USP22 is required for human endometrial stromal cell proliferation and decidualization by deubiquitinating FoxM1.

Despite significant advances in assisted reproductive technology (ART), recurrent implantation failure (RIF) still occurs in some patients. Poor endometrial receptivity and abnormal human endometrial stromal cell (HESC) proliferation and decidualization have been identified as the major causes. Ubiquitin-specific protease 22 (USP22) has been reported to participate in the decidualization of endometrial stromal cells in mice. However, the role of USP22 in HESC function and RIF development remains unknown. In this study, clinical endometrial tissue samples were gathered to investigate the involvement of USP22 in RIF, and HESCs were utilized to examine the molecular mechanisms of USP22 and Forkhead box M1 (FoxM1). The findings indicated a high expression of USP22 in the secretory phase of the endometrium. Knockdown of USP22 led to a notable reduction in the proliferation and decidualization of HESCs, along with a decrease in FoxM1 expression, while overexpression of USP22 yielded opposite results. Furthermore, USP22 was found to deubiquitinate FoxM1 in HESCs. Moreover, both USP22 and FoxM1 were downregulated in the endometria of patients with RIF. In conclusion, these results suggest that USP22 may have a significant impact on HESCs proliferation and decidualization through its interaction with FoxM1, potentially contributing to the underlying mechanisms of RIF pathogenesis.

ß-Sitosterol suppresses hepatocellular carcinoma growth and metastasis via FOXM1-regulated Wnt/ß-catenin pathway.

ß-Sitosterol is a natural compound with demonstrated anti-cancer properties against various cancers. However, its effects on hepatocellular carcinoma (HCC) and the underlying mechanisms are not well understood. This study aims to investigate the impact of ß-sitosterol on HCC. In this study, we investigated the effects of ß-sitosterol on HCC tumour growth and metastasis using a xenograft mouse model and a range of molecular analyses, including bioinformatics, real-time PCR, western blotting, lentivirus transfection, CCK8, scratch and transwell assays. The results found that ß-sitosterol significantly inhibits HepG2 cell proliferation, migration and invasion both in vitro and in vivo. Bioinformatics analysis identifies forkhead box M1 (FOXM1) as a potential target for ß-sitosterol in HCC treatment. FOXM1 is upregulated in HCC tissues and cell lines, correlating with poor prognosis in patients. ß-Sitosterol downregulates FOXM1 expression in vitro and in vivo. FOXM1 overexpression mitigates ß-sitosterol's inhibitory effects on HepG2 cells. Additionally, ß-sitosterol suppresses epithelial-mesenchymal transition (EMT) in HepG2 cells, while FOXM1 overexpression promotes EMT. Mechanistically, ß-sitosterol inhibits Wnt/ß-catenin signalling by downregulating FOXM1, regulating target gene transcription related to HepG2 cell proliferation and metastasis. ß-Sitosterol shows promising potential as a therapeutic candidate for inhibiting HCC growth and metastasis through FOXM1 downregulation and Wnt/ß-catenin signalling inhibition.

Comprehensive clinicopathological significance and putative transcriptional mechanisms of Forkhead box M1 factor in hepatocellular carcinoma.

BACKGROUND: The Forkhead box M1 factor (FOXM1) is a crucial activator for cancer cell proliferation. While FOXM1 has been shown to promote hepatocellular carcinoma (HCC) progression, its transcriptional mechanisms remain incompletely understood. METHODS: We performed an in-house tissue microarray on 313 HCC and 37 non-HCC tissue samples, followed by immunohistochemical staining. Gene chips and high throughput sequencing data were used to assess FOXM1 expression and prognosis. To identify candidate targets of FOXM1, we comprehensively reanalyzed 41 chromatin immunoprecipitation followed by sequencing (ChIP-seq) data sets. We predicted FOXM1 transcriptional targets in HCC by intersecting candidate FOXM1 targets with HCC overexpressed genes and FOXM1 correlation genes. Enrichment analysis was employed to address the potential mechanisms of FOXM1 underlying HCC. Finally, single-cell RNA sequencing analysis was performed to confirm the transcriptional activity of FOXM1 on its predicted targets. RESULTS: This study, based on 4235 HCC tissue samples and 3461 non-HCC tissue samples, confirmed the upregulation of FOXM1 in HCC at mRNA and protein levels (standardized mean difference = 1.70 [1.42, 1.98]), making it the largest multi-centered study to do so. Among HCC patients, FOXM1 was increased in Asian and advanced subgroups, and high expression of FOXM1 had a strong ability to differentiate HCC tissue from non-HCC tissue (area under the curve = 0.94, sensitivity = 88.72%, specificity = 87.24%). FOXM1 was also shown to be an independent exposure risk factor for HCC, with a pooled hazard ratio of 2.00 [1.77, 2.26]. The predicted transcriptional targets of FOXM1 in HCC were predominantly enriched in nuclear division, chromosomal region, and catalytic activity acting on DNA. A gene cluster encoding nine transcriptional factors was predicted to be positively regulated by FOXM1, promoting the cell cycle signaling pathway in HCC. Finally, the transcriptional activity of FOXM1 and its targets was supported by single-cell analysis of HCC cells. CONCLUSIONS: This study not only confirmed the upregulation of FOXM1 in HCC but also identified it as an independent risk factor. Moreover, our findings enriched our understanding of the complex transcriptional mechanisms underlying HCC pathogenesis, with FOXM1 potentially promoting HCC progression by activating other transcription factors within the cell cycle pathway.

