Objective To explore the mechanism of ubiquitin specific peptidase 21 (USP21) increasing the stability of forkhead box protein M1 (FOXM1) and promoting M2 polarization of macrophages in endometriosis (EM). Methods Eutopic endometrial stromal cells (EESC) collected from patients and normal endometrial stromal cells (NESC) from routine health examiners were cultured in vitro, and the expression levels of USP21 and FOXM1 were detected using RT-qPCR and Western blot. EESCs were co-cultured with macrophages. M1 polarization markers of interleukin 6 (IL-6) and CXC chemokine ligand 10 (CXCL10) and M2 polarization markers of CD206 and fibronectin 1 (FN1) were tested using RT-qPCR. M2 marker CD206 was further detected by flow cytometry. IL-6, tumor necrosis factor-alpha (TNF-α), IL-10, and transforming growth factor-beta (TGF-β) levels in cell supernatant were detected by ELISA. Co-immunoprecipitation was used to assess the interaction between USP21 and FOXM1, and the ubiquitination level of FOXM1. FOXM1 protein stability was detected through cycloheximide (CHX) assay. Results USP21 and FOXM1 expression levels in the EESC group were significantly increased compared with those in the NESC group; compared with the NESC + M0 group, the EESC + M0 group showed no significant difference in the expression of M1 polarization markers (IL-6 and CXCL10), but increased expression of M2 polarization markers (CD206 and FN1), along with notably increased number of M2 macrophages; there was no significant difference in IL-6 and TNF-α levels, but increased levels of IL-10 and TGF-β in the cell supernatant. The above findings indicated that the deubiquitinase USP21 was highly expressed in EM, promoting M2 polarization of macrophages. Knocking down USP21 or FOXM1 can inhibit M2 polarization of EM macrophages. USP21 interacted with FOXM1 in EESC, leading to a decrease in FOXM1 ubiquitination level and an increase in FOXM1 protein stability. Overexpression of FOXM1 reversed the inhibitory effect of knocking down USP21 on M2 polarization of EM macrophages. Conclusion The deubiquitinase USP21 interacts with FOXM1 to increase the stability of FOXM1 and promote M2 polarization of EM macrophages.
Humans ; Forkhead Box Protein M1/genetics* ; Female ; Macrophages/cytology* ; Endometriosis/genetics* ; Ubiquitin Thiolesterase/genetics* ; Cells, Cultured ; Endometrium/metabolism* ; Ubiquitination ; Adult ; Interleukin-10/metabolism* ; Interleukin-6/metabolism* ; Protein Stability ; Stromal Cells/metabolism*

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.
Humans ; bcl-2-Associated X Protein/metabolism* ; Carcinoma ; Cell Line, Tumor ; Cell Proliferation/genetics* ; Drug Resistance ; Forkhead Box Protein M1/metabolism* ; Gene Expression Regulation, Neoplastic ; MicroRNAs/genetics* ; Proliferating Cell Nuclear Antigen/metabolism* ; Receptor, Notch1/metabolism* ; RNA, Small Interfering/genetics* ; Stomach Neoplasms/genetics*

OBJECTIVETo construct a recombinant lentiviral vector that co-express green fluorescent protein (GFP) and FoxM1 shRNA and establish a prostate cancer cell line with stable FoxM1 down-regulation.

METHODSThree interfering sequences targeting FoxM1 were designed and inserted into the lentiviral vector pHBLV-U6-ZsGreen-Puro. After identification by DNA sequencing, the lentiviral vectors carrying Foxm1 shRNA were packaged in 293 cells. The lentiviral particles were collected to infect human prostate cancer DU-145 cells, and the transfection efficiency was observed under fluorescence microscope; the interference efficiency was assessed using real-time PCR. DU-145 cells with stable FoxM1 down-regulation were screened with puromycin, and the expression level of FoxM1 was detected by Western blotting and the cell growth was observed using MTT assay. The stably transfected cells were examined for cell apoptosis and cell clone formation capacity with flow cytometry and colony formation assay.

RESULTSDNA sequencing demonstrated successful construction of the 3 FoxM1 shRNA lentivirus vectors. Real-time PCR showed a high interference efficiency of FoxM1 shRNA1 vector, which resulted in obvious down-regulation of FoxM1 in DU-145 cells. Western blotting showed that the expression of FoxM1 protein was decreased in FoxM1 shRNA1 lentivirus-transfected cells, which displayed a suppressed cell proliferation, increased apoptosis rate, and attenuated clonogenic ability.

CONCLUSIONWe have successfully established a prostate cancer cell model with stable FoxM1 down-regulation, which shows lowered proliferative and clonogenic activities with increased cell apoptosis.

Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Forkhead Box Protein M1 ; Forkhead Transcription Factors ; genetics ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; Humans ; Lentivirus ; Male ; Prostatic Neoplasms ; genetics ; RNA, Small Interfering ; genetics ; Real-Time Polymerase Chain Reaction ; Transfection

