Participation of Long Noncoding RNA FOXP4-AS1 in the Development and Progression of Endometrioid Carcinoma with Epigenetically Silencing DUSP5.

Background: Long noncoding RNAs (lncRNAs), as emerging regulators of a wide variety of biological processes via diverse mechanisms, have been demonstrated to be of increasing importance in biology. Genome-wide association studies of tumor samples have identified several lncRNAs as either oncogenes or tumor suppressors in various types of cancers. In recent years, the importance of lncRNAs, especially in endometrioid cancer (EEC), has become increasingly well understood. The lncRNA Forkhead box P4 antisense RNA 1 (FOXP4-AS1) has been reported to fulfill roles in several types of cancers; however, the main biological function and associated underlying molecular mechanism of FOXP4-AS1 in EEC have yet to be fully elucidated. Materials and Methods: The present study therefore aimed to investigate how RNA FOXP4-AS1 may participate in the development and progression of endometrioid carcinoma tissues. To meet this aim, in the present study, the expression level of FOXP4-AS1 was investigated in endometrioid carcinoma tissues and matching nearby normal endometrial tissues collected from patients receiving surgery at the hospital, and a series of molecular biological assays were performed to investigate the effect of FOXP4-AS1 on cell proliferation, cell migration, and cell invasion, and so on. Results: An increased concentration of FOXP4-AS1 was identified in endometrioid carcinoma samples and cell lines compared with the corresponding controls, and this lncRNA was found to be positively correlated with advanced FIGO stages in patients with endometrial cancer. Furthermore, knocking down endogenous FOXP4-AS1 led to a significant reduction in the colony formation number and a significant inhibition of cell proliferation, cell migration, and cell invasion in endometrioid carcinoma cells. Moreover, dual-specificity phosphatase 5 (DUSP5), which is lowly expressed in endometrioid carcinoma tissues cells and negatively modulated by FOXP4-AS1, was identified as the downstream target molecule of FOXP4-AS1. Subsequently, the mechanistic experiments confirmed that, through binding to enhancer of zeste homolog 2 (EZH2; one of the catalytic subunits of polycomb repressive complex 2 [PRC2]), FOXP4-AS1 could epigenetically suppress the expression of DUSP5. Finally, the oncogenic function of the FOXP4-AS1/EZH2/DUSP5 axis in endometrioid carcinoma was confirmed via rescue assays. Conclusions: The findings of the present study have highlighted how FOXP4-AS1 fulfills an oncogenic role in endometrioid carcinoma, and targeting FOXP4-AS1 and its pathway may provide new biomarkers for patients with endometrioid carcinoma.

FOXP4 promotes proliferation of human spermatogonial stem cells.

Continuous self-renewal and differentiation of spermatogonial stem cells (SSCs) is vital for maintenance of adult spermatogenesis. Although several spermatogonial stem cell regulators have been extensively investigated in rodents, regulatory mechanisms of human SSC self-renewal and differentiation have not been fully established. We analyzed single-cell sequencing data from the human testis and found that forkhead box P4 (FOXP4) expression gradually increased with development of SSCs. Further analysis of its expression patterns in human testicular tissues revealed that FOXP4 specifically marks a subset of spermatogonia with stem cell potential. Conditional inactivation of FOXP4 in human SSC lines suppressed SSC proliferation and significantly activated apoptosis. FOXP4 expressions were markedly suppressed in tissues with dysregulated spermatogenesis. These findings imply that FOXP4 is involved in human SSC proliferation, which will help elucidate on the mechanisms controlling the fate decisions in human SSCs.

FOXP4 promotes proliferation of human spermatogonial stem cells / 亚洲男科学杂志(英文版)

Continuous self-renewal and differentiation of spermatogonial stem cells (SSCs) is vital for maintenance of adult spermatogenesis. Although several spermatogonial stem cell regulators have been extensively investigated in rodents, regulatory mechanisms of human SSC self-renewal and differentiation have not been fully established. We analyzed single-cell sequencing data from the human testis and found that forkhead box P4 (FOXP4) expression gradually increased with development of SSCs. Further analysis of its expression patterns in human testicular tissues revealed that FOXP4 specifically marks a subset of spermatogonia with stem cell potential. Conditional inactivation of FOXP4 in human SSC lines suppressed SSC proliferation and significantly activated apoptosis. FOXP4 expressions were markedly suppressed in tissues with dysregulated spermatogenesis. These findings imply that FOXP4 is involved in human SSC proliferation, which will help elucidate on the mechanisms controlling the fate decisions in human SSCs.