[Forkhead Box M1 Regulates the Proliferation,Invasion,and Drug Resistance of Gastric Cancer Cells via circ_NOTCH1].

Objective To investigate the impacts of forkhead box M1(FOXM1)on the proliferation,invasion,and drug resistance of gastric cancer cells by regulating the circular RNA circ_NOTCH1.Methods Western blotting and real-time quantitative PCR were performed to determine the expression of FOXM1 protein and circ_NOTCH1,respectively,in the gastric cancer tissue,para-carcinoma tissue,human normal gastric mucosa epithelial cell line GES-1 and gastric cancer cell lines MGC-803,HGC-27,and BGC-823.BGC-823 cells were classified into the following groups:control,short hairpin RNA FOXM1(sh-FOXM1)and negative control(sh-NC),small interfering RNA circ_NOTCH1(si-circ_NOTCH1)and negative control(si-NC),and sh-FOXM1+circ_NOTCH1 overexpression plasmid(sh-FOXM1+pcDNA-circ_NOTCH1)and sh-FOXM1+negative control(sh-FOXM1+pcDNA).CCK-8 assay and clone formation assay were employed to measure the cell proliferation,and Transwell assay to measure cell invasion.After treatment with 1.0 mg/L adriamycin for 48 h,the cell resistance in each group was analyzed.Western blotting was employed to determine the expression levels of FOXM1,proliferating cell nuclear antigen(PCNA),Bax,multi-drug resistance-associated protein 1(MRP1),and multi-drug resistance gene 1(MDR1).RNA pull-down and RNA immunoprecipitation were employed to examine the binding of circ_NOTCH1 to FOXM1 protein.Results Compared with those in the para-carcinoma tissue,the expression levels of FOXM1 protein and circ_NOTCH1 in the gastric cancer tissue were up-regulated(all P<0.001).Compared with GES-1 cells,MGC-803,HGC-27,and BGC-823 cells showed up-regulated expression levels of FOXM1 protein and circ_NOTCH1(all P<0.001).Compared with the control group and sh-NC group,the sh-FOXM1 group with down-regulated expression of FOXM1 protein and circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with the control group and the si-NC group,the si-circ_NOTCH1 group with down-regulated expression of circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with sh-FOXM1 group and sh-FOXM1+pcDNA group,the sh-FOXM1+pcDNA-circ_NOTCH1 group with up-regulated expression of circ_NOTCH1 showed increased optical density value,clone formation rate,cell invasion number,and cell viability,up-regulated expression of PCNA,MRP1,and MDR1,and down-regulated expression of Bax protein(all P<0.001).FOXM1 protein was able to interact with circ_NOTCH1.Conclusion Interference with FOXM1 may inhibit the proliferation,invasion,and drug resistance of gastric cancer cells by silencing circ_NOTCH1 expression.

Clinical implications of forkhead box M1, cyclooxygenase-2, and glucose-regulated protein 78 in breast invasive ductal carcinoma.

BACKGROUND: Breast infiltrating ductal carcinoma (BIDC) represents the largest heterotypic tumor group, and an in-depth understanding of the pathogenesis of BIDC is key to improving its prognosis. AIM: To analyze the expression profiles and clinical implications of forkhead box M1 (FOXM1), cyclooxygenase-2 (COX-2), and glucose-regulated protein 78 (GRP78) in BIDC. METHODS: A total of 65 BIDC patients and 70 healthy controls who presented to our hospital between August 2019 and May 2021 were selected for analysis. The peripheral blood FOXM1, COX-2, and GRP78 levels in both groups were measured and the association between their expression profiles in BIDC was examined. Additionally, we investigated the diagnostic value of FOXM1, COX-2, and GRP78 in patients with BIDC and their correlations with clinicopathological features. Furthermore, BIDC patients were followed for 1 year to identify factors influencing patient prognosis. RESULTS: The levels of FOXM1, COX-2, and GRP78 were significantly higher in BIDC patients compared to healthy controls (P < 0.05), and a positive correlation was observed among them (P < 0.05). Receiver operating characteristic analysis demonstrated that FOXM1, COX-2, and GRP78 had excellent diagnostic value in predicting the occurrence of BIDC (P < 0.05). Subsequently, we found significant differences in FOXM1, COX-2, and GRP78 levels among patients with different histological grades and metastasis statuses (with vs without) (P < 0.05). Cox analysis revealed that FOXM1, COX-2, GRP78, increased histological grade, and the presence of tumor metastasis were independent risk factors for prognostic death in BIDC (P < 0.001). CONCLUSION: FOXM1, COX-2, and GRP78 exhibit abnormally high expression in BIDC, promoting malignant tumor development and closely correlating with prognosis. These findings hold significant research implications for the future diagnosis and treatment of BIDC.

