palate for nine single nucleotide polymorphisms (SNPs) in and around FOXF2 in both Asian and European trios using PLINK. RESULTS: Statistically significant evidence of linkage and association was shown for two SNPs (rs1711968 and rs732835) in 216 Asian trios where the empiric P values with permutation tests were 0.0016 and 0.005, respectively. The corresponding estimated odds ratios for carrying the minor allele at these SNPs were 2.05 (95% confidence interval = 1.41, 2.98) and 1.77 (95% confidence interval = 1.26, 2.49), respectively. CONCLUSION: Our results provided statistical evidence of linkage and association between FOXF2 and nonsyndromic cleft lip with or without cleft palate.

Troxerutin improves cognitive function and forkhead box F2 expression in the hippocampus via modulating the microbial composition and the intestinal barrier function in diabetes mellitus mice.

Recent studies have found that gut microbes may affect blood-brain barrier (BBB) integrity. This study was to investigate the relationship between gut microbes and forkhead box F2 (FOXF2) and the mechanism of troxerutin improving diabetic cognitive dysfunction (DCD). Diabetic mice were used in this study for the prophylactic application of troxerutin (60 mg/kg/d) for 8 weeks. The cognitive function was assessed using the Morris water maze (MWM) and novel object recognition (NOR) tasks, and the changes of intestinal microbial composition were observed through 16S rRNA gene sequencing. The content of short-chain fatty acids (SCFAs) in feces was determined by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and the intestinal barrier function was assessed by enzyme-linked immunosorbent assay (ELISA) and western blotting. Troxerutin up-regulated FOXF2 expression in the hippocampus of mice, improving DCD. Meanwhile, it reversed the intestinal microbial composition (increased the abundance of the phylum Bacteroidota, as well as fecal propionic acid and butyric acid levels) and improved the intestinal barrier (increased the level of claudin-1 and significantly reduced the circulating lipopolysaccharide binding protein (LBP) levels). When intestinal microorganisms were removed with an antibiotic cocktail, the improvement of hippocampal FOXF2 expression and DCD by troxerutin attenuated accordingly, suggesting that troxerutin improved DCD by up-regulating the expression of hippocampal FOXF2 through the regulation of intestinal microbial composition and the intestinal barrier. In summary, troxerutin improved DCD by up-regulating the expression of hippocampal FOXF2 through the regulation of intestinal microbial composition and the intestinal barrier.

FOXF2 Regulates PRUNE2 Transcription in the Pathogenesis of Colorectal Cancer.

Background: Forkhead box F2, a member of the Forkhead box transcription factor superfamily, plays an important role in several types of cancer. However, the mechanisms of Forkhead box F2 in the progression of colorectal cancer remain unclear. PRUNE2 is closely associated with prostate cancer, neuroblastoma, glioblastoma, and melanoma. The relationship between Forkhead box F2 and PRUNE2 in colorectal cancer remains unknown. Method: We investigated the effects of Forkhead box F2 upregulation on colorectal cancer cell behavior in vitro using Cell Counting Kit-8, colony formation, flow cytometry, Transwell, reverse transcription quantitative polymerase chain reaction and Western blot analyses. Nude mouse xenografts were established to investigate the effect of Forkhead box F2 upregulation on the growth of colorectal cancer cells. Dual-luciferase reporter assays were performed to confirm the Forkhead box F2 regulation of PRUNE2 transcription. A series of in vitro assays was performed in cells with Forkhead box F2 upregulation and PRUNE2 knockdown to elucidate the function and regulatory effects of Forkhead box F2 on PRUNE2 transcription in colorectal cancer. Results: Forkhead box F2 was downregulated in colorectal cancer tissues compared with adjacent tissues. Forkhead box F2 overexpression significantly suppressed the proliferation and invasion of colorectal cancer cells in vitro and in vivo. Moreover, Forkhead box F2 directly targeted PRUNE2 to promote its transcription in colorectal cancer cells. Furthermore, PRUNE2 mediated the Forkhead box F2-regulated proliferation and invasion of colorectal cancer cells. Additionally, we demonstrated a significant positive correlation between Forkhead box F2 and PRUNE2 mRNA levels in colorectal cancer tissues. Conclusion: These results indicated that Forkhead box F2 and PRUNE2 in combination may serve as a prognostic biomarker for colorectal cancer and that Forkhead box F2 upregulation inhibits the proliferation and invasion of colorectal cancer cells by upregulating PRUNE2.

