Oct4 redox sensitivity potentiates reprogramming and differentiation.

The transcription factor Oct4/Pou5f1 is a component of the regulatory circuitry governing pluripotency and is widely used to induce pluripotency from somatic cells. Here we used domain swapping and mutagenesis to study Oct4's reprogramming ability, identifying a redox-sensitive DNA binding domain, cysteine residue (Cys48), as a key determinant of reprogramming and differentiation. Oct4 Cys48 sensitizes the protein to oxidative inhibition of DNA binding activity and promotes oxidation-mediated protein ubiquitylation. Pou5f1 C48S point mutation has little effect on undifferentiated embryonic stem cells (ESCs) but upon retinoic acid (RA) treatment causes retention of Oct4 expression, deregulated gene expression, and aberrant differentiation. Pou5f1 C48S ESCs also form less differentiated teratomas and contribute poorly to adult somatic tissues. Finally, we describe Pou5f1 C48S (Janky) mice, which in the homozygous condition are severely developmentally restricted after E4.5. Rare animals bypassing this restriction appear normal at birth but are sterile. Collectively, these findings uncover a novel Oct4 redox mechanism involved in both entry into and exit from pluripotency.

IκBζ is a dual-use coactivator of NF-κB and POU transcription factors.

OCA-B, OCA-T1, and OCA-T2 belong to a family of coactivators that bind to POU transcription factors (TFs) to regulate gene expression in immune cells. Here, we identify IκBζ (encoded by the NFKBIZ gene) as an additional coactivator of POU TFs. Although originally discovered as an inducible regulator of NF-κB, we show here that IκBζ shares a microhomology with OCA proteins and uses this segment to bind to POU TFs and octamer-motif-containing DNA. Our functional experiments suggest that IκBζ requires its interaction with POU TFs to coactivate immune-related genes. This finding is reinforced by epigenomic analysis of MYD88L265P-mutant lymphoma cells, which revealed colocalization of IκBζ with the POU TF OCT2 and NF-κB:p50 at hundreds of DNA elements harboring octamer and κB motifs. These results suggest that IκBζ is a transcriptional coactivator that can amplify and integrate the output of NF-κB and POU TFs at inducible genes in immune cells.

Diallyl disulfide induces DNA damage and growth inhibition in colorectal cancer cells by promoting POU2F1 ubiquitination.

Previous studies have demonstrated that diallyl disulfide (DADS) exhibits potent anti-tumor activity. However, the pharmacological actions of DADS in inhibiting the growth of colorectal cancer (CRC) cells have not been clarified. Herein, we show that DADS treatment impairs the activation of the pentose phosphate pathway (PPP) to decrease PRPP (5-phosphate ribose-1-pyrophosphate) production, enhancing DNA damage and cell apoptosis, and inhibiting the growth of CRC cells. Mechanistically, DADS treatment promoted POU2F1 K48-linked ubiquitination and degradation by attenuating the PI3K/AKT signaling to up-regulate TRIM21 expression in CRC cells. Evidently, TRIM21 interacted with POU2F1, and induced the K272 ubiquitination of POU2F1. The effects of DADS on the enhanced K272 ubiquitination of POU2F1, the PPP flux, PRPP production, DNA damage and cell apoptosis as well as the growth of CRC tumors in vivo were significantly mitigated by TRIM21 silencing or activating the PI3K signaling in CRC cells. Conversely, the effects of DADS were enhanced by TRIM21 over-expression or inhibiting the PI3K/AKT signaling in CRC cells. Collectively, our findings reveal a novel mechanism by which DADS suppresses the growth of CRC by promoting POU2F1 ubiquitination, and may aid in design of novel therapeutic intervention of CRC.

HMGB1 in exosomes derived from gastric cancer cells induces M2-like macrophage polarization by inhibiting the NF-κB signaling pathway.