Tissue transcriptome profiling and pathway analyses revealed novel potential biomarkers in the tumor progression of canine oral melanoma.

Canine oral melanoma (COM) is an aggressive oral malignancy in dogs, mostly with metastasis. However, the understanding of total gene expression of oral melanoma (OM) at different clinical stages has been limited. The objective of this study was to identify novel mRNA biomarkers of early-stage OM (EOM) and late-stage OM (LOM). Transcriptome sequencing of 3 EOM, 5 LOM and 4 normal gingival tissues (controls) was performed. Selected transcriptome results were validated by quantitative reverse transcription-PCR (qRT-PCR) using 12 LOM and 10 controls. We found 534 differentially expressed in EOM compared with controls, whereas 696 genes in LOM were differentially expressed compared with controls (P < 0.05). Moreover, 27 genes were differentially expressed in LOM compared with EOM (P < 0.05). The genes expressed in COM were involved in the molecular mechanism of cancer and melanocyte development pathways, promoting melanoma progression. qRT-PCR confirmed an increased expression of genes encoding an important protein in chemotherapy resistance (dopachrome tautomerase, DCT) and tumor progression (forkhead box M1, FOXM1), and decreased expression of a tumor suppression gene (N-myc downstream-regulated gene 2, NDRG2) in LOM, concordant with transcriptome results. In conclusion, this study revealed the comprehensive transcriptome from COM tissues, and increased DCT and FOXM1 and decreased NDRG2 gene expression indicated the potential candidate biomarkers in COM progression.

Role of transcription factor FOXM1 in diabetes and its complications (Review).

Diabetes mellitus is a chronic metabolic disease commonly associated with complications such as cardiovascular disease, nephropathy and neuropathy, the incidence of which is increasing yearly. Transcription factor forkhead box M1 (FOXM1) serves an important role in development of diabetes and its complications. The present study aimed to review the association between FOXM1 with pathogenesis of diabetes and its complications. FOXM1 may be involved in development and progression of diabetes and its complications by regulating cell biological processes such as cell cycle, DNA damage repair, cell differentiation and epithelial­mesenchymal transition. FOXM1 is involved in regulation of insulin secretion and insulin resistance, and FOXM1 affects insulin secretion by regulating expression of insulin­related genes and signaling pathways; FOXM1 is involved in the inflammatory response in diabetes, and FOXM1 can regulate key genes associated with inflammatory response and immune cells, which in turn affects occurrence and development of the inflammatory response; finally, FOXM1 is involved in the regulation of diabetic complications such as cardiovascular disease, nephropathy and neuropathy. In summary, the transcription factor FOXM1 serves an important role in development of diabetes and its complications. Future studies should explore the mechanism of FOXM1 in diabetes and find new targets of FOXM1 as a potential treatment for diabetes and its complications.

Overexpressed FOXM1 collaborates with MMB to increase WEE1 inhibitor sensitivity in NSCLC.

Background: Non-small cell lung cancer (NSCLC) is a common lung tumor with high mortality. The complex formed by MYB-MuvB complex (MMB) and forkhead box M1 (FOXM1) (MMB-FOXM1) plays a vital role in cell cycle progression to affect the progression of diseases. The role of the FOXM1-MMB complex in Wee1-like protein kinase (WEE1) inhibitor sensitivity in NSCLC keeps unclear. Methods: The reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed to measure the mRNA levels of FOXM1, LIN54, Replication Protein A (RPA), gammaH2AX (γH2AX) and Cyclin B (CCNB). The western blot was performed to examine the corresponding protein expressions. The Cell Counting Kit-8 (CCK-8) assay was performed to test cell survival. Result: It was demonstrated that after AZD-1775 treatment, the decrease in cell survival mediated by FOXM1 overexpression (P<0.001) could be reversed by LIN54 knockdown (P<0.01) and that cell survival in the control group did not differ obviously from that in the pcDNA3.1-FOXM1+siLIN54 group, indicating that the FOXM1-MMB complex was necessary for WEE1 inhibitor sensitivity. Moreover, the mRNA and protein expression levels of RPA and γH2AX were increased after AZD-1775 treatment and FOXM1 overexpression (P<0.01), suggesting that FOXM1 upregulation enhanced DNA replication stress and DNA damage. Finally, we found that the increases in the mRNA and protein expression levels of CCNB mediated by FOXM1 (P<0.01) could be rescued by silencing LIN54 (P<0.001) and that CCNB expression in the control group did not differ obviously from that in the pcDNA3.1-FOXM1+siLIN54 group. These findings revealed that the FOXM1-MMB complex activated G2/M checkpoints. In our work, it was discovered that FOXM1 overexpression increased DNA replication stress, which increased DNA replication and pressure on the WEE1 checkpoint. On the other hand, FOXM1 can enhance CCNB expression, increase the threshold content of the CCNB/CDK1 complex, facilitate mitosis, and promote WEE1 dephosphorylation. Under these two conditions, sensitivity to the WEE1 inhibitor AZD-1775 is increased, which leads to the accumulation of DNA damage and drives the activation of apoptosis. Conclusions: Overexpressed FOXM1 collaborates with MMB to increase WEE1 inhibitor sensitivity in NSCLC. This discovery might highlight the regulatory function of FOXM1/MMB in the treatment of NSCLC patients.