Aberrant DNA Methylation-Mediated FOXF2 Dysregulation Is a Prognostic Risk Factor for Gastric Cancer.

Background: The prognosis of gastric cancer (GC) patients is poor. The effect of aberrant DNA methylation on FOXF2 expression and the prognostic role of FOXF2 methylation in GC have not yet been identified. Methods: The RNA-Seq and gene methylation HM450 profile data were used for analyzing FOXF2 expression in GC and its association with methylation level. Bisulfite sequencing PCR (BSP) was performed to measure the methylation level of the FOXF2 promoter region in GC cell lines and normal GES-1 cells. The cells were treated with the demethylation reagent 5-Aza-dC, and the mRNA and protein expression levels of FOXF2 were then measured by qRT-PCR and western blot assays. The risk score system from SurvivalMeth was calculated by integrating the methylation level of the cg locus and the corresponding Cox regression coefficient. Results: FOXF2 was significantly downregulated in GC cells and tissues. On the basis of RNA-Seq and Illumina methylation 450 data, FOXF2 expression was significantly negatively correlated with the FOXF2 methylation level (Pearson's R = -0.42, p < 2.2e-16). The FOXF2 methylation level in the high FOXF2 expression group was lower than that in the low FOXF2 expression group. The BSP assay indicated that the methylation level of the FOXF2 promoter region in GC cell lines was higher than that in GES-1 cells. The qRT-PCR and western blot assay showed that FOXF2 mRNA and protein levels were increased in GC cells following treatment with 5-Aza-Dc. The methylation risk score model indicated that patients in the high risk group had poorer survival probability than those in the low risk group (HR = 1.84 (1.11-3.07) and p = 0.0068). FOXF2 also had a close transcriptional regulation network with four miRNAs and their corresponding target genes. Functional enrichment analysis of the target genes revealed that these genes were significantly related to several important signaling pathways. Conclusion: FOXF2 was downregulated due to aberrant DNA methylation in GC, and the degree of methylation in the promoter region of FOXF2 was related to the prognosis of patients. The FOXF2/miRNAs/target genes axis may play a vital biological regulation role in GC.

CircGDI2 Regulates the Proliferation, Migration, Invasion and Apoptosis of OSCC via miR-454-3p/FOXF2 Axis.

BACKGROUND: Aberrant expression of circular RNA (circRNA) is involved in the occurrence and development of multifarious cancers, including oral squamous cell carcinoma (OSCC). However, the biological role of circGDI2 and the action mechanism in OSCC remain largely unclear. METHODS: The expression levels of circGDI2, miR-454-3p and forkhead box F2 (FOXF2) were examined by quantitative real-time PCR (qRT-PCR) or Western blot. The stability of circGDI2 was confirmed by Ribonuclease R (RNase R) assay. Cell Counting Kit 8 (CCK8) assay, colony formation and transwell assay were used to detect cell proliferation, migration or invasion. Cell apoptosis was tested by flow cytometry. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were employed to verify the interaction between miR-454-3p and circGDI2 or FOXF2. Moreover, xenograft mouse models were constructed to assess tumor growth in vivo. RESULTS: CircGDI2 was a stable circRNA and was low expressed in OSCC tissues and cells. CircGDI2 overexpression could effectively inhibit the proliferation, migration, invasion and promote apoptosis in OSCC cells, and suppress OSCC tumor growth in nude mice in vivo. MiR-454-3p could be sponged by circGDI2, and its overexpression could mitigate the suppressive effects of circGDI2 overexpression on OSCC progression. In addition, FOXF2 was a target of miR-454-3p, and miR-454-3p silence could impede the cell growth of OSCC cells by enhancing FOXF2 expression. Meanwhile, circGDI2 positively regulated FOXF2 expression by targeting miR-454-3p. CONCLUSION: CircGDI2 served as a repressor to restrain OSCC malignancy via miR-454-3p/FOXF2 axis, which might be a novel biomarker for targeted OSCC therapy.