Downregulation of microRNA­423­5p suppresses TGF­ß1­induced EMT by targeting FOXP4 in airway fibrosis.

Airway fibrosis (AF) is a common disease that can severely affect patient prognosis. Epithelial­mesenchymal transition (EMT) participates in the pathophysiological development of AF and several studies have demonstrated that some microRNAs (miRNAs) contribute to the development of EMT. The aim of this study was to investigate the function of miR­423­5p in the EMT process and its possible underlying mechanism in BEAS­2B cells. The present study utilized the BEAS­2B cell line to model EMT in AF. Online tools, fluorescence in situ hybridization analysis and an RNA pull­down assay were used to identify potential target genes of miR­423­5p. In addition, immunohistochemistry, wound healing assays, Transwell migration assays, flow cytometry, enzyme­linked immunosorbent assay, reverse transcription­quantitative PCR, western blot analysis and immunofluorescence staining were used to determine the function of miR­423­5p and its target gene in the EMT process in AF. The results indicated that the miR­423­5p expression in AF tissues and BEAS­2B cells stimulated with 10 ng/ml TGF­ß1 for 24 h was significantly increased compared with that in the control group. Overexpression of miR­423­5p facilitated TGF­ß1­induced EMT in BEAS­2B cells; by contrast, downregulation of miR­423­5p suppressed TGF­ß1­induced EMT in BEAS­2B cells. Furthermore, forkhead box p4 (FOXP4) was identified as a potential target gene of miR­423­5p and changes in the miR­423­5p and FOXP4 expression were shown to significantly affect the expression of PI3K/AKT/mTOR pathway members. In summary, overexpression of miR­423­5P promoted the EMT process in AF by downregulating FOXP4 expression and the underlying mechanism may partly involve activation of the PI3K/AKT/mTOR pathway.

Long non-coding RNA FOXP4-AS1 facilitates the biological functions of hepatocellular carcinoma cells via downregulating ZC3H12D by mediating H3K27me3 through recruitment of EZH2.

BACKGROUND: Some studies have reported the effect of long non-coding RNA forkhead box P4 antisense RNA 1 (lncRNA FOXP4-AS1) on hepatocellular carcinoma (HCC). Here, we aimed to discuss the effects of FOXP4-AS1/enhancer of zeste homolog 2 (EZH2)/trimethylation of lysine 27 on histone H3 (H3K27me3)/zinc finger CCCH-type containing 12D (ZC3H12D) axis on HCC. METHODS: The expression of FOXP4-AS1, EZH2, and ZC3H12D, and abundance of H3K27me3 in HCC tissues and cells were tested. The relationship between FOXP4-AS1 expression and prognosis of HCC patients was analyzed. The biological functions of HCC cells were detected via loss- and gain-of-function assays. The tumor weight and volume in vivo were tested. The interaction between FOXP4-AS1 and EZH2 as well as that between EZH2 and H3K27me3 was verified. RESULTS: FOXP4-AS1 and EZH2 expression and H3K27me3 abundance were enhanced while ZC3H12D expression was depressed in HCC tissues and cells. Knockdown of FOXP4-AS1 suppressed biological functions of HCC cells as well as the weight and volume of HCC transplanted tumor. Depleting ZC3H12D reversed the effect of downregulated FOXP4-AS1 on HCC cells. FOXP4-AS1 suppressed ZC3H12D expression via mediating H3K27me3 by recruitment of EZH2. CONCLUSION: The key findings of the present study demonstrate that FOXP4-AS1 suppresses ZC3H12D expression via mediating H3K27me3 by recruitment of EZH2, thus promoting the progression of HCC.

FOXP4 promotes laryngeal squamous cell carcinoma progression through directly targeting LEF­1.