α-catenin interaction with YAP/FoxM1/TEAD-induced CEP55 supports liver cancer cell migration.

BACKGROUND: Adherens junctions (AJs) facilitate cell-cell contact and contribute to cellular communication as well as signaling under physiological and pathological conditions. Aberrant expression of AJ proteins is frequently observed in human cancers; however, how these factors contribute to tumorigenesis is poorly understood. In addition, for some factors such as α-catenin contradicting data has been described. In this study we aim to decipher how the AJ constituent α-catenin contributes to liver cancer formation. METHODS: TCGA data was used to detect transcript changes in 23 human tumor types. For the detection of proteins, liver cancer tissue microarrays were analyzed by immunohistochemistry. Liver cancer cell lines (HLF, Hep3B, HepG2) were used for viability, proliferation, and migration analyses after RNAinterference-mediated gene silencing. To investigate the tumor initiating potential, vectors coding for α-catenin and myristoylated AKT were injected in mice by hydrodynamic gene delivery. A BioID assay combined with mass spectrometry was performed to identify α-catenin binding partners. Results were confirmed by proximity ligation and co-immunoprecipitation assays. Binding of transcriptional regulators at gene promoters was investigated using chromatin-immunoprecipitation. RESULTS: α-catenin mRNA was significantly reduced in many human malignancies (e.g., colon adenocarcinoma). In contrast, elevated α-catenin expression in other cancer entities was associated with poor clinical outcome (e.g., for hepatocellular carcinoma; HCC). In HCC cells, α-catenin was detectable at the membrane as well as cytoplasm where it supported tumor cell proliferation and migration. In vivo, α-catenin facilitated moderate oncogenic properties in conjunction with AKT overexpression. Cytokinesis regulator centrosomal protein 55 (CEP55) was identified as a novel α-catenin-binding protein in the cytoplasm of HCC cells. The physical interaction between α-catenin and CEP55 was associated with CEP55 stabilization. CEP55 was highly expressed in human HCC tissues and its overexpression correlated with poor overall survival and cancer recurrence. Next to the α-catenin-dependent protein stabilization, CEP55 was transcriptionally induced by a complex consisting of TEA domain transcription factors (TEADs), forkhead box M1 (FoxM1), and yes-associated protein (YAP). Surprisingly, CEP55 did not affect HCC cell proliferation but significantly supported migration in conjunction with α-catenin. CONCLUSION: Migration-supporting CEP55 is induced by two independent mechanisms in HCC cells: stabilization through interaction with the AJ protein α-catenin and transcriptional activation via the FoxM1/TEAD/YAP complex.

Cell­cell contact in epithelial cells is important for cell polarity, cellular compartmentalisation, as well as tissue architecture during development, homeostasis, and regeneration of adult tissues in metazoans. In this context, adherens junctions (AJs) mechanically sense cell contact information with direct impact on cytoskeletal remodelling, the regulation of signalling pathways, and eventually cell biology. Indeed, the loss of cell­cell contact and cellular polarity are key features in human carcinogenesis and important pathological parameters for the identification of many epithelial tumors.We demonstrate in this study, that overexpression of the AJ constituent α­catenin is frequently observed in human hepatocellular carcinoma (HCC). α­catenin supports HCC cell proliferation and migration. Together with the oncogene AKT, α­catenin moderately facilitates tumor initiation in mouse livers. Using mass spectrometry, we identified several new α­catenin interaction partners in the cytosol of liver cancer cells, including the cytokinesis regulator centrosomal protein 55 (CEP55). CEP55 mediates pro-migratory effects and its overexpression in HCC cells is controlled by two molecular mechanisms: α­catenin-dependent protein stabilization and transcriptional induction by the TEA domain transcription factors (TEADs)/forkhead box M1 (FoxM1)/yes-associated protein (YAP) complex.In summary, we here describe a new mechanism how changes in cell­cell contact support liver cancer formation and progression. This study demonstrates that dysregulation of the AJ component α­catenin contributes to liver carcinogenesis via distinct molecular mechanisms. Video Abstract.

FOXM1-Mediated Regulation of Reactive Oxygen Species and Radioresistance in Oral Squamous Cell Carcinoma Cells.