The regulatory roles and mechanisms of the transcription factor FOXF2 in human diseases.

Many studies have focused on the relationship between transcription factors and a variety of common pathological conditions, such as diabetes, stroke, and cancer. It has been found that abnormal transcription factor regulation can lead to aberrant expression of downstream genes, which contributes to the occurrence and development of many diseases. The forkhead box (FOX) transcription factor family is encoded by the FOX gene, which mediates gene transcription and follow-up functions during physiological and pathological processes. FOXF2, a member of the FOX transcription family, is expressed in various organs and tissues while maintaining their normal structural and functional development during the embryonic and adult stages. Multiple regulatory pathways that regulate FOXF2 may also be controlled by FOXF2. Abnormal FOXF2 expression induced by uncontrollable regulatory signals mediate the progression of human diseases by interfering with the cell cycle, proliferation, differentiation, invasion, and metastasis. FOXF2 manipulates downstream pathways and targets as both a pro-oncogenic and anti-oncogenic factor across different types of cancer, suggesting it may be a new potential clinical marker or therapeutic target for cancer. However, FOXF2's biological functions and specific roles in cancer development remain unclear. In this study, we provide an overview of FOXF2's structure, function, and regulatory mechanisms in the physiological and pathological conditions of human body. We also discussed the possible reasons why FOXF2 performs the opposite function in the same types of cancer.

Cis-Repression of Foxq1 Expression Affects Foxf2-Mediated Gene Expression in Palate Development.

Disruption of FOXF2, encoding a member of the Forkhead family transcription factors, has been associated with cleft palate in humans and mice. FOXF2 is located in a conserved gene cluster containing FOXQ1, FOXF2, and FOXC1. We found that expression of Foxq1 is dramatically upregulated in the embryonic palatal mesenchyme in Foxf2 -/- mouse embryos. We show here that the Foxf2 promoter-deletion mutation caused dramatically increased expression of the cis-linked Foxq1 allele but had little effect on the Foxq1 allele in trans. We analyzed effects of the Foxf2 mutation on the expression of other neighboring genes and compared those effects with the chromatin domain structure and recently identified enhancer-promoter associations as well as H3K27ac ChIP-seq data. We show that the Foxf2 mutation resulted in significantly increased expression of the Foxq1 and Exoc2 genes located in the same topologically associated domain with Foxf2 but not the expression of the Foxc1 and Gmds genes located in the adjacent chromatin domain. We inactivated the Foxq1 gene in mice homozygous for a Foxf2 conditional allele using CRISPR genome editing and generated (Foxf2/Foxq1)+/- mice with loss-of-function mutations in Foxf2 and Foxq1 in cis. Whereas the (Foxf2/Foxq1)-/- mice exhibited cleft palate at birth similar as in the Foxf2 -/- mice, systematic expression analyses of a large number of Foxf2-dependent genes revealed that the (Foxf2/Foxq1)-/- embryos exhibited distinct effects on the domain-specific expression of several important genes, including Foxf1, Shox2, and Spon1, in the developing palatal shelves compared with Foxf2 -/- embryos. These results identify a novel cis-regulatory effect of the Foxf2 mutation and demonstrate that cis-regulation of Foxq1 contributed to alterations in palatal gene expression in Foxf2 -/- embryos. These results have important implications for interpretation of results and mechanisms from studies of promoter- or gene-deletion alleles. In addition, the unique mouse lines generated in this study provide a valuable resource for understanding the cross-regulation and combinatorial functions of the Foxf2 and Foxq1 genes in development and disease.