Forkhead box (FOX) proteins are multifaceted transcription factors that have been shown to be involved in cell cycle progression, proliferation and metastasis. FOXP4, a member of the FOX family, has been implicated in diverse biological processes in tumor initiation and progression. However, the molecular mechanisms of FOXP4 in laryngeal squamous cell carcinoma (LSCC) remain unknown. In the present study, differentially expressed transcripts in transforming growth factor­ß­treated TU177 cells were screened using microarrays and it was found that FOXP4 was significantly upregulated. The high expression of FOXP4 was detected in LSCC tissues and cells, and predicted poor prognosis. The role of FOXP4 in laryngeal cancer cell proliferation, migration and invasion was determined by gain­ and loss­of­function assays. Besides, FOXP4 was demonstrated to participate in the epithelial­mesenchymal transition process at the mRNA and protein levels. Mechanically, FOXP4 directly bound to the promoter of lymphoid enhancer­binding factor 1 and activated Wnt signaling pathway, which was confirmed via chromatin immunoprecipitation and luciferase reporter assays. Consequently, these findings provided novel mechanisms of FOXP4 in LSCC progression, which may be considered as potential therapeutic and prognostic targets for LSCC.

miR-491-5p inhibits the proliferation and migration of A549 cells by FOXP4.

Aberrant expression of microRNAs (miRNAs/miRs) plays a key role in the development of non-small cell lung cancer (NSCLC). In the present study, lower miRNA (miR)-491-5p levels and a higher forkhead box P4 (FOXP4) mRNA level were observed in NSCLC tissues and cell lines, compared to adjacent tissues and the normal human lung epithelial cell line BEAS-2B, respectively. A549 cell proliferation and migration were inhibited upon transfection of miR-491-5p mimics compared to miR-negative control (NC) mimics. In addition, compared to miR-NC mimics, overexpression of miR-491-5p decreased FOXP4 expression, while downregulation of miR-491-5p increased FOXP4 expression in A549 cells. The dual luciferase assay confirmed that the 3'untranslated region of FOXP4 was a target for miR-491-5p in A549 cells. Moreover, compared with the control short hairpin (sh)RNA, there was lower expression levels of TGF-ß and its downstream targets (MMP-2 and MMP-9) in the FOXP4 shRNA group. Similarly, compared to miR-NC mimics, overexpression of miR-491-5p decreased MMP-2 and MMP-9 expression levels. In FOXP4-knockdown A549 cells, overexpression of miR-491-5p showed little effect on cell proliferation/migration. In A549 cells, overexpression of FOXP4 partially reversed the miR-491-5p mimics-induced inhibition on the cell proliferation and migration. These results may provide new insights into the role of miR-491-5p in NSCLC.

circ­0000212 promotes cell proliferation of colorectal cancer by sponging miR­491 and modulating FOXP4 expression.

Colorectal cancer (CRC) is a lethal and common malignancy worldwide. Non­coding (nc)RNAs have been shown to modulate tumor progression in several types of cancer. The present study aimed to investigate the role of hsa_circ_0000212 in CRC, as a sponge of microRNA (miR)­491. The expression levels of miR­491 and forkhead box P4 (FOXP4) were analyzed using data from The Cancer Genome Atlas. The association between miR­491 and FOXP4 and the clinicopathological characteristics were also analyzed. A novel circular (circ)RNA, hsa_circ_0000212, was found to sponge miR­491 based on bioinformatics analysis. The potential binding site between miR­491 and FOXP4 or circ­0000212 was validated using luciferase and RNA immunoprecipitation assays. The expression levels and distribution of circ­0000212 was also determined. Cell Counting Kit­8 and colony formation assays were performed to determine the role of miR­491 or circ­0000212 on the proliferation of the CRC cells. Decreased miR­491 or increased FOXP4 expression levels were associated with the pathological stage in patients with CRC. In addition, miR­491 inhibited cell proliferation by targeting FOXP4. circ­0000212 was increased in CRC tissues and was predominantly localized in the cytoplasm. Furthermore, circ­0000212 augmented viability of the CRC cells by sponging miR­491 and modulating FOXP4. In conclusion, circ­0000212 may serve as a novel tumor­promoter and drug target in CRC.