Radioresistance is a major obstacle to the successful treatment of oral squamous cell carcinoma (OSCC). To help overcome this issue, we have developed clinically relevant radioresistant (CRR) cell lines generated by irradiating parental cells over time, which are useful for OSCC research. In the present study, we conducted gene expression analysis using CRR cells and their parental lines to investigate the regulation of radioresistance in OSCC cells. Based on gene expression changes over time in CRR cells and parental lines subjected to irradiation, forkhead box M1 (FOXM1) was selected for further analysis in terms of its expression in OSCC cell lines, including CRR cell lines and clinical specimens. We suppressed or upregulated the expression of FOXM1 in OSCC cell lines, including CRR cell lines, and examined radiosensitivity, DNA damage, and cell viability under various conditions. The molecular network regulating radiotolerance was also investigated, especially the redox pathway, and the radiosensitizing effect of FOXM1 inhibitors was examined as a potential therapeutic application. We found that FOXM1 was not expressed in normal human keratinocytes but was expressed in several OSCC cell lines. The expression of FOXM1 was upregulated in CRR cells compared with that detected in the parental cell lines. In a xenograft model and clinical specimens, FOXM1 expression was upregulated in cells that survived irradiation. FOXM1-specific small interfering RNA (siRNA) treatment increased radiosensitivity, whereas FOXM1 overexpression decreased radiosensitivity, and DNA damage was altered significantly under both conditions, as well as the levels of redox-related molecules and reactive oxygen species production. Treatment with the FOXM1 inhibitor thiostrepton had a radiosensitizing effect and overcame radiotolerance in CRR cells. According to these results, the FOXM1-mediated regulation of reactive oxygen species could be a novel therapeutic target for the treatment of radioresistant OSCC; thus, treatment strategies targeting this axis might overcome radioresistance in this disease.

Forkhead Box M1 Regulates the Proliferation,Invasion,and Drug Resistance of Gastric Cancer Cells via circ_NOTCH1 / 中国医学科学院学报

Objective To investigate the impacts of forkhead box M1(FOXM1)on the proliferation,invasion,and drug resistance of gastric cancer cells by regulating the circular RNA circ_NOTCH1.Methods Western blotting and real-time quantitative PCR were performed to determine the expression of FOXM1 protein and circ_NOTCH1,respectively,in the gastric cancer tissue,para-carcinoma tissue,human normal gastric mucosa epithelial cell line GES-1 and gastric cancer cell lines MGC-803,HGC-27,and BGC-823.BGC-823 cells were classified into the following groups:control,short hairpin RNA FOXM1(sh-FOXM1)and negative control(sh-NC),small interfering RNA circ_NOTCH1(si-circ_NOTCH1)and negative control(si-NC),and sh-FOXM1+circ_NOTCH1 overexpression plasmid(sh-FOXM1+pcDNA-circ_NOTCH1)and sh-FOXM1+negative control(sh-FOXM1+pcDNA).CCK-8 assay and clone formation assay were employed to measure the cell proliferation,and Transwell assay to measure cell invasion.After treatment with 1.0 mg/L adriamycin for 48 h,the cell resistance in each group was analyzed.Western blotting was employed to determine the expression levels of FOXM1,proliferating cell nuclear antigen(PCNA),Bax,multi-drug resistance-associated protein 1(MRP1),and multi-drug resistance gene 1(MDR1).RNA pull-down and RNA immunoprecipitation were employed to examine the binding of circ_NOTCH1 to FOXM1 protein.Results Compared with those in the para-carcinoma tissue,the expression levels of FOXM1 protein and circ_NOTCH1 in the gastric cancer tissue were up-regulated(all P<0.001).Compared with GES-1 cells,MGC-803,HGC-27,and BGC-823 cells showed up-regulated expression levels of FOXM1 protein and circ_NOTCH1(all P<0.001).Compared with the control group and sh-NC group,the sh-FOXM1 group with down-regulated expression of FOXM1 protein and circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with the control group and the si-NC group,the si-circ_NOTCH1 group with down-regulated expression of circ_NOTCH1 showed decreased optical density value,clone formation rate,cell invasion number,and cell viability,down-regulated expression of PCNA,MRP1,and MDR1,and up-regulated expression of Bax protein in BGC-823 cells(all P<0.001).Compared with sh-FOXM1 group and sh-FOXM1+pcDNA group,the sh-FOXM1+pcDNA-circ_NOTCH1 group with up-regulated expression of circ_NOTCH1 showed increased optical density value,clone formation rate,cell invasion number,and cell viability,up-regulated expression of PCNA,MRP1,and MDR1,and down-regulated expression of Bax protein(all P<0.001).FOXM1 protein was able to interact with circ_NOTCH1.Conclusion Interference with FOXM1 may inhibit the proliferation,invasion,and drug resistance of gastric cancer cells by silencing circ_NOTCH1 expression.

PVECs-Derived Exosomal microRNAs Regulate PASMCs via FoxM1 Signaling in IUGR-induced Pulmonary Hypertension.