Unbiased identification of novel transcription factors in striatal compartmentation and striosome maturation.

Many diseases are linked to dysregulation of the striatum. Striatal function depends on neuronal compartmentation into striosomes and matrix. Striatal projection neurons are GABAergic medium spiny neurons (MSNs), subtyped by selective expression of receptors, neuropeptides, and other gene families. Neurogenesis of the striosome and matrix occurs in separate waves, but the factors regulating compartmentation and neuronal differentiation are largely unidentified. We performed RNA- and ATAC-seq on sorted striosome and matrix cells at postnatal day 3, using the Nr4a1-EGFP striosome reporter mouse. Focusing on the striosome, we validated the localization and/or role of Irx1, Foxf2, Olig2, and Stat1/2 in the developing striosome and the in vivo enhancer function of a striosome-specific open chromatin region 4.4 Kb downstream of Olig2. These data provide novel tools to dissect and manipulate the networks regulating MSN compartmentation and differentiation, including in human iPSC-derived striatal neurons for disease modeling and drug discovery.

si-SNHG5-FOXF2 inhibits TGF-ß1-induced fibrosis in human primary endometrial stromal cells by the Wnt/ß-catenin signalling pathway.

BACKGROUND: Intrauterine adhesions (IUAs) are manifestations of endometrial fibrosis characterized by inflammation and fibrinogen aggregation in the extracellular matrix (ECM). The available therapeutic interventions for IUA are insufficiently effective in the clinical setting for postoperative adhesion recurrence and infertility problems. In this study, we investigated whether si-SNHG5-FOXF2 can serve as a molecular mechanism for the inhibition of IUA fibrosis ex vivo. METHODS: FOXF2, TGF-ß1 and collagen expression levels were measured by microarray sequencing analysis in three normal endometrium groups and six IUA patients. We induced primary human endometrial stromal cells (HESCs) into myofibroblasts (MFs) to develop an IUA cell model with various concentrations of TGF-ß1 at various times. Downstream target genes of FOXF2 were screened by chromatin immunoprecipitation combined with whole-genome high-throughput sequencing (ChIP-seq). We investigated ECM formation, cell proliferation and Wnt/ß-catenin signalling pathway-related proteins in primary HESCs with FOXF2 downregulation by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), western blotting (WB), immunohistochemistry (IHC), flow cytometry, ethylenediurea (EdU) and CCK8 assays. We identified long noncoding RNAs (lncRNA) SNHG5 as the upstream regulatory gene of FOXF2 through RNA immunoprecipitation (RIP), RNA pulldown and fluorescence in situ hybridization (FISH). Finally, we examined FOXF2 expression, ECM formation, cell proliferation and Wnt/ß-catenin signalling pathway-related proteins in primary HESCs upon FOXF2 downregulation. RESULTS: FOXF2 was highly expressed in the endometrium of patients with IUA. Treatment of primary HESCs with 10 ng/ml TGF-ß1 for 72 h was found to be most effective for developing an IUA cell model. FOXF2 regulated multiple downstream target genes, including collagen, vimentin (VIM) and cyclin D2/DK4, by ChIP-seq and ChIP-PCR. FOXF2 downregulation inhibited TGF-ß1-mediated primary HESC fibrosis, including ECM formation, cell proliferation and Wnt/ß-catenin signalling pathway-related protein expression. We identified lncRNA SNHG5 as an upstream gene that directly regulates FOXF2 by RIP-seq, qRT-PCR, WB and FISH. SNHG5 downregulation suppressed FOXF2 expression in the IUA cell model, resulting in synergistic repression of the Wnt/ß-catenin pathway, thereby altering TGF-ß1-mediated ECM aggregation in endometrial stromal cells ex vivo. CONCLUSIONS: Regulation of the Wnt/ß-catenin signalling pathway and ECM formation by si-SNHG5-FOXF2 effectively inhibited the profibrotic effect of TGF-ß1 on primary HESCs. This finding can provide a molecular basis for antagonizing TGF-ß1-mediated fibrosis in primary HESCs.