This study aimed to identify the differentially expressed genes after silencing of β-catenin in multiple myeloma transduced with β-catenin shRNA. The DNA microarray dataset GSE17385 was downloaded from Gene Expression Omnibus, including 3 samples of MM1.S (human multiple myeloma cell lines) cells transduced with control shRNA and 3 samples of MM1.S cells transduced with β-catenin shRNA. Then the differentially expressed genes (DEGs) were screened by using Limma. Their underlying functions were analyzed by employing Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses. Moreover, DEGs annotation was conducted based on the databases of tumor associated genes, tumor suppressed genes and the transcriptional regulation from patterns to profiles. Furthermore, the protein-protein interaction (PPI) relationship was obtained from STRING and the protein-protein interaction network and the functional modules were visualized by Cytoscape. Then, the pathway enrichment for the DEGs in the functional module was performed. A total of 301 DEGs, including 124 up-regulated and 117 down-regulated DEGs, were screened. Functional enrichment showed that CCNB1 and CDK1 were significantly related to the function of cell proliferation. FOS and JUN were related to innate immune response-activating signal transduction. Pathway enrichment analysis indicated that CCNB1 and CDK1 were most significantly enriched in the pathway of cell cycle. Besides, FOS and JUN were significantly enriched in the Toll-like receptor signaling pathway. FOXM1 was identified as a transcription factor. Moreover, there existed interactions among CCNB1, FOXM1 and CDK1 in PPI network. The expression of FOS, JUN, CCNB1, FOXM1 and CDK1 may be affected by β-catenin in multiple myeloma.
CDC2 Protein Kinase ; Cyclin B1 ; genetics ; Cyclin-Dependent Kinases ; genetics ; Forkhead Box Protein M1 ; Forkhead Transcription Factors ; genetics ; Gene Expression Profiling ; methods ; Gene Expression Regulation, Neoplastic ; Gene Regulatory Networks ; Gene Silencing ; Humans ; Multiple Myeloma ; genetics ; Oncogene Proteins v-fos ; genetics ; Protein Interaction Maps ; Proto-Oncogene Proteins c-jun ; genetics ; beta Catenin ; genetics

The FOXO3a and FOXM1 forkhead transcription factors are key players in cancer initiation, progression, and drug resistance. Recent research shows that FOXM1 is a direct transcriptional target of FOXO3a, a vital downstream effector of the PI3K-AKT-FOXO signaling cascade. In addition, FOXM1 and FOXO3a also antagonize each other's activity by competitively binding to the same target genes, which are involved in chemotherapeutic drug sensitivity and resistance. Understanding the role and regulation of the FOXO-FOXM1 axis will provide insight into chemotherapeutic drug action and resistance in patients, and help to identify novel therapeutic approaches as well as diagnostic and predictive biomarkers.
Antineoplastic Agents ; pharmacology ; therapeutic use ; Carcinogenesis ; genetics ; metabolism ; Drug Resistance, Neoplasm ; Forkhead Box Protein M1 ; Forkhead Box Protein O3 ; Forkhead Transcription Factors ; genetics ; metabolism ; Humans ; Neoplasms ; drug therapy ; metabolism ; pathology ; Phosphatidylinositol 3-Kinases ; metabolism ; Proto-Oncogene Proteins c-akt ; metabolism ; Signal Transduction

OBJECTIVETo explore the effects of EGFR-TKI AG1478 on the expression of FoxMl and FOXO3a genes in non-small cell cancer (NSCLC) cell lines, and explore the effect on cell proliferation and drug sensitivity to AG1478 after down-regulation of FOXMl and FOXO3a expression by RNAi technique.

METHODSHuman lung cancer cells were treated with AG1478 at different concentrations. RT-PCR and Western blot were used to examine the expression of P-EGFR, FOXM1, FOXO3a mRNA and protein. After transient transfection of FOXM1 and FOXO3a siRNA, RT-PCR and Western blot were employed to determine the transfection efficiency and expression of the related proteins. CCK-8 assay, colony formation assay and flow cytometry were performed to evaluate the cell proliferation, colony formation ability and the changes in cell cycle distribution.

RESULTSThe expressions of FOXM1 mRNA and protein were inhibited by AG1478 in a dose-dependent manner (both P < 0.05). After transfection with FOXM1 siRNA, the expressions of FOXM1 mRNA and protein, and proteins of cyclin B1, c-Myc, and Bcl-2 were significantly down-regulated, and the expressions of p21 and cleaved-PARP proteins were significantly up-regulated (all P < 0.05). The colony number of FOXM1siRNA transfection group was 37.3 ± 8.6, significantly lower than that of the blank control (135.3 ± 7.0) and negative control group (125.3 ± 7.5, P < 0.05). The colony formation inhibition rate was (7.40 ± 0.94)% in the negative control group and (72.4 ± 6.09)% in the FOXM1 siRNA transfection group. FOXM1siRNA transfection induced cell cycle arrest at G2/M phase with a percentage of (55.6 ± 4.83)%, significantly higher than that of the blank control [(24.30 ± 1.95)%] and negative control group [(21.3 ± 2.06)%, P < 0.05]. Additionally, the FOXM1siRNA transfection significantly increased the chemosensitivity of A549 cells to AG1478 (P < 0.05). Besides, AG1478 induced expression and nuclear relocation of FOXO3a. After the FOXO3a siRNA transfection, the expression of FOXM1 protein was significantly up-regulated, and resulted in a reduction of AG1478-induced inhibition of FOXM1.

CONCLUSIONSThe expression of FOXM1 is down-regulated by AG1478 via FOXO3a in the NSCLC cell lines, and then increases the chemosensitivity of A549 cells to AG1478. It suggests that FOXM1 could be a potential target for the therapy and drug exploitation for NSCLC.

Adenocarcinoma ; metabolism ; pathology ; Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Cell Proliferation ; Dose-Response Relationship, Drug ; Down-Regulation ; Forkhead Box Protein M1 ; Forkhead Box Protein O3 ; Forkhead Transcription Factors ; genetics ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Quinazolines ; administration & dosage ; pharmacology ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Receptor, Epidermal Growth Factor ; antagonists & inhibitors ; Transfection ; Tyrphostins ; administration & dosage ; pharmacology