Background Intrauterine growth restriction (IUGR) is closely related to systemic or pulmonary hypertension (PH) in adulthood. Aberrant crosstalk between pulmonary vascular endothelial cells (PVECs) and pulmonary arterial smooth muscle cells (PASMCs) that is mediated by exosomes plays an essential role in the progression of PH. FoxM1 (Forkhead box M1) is a key transcription factor that governs many important biological processes. Methods and Results IUGR-induced PH rat models were established. Transwell plates were used to coculture PVECs and PASMCs. Exosomes were isolated from PVEC-derived medium, and a microRNA (miRNA) screening was proceeded to identify effects of IUGR on small RNAs enclosed within exosomes. Dual-Luciferase assay was performed to validate the predicted binding sites of miRNAs on FoxM1 3' untranslated region. FoxM1 inhibitor thiostrepton was used in IUGR-induced PH rats. In this study, we found that FoxM1 expression was remarkably increased in IUGR-induced PH, and PASMCs were regulated by PVECs through FoxM1 signaling in a non-contact way. An miRNA screening showed that miR-214-3p, miR-326-3p, and miR-125b-2-3p were downregulated in PVEC-derived exosomes of the IUGR group, which were associated with overexpression of FoxM1 and more significant proliferation and migration of PASMCs. Dual-Luciferase assay demonstrated that the 3 miRNAs directly targeted FoxM1 3' untranslated region. FoxM1 inhibition blocked the PVECs-PASMCs crosstalk and reversed the abnormal functions of PASMCs. In vivo, treatment with thiostrepton significantly reduced the severity of PH. Conclusions Transmission of exosomal miRNAs from PVECs regulated the functions of PASMCs via FoxM1 signaling, and FoxM1 may serve as a potential therapeutic target in IUGR-induced PH.

The circular RNA circ_0099630/miR-940/receptor-associated factor 6 regulation cascade modulates the pathogenesis of periodontitis.

Background/purpose: Periodontitis is one of the highly prevalent chronic inflammatory conditions in adults. The importance of circular RNAs (circRNAs) in the regulation of inflammation has been gradually reported in recent years, but the role of circRNA circ_0099630 in periodontitis has not been reported. Materials and methods: The contents of circ_0099630, microRNA-940 (miR-940) and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) were measured using quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot. Inflammatory factor secretion, cell proliferation, and apoptosis were analyzed under the application of Enzyme-linked immunosorbent assay (ELISA), Cell Counting Kit-8 (CCK8), 5-ethynyl-2'-deoxyuridine (EdU) and flow cytometry, respectively. The Western blot also analyzed the phosphorylation levels of RELA proto-oncogene (P65) and IkappaBalpha (IκBα), key molecules of the nuclear factor kappa-B (NF-κB) pathway. The relationship between miR-940 and circ_0099630 or TRAF6 was verified by luciferase reporter system and RNA immunoprecipitation (RIP) assay. Results: Higher abundance of circ_0099630 and TRAF6 and lower miR-940 expression were observed in periodontitis, and circ_0099630 knockdown attenuated the damage of human PDL cells (PDLCs) induced by lipopolysaccharides (LPS). The relationship between miR-940 and circ_0099630 or TRAF6 was evidenced, while miR-940 downregulation diminished the repair effect of si-circ_0099630 on overexpression LPS-induced damage in PDLCs. Similarly, TRAF6 upregulation impaired the mitigating effect of miR-940 overexpression on LPS-induced injury in PDLCs. Circ_0099630 silencing evidently curbed the phosphorylation levels of P65 and IκBα and thus attenuating the inflammatory response by acting on the miR-940/TRAF6 axis. Conclusion: Silencing circ_0099630 alleviates LPS-induced periodontal ligament cell injury via targeting miR-940/TRAF6/NF-κB in periodontitis.

Activation of Autophagy Induces Monocrotaline-Induced Pulmonary Arterial Hypertension by FOXM1-Mediated FAK Phosphorylation.

PURPOSE: It has been shown that activation of autophagy promotes the development of pulmonary arterial hypertension (PAH). Meanwhile, forkhead box M1 (FOXM1) has been found to induce autophagy in several types of cancer. However, it is still unclear whether FOXM1 mediates autophagy activation in PAH, and detailed mechanisms responsible for these processes are indefinite. METHOD: PAH was induced by a single intraperitoneal injection of monocrotaline (MCT) to rats. The right ventricle systolic pressure (RVSP), right ventricular hypertrophy index (RVHI), percentage of medial wall thickness (%MT), α-smooth muscle actin (α-SMA) staining, and Ki67 staining were performed to evaluate the development of PAH. The protein levels of FOXM1, phospho-focal adhesion kinase (p-FAK), FAK, and LC3B were determined by immunoblotting or immunohistochemistry. RESULTS: FOXM1 protein level and FAK activity were significantly increased in MCT-induced PAH rats, this was accompanied with the activation of autophagy. Pharmacological inhibition of FOXM1 or FAK suppressed MCT-induced autophagy activation, decreased RVSP, RVHI and %MT in MCT-induced PAH rats, and inhibited the proliferation of pulmonary arterial smooth muscle cells and pulmonary vessel muscularization in MCT-induced PAH rats. CONCLUSION: FOXM1 promotes the development of PAH by inducing FAK phosphorylation and subsequent activation of autophagy in MCT-treated rats.