Gastric cancer (GC) seriously threatens human health. High mobility group protein B1 (HMGB1) and M2-like macrophages are closely associated with core events about human cancers, such as invasion, and metastasis, and cancer microenvironment. This study mainly determined the regulatory effect of HMGB1 in GC cell-derived exosomes on M2-like macrophage polarization as well as the underlying mechanism. HMGB1 was found to be highly expressed in gastric tissue specimens, which might lead to the poor prognosis of GC. High levels of HMGB1 were also observed in the plasma of GC patients, indicating the possibility that it regulates the immune microenvironment via exosomes. Further study revealed and confirmed the regulatory effect of exosomes derived from GC cells with high HMGB1 level on inducing M2-like macrophage polarization. Mechanistically, by interacting with the transcription factor POU2F1, exosomal HMGB1 inhibited the transcriptional activity of p50, resulting in the inactivation of NF-κB signaling pathway and thereby inducing M2-like macrophage polarization. Moreover, instead of promoting the proliferation of GC cells, exosomes with high HMGB1 levels induced M2-like macrophage polarization and promoted GC progression. This study reveals a novel mechanism by which HMGB1 promotes GC progression, which may provide new insights for improving the efficacy of cancer immunotherapy.

HSV-1 selectively packs the transcription factor Oct-1 into EVs to facilitate its infection.

HSV-1 hijacks the cellular vesicular secretion system and promotes the secretion of extracellular vesicles (EVs) from infected cells. This is believed to facilitate the maturation, secretion, intracellular transportation and immune evasion of the virus. Intriguingly, previous studies have shown that noninfectious EVs from HSV-1-infected cells exert antiviral effects on HSV-1 and have identified host restrictive factors, such as STING, CD63, and Sp100 packed in these lipid bilayer-enclosed vesicles. Octamer-binding transcription factor-1 (Oct-1) is shown here to be a pro-viral cargo in non-virion-containing EVs during HSV-1 infection and serves to facilitate virus dissemination. Specifically, during HSV-1 infection, the nuclear localized transcription factor Oct-1 displayed punctate cytosolic staining that frequently colocalized with VP16 and was increasingly secreted into the extracellular space. HSV-1 grown in cells bereft of Oct-1 (Oct-1 KO) was significantly less efficient at transcribing viral genes during the next round of infection. In fact, HSV-1 promoted increased exportation of Oct-1 in non-virion-containing EVs, but not the other VP16-induced complex (VIC) component HCF-1, and EV-associated Oct-1 was promptly imported into the nucleus of recipient cells to facilitate the next round of HSV-1 infection. Interestingly, we also found that EVs from HSV-1-infected cells primed cells for infection by another RNA virus, vesicular stomatitis virus. In summary, this investigation reports one of the first pro-viral host proteins packed into EVs during HSV-1 infection and underlines the heterogenetic nature and complexity of these noninfectious double-lipid particles.

High expression of transcription factor POU2F1 confers improved survival on smokers with lung adenocarcinoma: a retrospective study of two cohorts.

Background: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related mortality worldwide and its most important risk factor is tobacco smoking. While smoking is associated with inferior outcome in NSCLC patients, smoking also correlates with a higher tumor mutational burden. In contrast to adenocarcinomas (ADC) of non-smokers, that frequently harbor targetable gain-of-function mutations, NSCLC smokers largely present with non-targetable loss-of-function mutations of genes associated with DNA-damage repair. The transcription factor Pit-1, Oct1/2, Unc-86 (POU) domain class 2 transcription factor 1 (POU2F1) is a widely expressed bipotential stabilizer of repressed and inducible transcriptional states and frequently deregulated in cancer. Methods: Via immunohistochemistry, we evaluated POU2F1 protein expression on a tissue micro array of 217 operable stage I-III NSCLC patients. Findings were reproduced in a gene expression database of 1144 NSCLC patients, filtered for POU2F1 mRNA expression. After retroviral overexpression of POU2F1 in A549 cells, we evaluated for clonogenic growth and proliferation. Additionally, CRISPR-Cas9 mediated POU2F1 knockdown in A549 cells was likewise analyzed. Results: High protein expression of POU2F1 in 217 NSCLC patients resulted in improved outcome of smokers with ADC [hazard ratio (HR) 0.30 (0.09-0.99), P=0.035]. Moreover, gene expression analysis confirmed favorable outcome of high POU2F1 mRNA expression in smokers with ADC [HR 0.41 (0.24-0.69), P<0.001]. Other than that, retrovirally induced overexpression of POU2F1 in A549 cells significantly reduced both, clonogenic growth as well as proliferation of NSCLC cells, whereas CRISPR-Cas9 mediated knockdown of the protein did not have any impact. Conclusions: Our data suggest that high expression of POU2F1 mediates a less aggressive cancer phenotype in smokers with ADC NSCLC. Pharmacological induction of genes and signaling pathways controlled by POU2F1 may provide novel avenues for future targeted NSCLC therapies in smokers.