E2F1 induced neuroblastoma cell migration and invasion via activation of CENPE/FOXM1 signaling pathway.

Background: Neuroblastoma (NB) is a common malignancy occurring in infants and young children. Centrosome-associated protein E (CENPE) is a kinetochore-related motor protein highly expressed in NB, with the mechanism largely unknown. This study is committed to investigating the role and mechanism of CENPE in NB.Method: Short hairpin RNAs targeting CENPE and E2F transcription factor 1 (shCENPE and shE2F1) and CENPE overexpression plasmid were transfected into IMR-32 and SK-N-SH cells. The mRNA expressions of CENPE, N-Cadherin, Vimentin, and proliferating cell nuclear antigen (PCNA) in NB cells were detected by qRT-PCR. The viability, migration, and invasion of cells were tested through cell function experiments. Western blot was applied to detect the protein levels of N-Cadherin, Vimentin, PCNA, CENPE and Forkhead box M1 (FOXM1). The relationship between CENPE and E2F1 was verified by dual-luciferase reporter assay, while the interaction between FOXM1 and CENPE in NB cells was analyzed by rescue experiments.Results: CENPE expression was upregulated in NB cells from metastatic sites. Silencing of CENPE suppressed the NB cell viability, migration, and invasion; and decreased N-Cadherin, Vimentin and PCNA expressions, while overexpressed CENPE did oppositely. E2F1 positively targeted CENPE and CENPE partly reversed the effects of shE2F1 on repressing NB cell viability, migration, invasion and the activation of CENPE/FOXM1 signaling pathway. In addition, silenced FOXM1 partly offset the effects of CENPE on promoting NB cell migration and invasion.Conclusion: E2F1 induces NB cell migration and invasion via activating CENPE/FOXM1 pathway.

FOXM1 Is a Novel Molecular Target of AFP-Positive Hepatocellular Carcinoma Abrogated by Proteasome Inhibition.

Alpha-fetoprotein (AFP) is an oncofetal protein that is elevated in a subset of hepatocellular carcinoma (HCC) with poor prognosis, but the molecular target activated in AFP-positive HCC remains elusive. Here, we demonstrated that the transcription factor forkhead box M1 (FOXM1) is upregulated in AFP-positive HCC. We found that FOXM1 expression was highly elevated in approximately 40% of HCC cases, and FOXM1-high HCC was associated with high serum AFP levels, a high frequency of microscopic portal vein invasion, and poor prognosis. A transcriptome and pathway analysis revealed the activation of the mitotic cell cycle and the inactivation of mature hepatocyte metabolism function in FOXM1-high HCC. The knockdown of FOXM1 reduced AFP expression and induced G2/M cell cycle arrest. We further identified that the proteasome inhibitor carfilzomib attenuated FOXM1 protein expression and suppressed cell proliferation in AFP-positive HCC cells. Carfilzomib in combination with vascular endothelial growth factor receptor 2 (VEGFR2) blockade significantly prolonged survival by suppressing AFP-positive HCC growth in a subcutaneous tumor xenotransplantation model. These data indicated that FOXM1 plays a pivotal role in the proliferation of AFP-positive liver cancer cells. Carfilzomib can effectively inhibit FOXM1 expression to inhibit tumor growth and could be a novel therapeutic option in patients with AFP-positive HCC who receive anti-VEGFR2 antibodies.

Research on the mechanism of berberine in the treatment of COVID-19 pneumonia pulmonary fibrosis using network pharmacology and molecular docking.

Purpose Pulmonary fibrosis caused by COVID-19 pneumonia is a serious complication of COVID-19 infection, there is a lack of effective treatment methods clinically. This article explored the mechanism of action of berberine in the treatment of COVID-19 (Corona Virus Disease 2019, COVID-19) pneumonia pulmonary fibrosis with the help of the network pharmacology and molecular docking. Methods We predicted the role of berberine protein targets with the Pharmmapper database and the 3D structure of berberine in the Pubchem database. And GeneCards database was used in order to search disease target genes and screen common target genes. Then we used STRING web to construct PPI interaction network of common target protein. The common target genes were analyzed by GO and KEGG by DAVID database. The disease-core target gene-drug network was established and molecular docking was used for prediction. We also analyzed the binding free energy and simulates molecular dynamics of complexes. Results Berberine had 250 gene targets, COVID-19 pneumonia pulmonary fibrosis had 191 gene targets, the intersection of which was 23 in common gene targets. Molecular docking showed that berberine was associated with CCl2, IL-6, STAT3 and TNF-α. GO and KEGG analysis reveals that berberine mainly plays a vital role by the signaling pathways of influenza, inflammation and immune response. Conclusion Berberine acts on TNF-α, STAT3, IL-6, CCL2 and other targets to inhibit inflammation and the activation of fibrocytes to achieve the purpose of treating COVID-19 pneumonia pulmonary fibrosis.

Dinaciclib inhibits the stemness of two subtypes of human breast cancer cells by targeting the FoxM1 and Hedgehog signaling pathway.

Cyclin­dependent kinase (CDK)4/6 inhibitors in combination with endocrine therapy are the current standard of care used in the first­line treatment of hormone receptor­positive/HER2­negative metastatic breast cancer (BC). Although CDK4/6 inhibitors mainly target the cell cycle, emerging evidence has indicated further potential roles of CDKs other than regulating cell cycle progression. The G1 and G2/M transition regulators, including cyclins D and E, as well as their catalytic partners, CDK2, CDK4 and CDK6, have been reported to play crucial roles in pluripotency maintenance and cell fate decisions of human pluripotent stem cells by controlling transcription factors, signaling pathways and epigenetic regulators. Dinaciclib, a CDK1/2/5/9 inhibitor, is currently being evaluated in clinical trials against various cancer types, including BC. However, the underlying molecular mechanisms of CDK1/2/5/9 inhibitors in regulating BC stemness remain poorly understood. The present study aimed to examine the stemness­inhibitory effects of dinaciclib in MCF­7 (luminal) and HCC­1806 (triple­negative) BC cells. We found that this drug not only effectively reduced the self­renewal abilities and other malignant properties, but also dose­dependently decreased the protein expression levels of three BC stem cell markers, CD44, aldehyde dehydrogenase 1 family member A1 (ALDH1A1) and BMI1 proto­oncogene, polycomb ring finger (Bmi1), as well as three embryonic stem cell markers, Oct4, Nanog and Sox2. Moreover, the dinaciclib­induced decrease of Oct4 and Nanog protein expression was able to be restored by co­treatment with MG­132, a proteasome inhibitor. Forkhead box M1 (FoxM1), both a stemness­stimulating transcription factor and a cell cycle regulator, along with the Hedgehog signaling pathway, were identified as the therapeutic targets of dinaciclib. Collectively, the present results demonstrated a novel role of dinaciclib in suppressing BC stemness and indicated its potential use for future cancer treatments.

Long noncoding RNA ELDR promotes cell cycle progression in normal oral keratinocytes through induction of a CTCF-FOXM1-AURKA signaling axis.

Long noncoding RNAs (lncRNAs) have gained widespread attention as a new layer of regulation in biological processes during development and disease. The lncRNA ELDR (EGFR long noncoding downstream RNA) was recently shown to be highly expressed in oral cancers as compared to adjacent nontumor tissue, and we previously reported that ELDR may be an oncogene as inhibition of ELDR reduces tumor growth in oral cancer models. Furthermore, overexpression of ELDR induces proliferation and colony formation in normal oral keratinocytes (NOKs). In this study, we examined in further detail how ELDR drives the neoplastic transformation of normal keratinocytes. We performed RNA-seq analysis on NOKs stably expressing ELDR (NOK-ELDR), which revealed that ELDR enhances the expression of cell cycle-related genes. Expression of Aurora kinase A and its downstream targets Polo-like kinase 1, cell division cycle 25C, cyclin-dependent kinase 1, and cyclin B1 (CCNB1) are significantly increased in NOK-ELDR cells, suggesting induction of G2/M progression. We further identified CCCTC-binding factor (CTCF) as a binding partner of ELDR in NOK-ELDR cells. We show that ELDR stabilizes CTCF and increases its expression. Finally, we demonstrate the ELDR-CTCF axis upregulates transcription factor Forkhead box M1, which induces Aurora kinase A expression and downstream G2/M transition. These findings provide mechanistic insights into the role of the lncRNA ELDR as a potential driver of oral cancer during neoplastic transformation of normal keratinocytes.

FOXM1 accelerates wound healing in diabetic foot ulcer by inducing M2 macrophage polarization through a mechanism involving SEMA3C/NRP2/Hedgehog signaling.

AIMS: The diabetic wound environment is accompanied with prolonged inflammation leading to impaired wound healing in diabetic foot ulcer (DFU). Our study illustrated the molecular mechanisms by which Forkhead box M1 (FOXM1) enhanced M2 polarization and wound healing of DFU. METHODS: Diabetes was modeled in vivo by streptozotocin injection in rats and in vitro by exposure to high glucose in human dermal fibroblasts (HDF). Macrophages were exposed to IL-4 to induce M2 phenotype polarization. Ectopic expression or knockdown of FOXM1 was performed to observe collagen deposition, angiogenesis, the proliferation and migration of HDF, as well as macrophage polarization. RESULTS: FOXM1 was lowly expressed in the wound tissue of DFU rats. In vitro experiments showed that silencing FOXM1 reversed the M2 polarization-induced promotion of HDF proliferation and migration. We further found that FOXM1 bound to the promoter region of SEMA3C to elevate its expression, and SEMA3C upregulated NRP2 and activated the Hedgehog signaling pathway. Silencing of SMO, a signal transducer in the Hedgehog pathway, negated the promoting effect of FOXM1 overexpression in M2 polarization and HDF proliferation. CONCLUSIONS: Thus, our results suggest that targeting transcription factor FOXM1 may provide a therapeutic target for promoting wound healing in DFU.