Exercise training attenuates angiotensin II-induced cardiac fibrosis by reducing POU2F1 expression.

BACKGROUND: Exercise training protects against heart failure. However, the mechanism underlying the protective effect of exercise training on angiotensin II (Ang II)-induced cardiac fibrosis remains unclear. METHODS: An exercise model involving C57BL/6N mice and 6 weeks of treadmill training was used. Ang II (1.44 mg/kg/day) was administered to induce cardiac fibrosis. RNA sequencing and bioinformatic analysis were used to identify the key factors mediating the effects of exercise training on cardiac fibrosis. Primary adult mouse cardiac fibroblasts (CFs) were used in vitro. Adeno-associated virus serotype 9 was used to overexpress POU domain, class 2, transcription factor 1 (POU2F1) in vivo. RESULTS: Exercise training attenuated Ang II-induced cardiac fibrosis and reversed 39 gene expression changes. The transcription factor regulating the largest number of these genes was POU2F1. Compared to controls, POU2F1 was shown to be significantly upregulated by Ang II, which is itself reduced by exercise training. In vivo, POU2F1 overexpression nullified the benefits of exercise training on cardiac fibrosis. In CFs, POU2F1 promoted cardiac fibrosis. CCAAT enhancer-binding protein ß (C/EBPß) was predicted to be the transcription factor of POU2F1 and verified using a dual-luciferase reporter assay. In vivo, exercise training activated AMP-activated protein kinase (AMPK) and alleviated the increase in C/EBPß induced by Ang II. In CFs, AMPK agonist inhibited the increase in C/EBPß and POU2F1 induced by Ang II, whereas AMPK inhibitor reversed this effect. CONCLUSION: Exercise training attenuates Ang II-induced cardiac fibrosis by reducing POU2F1. Exercise training inhibits POU2F1 by activating AMPK, which is followed by the downregulation of C/EBPß, the transcription factor of POU2F1.

[Reduced Expression of the Tissue-Specific Oct-IL Isoform Exerts an Antitumor Effect on Namalwa Burkitt's Lymphoma Cells].

Increased expression levels of the Oct-1 transcription factor is considered to be one of the key markers of poor cancer prognosis. In addition to the ubiquitous Oct-1A isoform, which is found in all cells, there also exists a tissue-specific Oct-1L isoform, which is expressed in hematopoietic cells. Oct-1L increases cell resistance to different stresses and also regulates the expression of genes controlling differentiation of hematopoietic and immune system cells. The tissue-specific Oct-1L isoform levels are significantly increased in the B-cell lymphoblastoma Namalwa and Raji lines and the T-cell lymphoblastoma Jurkat line compared to normal B and T cells. Apparently, aberrant Oct-1L overexpression not only enhances stress resistance but also leads to the disruption of developmental pathways in the cells promoting their malignant transformation. We report here that targeted suppression of the tissue-specific Oct-1L isoform expression reduces the proliferation rate of Namalwa B-lymphoblastic Burkitt's lymphoma cells, significantly increases cell death rate under hypoxic conditions, and makes cells more sensitive to chemotherapeutic agents such as docetaxel and doxorubicin. These results indicate that targeted therapy aimed at the suppression of the Oct-1 isoforms with increased expression levels in tumor cells rather than the total Oct-1, thus avoiding the traumatic effects of total Oct-1 knockdown, may be promising. Selective suppression of Oct-1 isoforms is a promising strategy in the treatment of lymphoid tumors and may contribute to mitigating the disease course and increasing survival rates in cancer patients.

5-Azacytidine Suppresses the Expression of Tissue-Specific Oct-1 Isoform in Namalwa Burkitt's Lymphoma Cell Culture.

Overexpression of the transcription factor POU2F1 (Oct-1) increases the malignant potential of the tumor and determines the unfavorable prognosis for both solid and hematological cases of the disease in human carcinogenesis. The Oct-1 level determines the rate of development of the disease in acute myelodysplastic leukemia (AML), and a decrease in its expression significantly delays the development of leukemia in mice; however, a complete knockout of Oct-1 leads to the death of the animals. POU2F1 (Oct-1) is expressed as several isoforms transcribed from alternative promoters. They include both ubiquitous and tissue-specific isoforms. It was shown that in Burkitt's lymphoma Namalwa cells 5-azacytidine specifically suppresses the expression of the tissue-specific isoform Oct-1L mRNA (level of Oct-1L is abnormally increased in these cells), while not causing changes in the amount of the ubiquitous isoform Oct-1A mRNA. These results show that it is possible to selectively reduce the transcription level of the Oct-1L isoform aberrantly expressed in human tumor cells.

The Emergence of a New Isoform of POU2F1 in Primates through the Use of Egoistic Mobile Genetic Elements.

The emergence of new genes and functions is of paramount importance in the emergence of new animal species. For example, the insertion of the mobile element Tigger 2 into the sequence of the functional gene POU2F1 in primates led to the formation of a new chimeric primate-specific isoform POU2F1Z, the translation of which is activated under cellular stress. Its mRNA was found in all species of monkeys, starting with macaques. Analysis of the fragments of the Tigger2 copy corresponding to the human exon Z showed that the splicing sites of exon Z are homologous in humans and in most monkeys, with the exception of lemurs and galagos. The stop codon introduced into the mRNA by the Tigger2 sequence is present in all primates, starting with macaques. The internal ATG codon is also present in all primates, with the exception of lemurs and galagos. In the course of evolution, other MGEs, mainly of the SINE type, were inserted into the Tigger2 copy. In the course of evolution, both the location and the number of mobile SINE elements within the POU2F1 gene changed. Starting with macaques, the pattern of the arrangement of SINE elements within the Tigger2 copy in the studied region of the POU2F1 gene was fixed and then remained unchanged in other primates and humans, which may indicate its functional significance.

The interaction between long non-coding RNA LINC01564 and POU2F1 promotes the proliferation and metastasis of gastric cancer.

BACKGROUND: An increasing number of studies have demonstrated that long non-coding RNAs (lncRNAs) serve as key regulators in tumor development and progression. However, only a few lncRNAs have been functionally characterized in gastric cancer (GC). METHODS: Bioinformatics analysis was conducted to find lncRNAs that are associated with GC metastasis. RNA FISH, RIP, and RNA pull down assays were used to study the complementary binding of LINC01564 complementary to the 3'UTR of transcription factor POU2F1. The transcription activation of LINC01564 by POU2F1 as a transcription factor was examined by ChIP assay. In vitro assays such as MTT, cell invasion assay, and clonogenic assay were conducted to examined the impacts of LINC01564 and POU2F1 on GC cell proliferation and invasion. Experiments in vivo were performed to access the impacts of LINC01564 and POU2F1 on GC metastasis. RESULTS: The results showed that LINC01564 complementary bound to the 3'UTR of POU2F1 to form an RNA duplex, whereby stabilizing POU2F1 mRNA and increasing the enrichment in cells. The level of LINC01564 was also increased by POU2F1 through transcription activation. In vitro assays showed that LINC01564 promoted the proliferation, invasion and migration of GC cells through increasing POU2F1. In vivo experiments indicate the promotion of GC proliferation and metastasis by the interaction between LINC01564 and POU2F1. CONCLUSION: Taken together, our results indicate that the interaction between LINC01564 and POU2F1 promotes the proliferation, migration and invasion of GC cells.

[POU2F1 (Oct-1) Differently Autoregulates the Alternative Promoters of Its Own Gene by Binding to Different Regulatory Sites].

The POU2F1 gene, which plays an important role in regulating the mammalian genome and development, has both a ubiquitous (U) and a tissue-specific (L) promoter and is subject to intricate regulation. Regions of POU2F1 gene were found to contain multiple binding sites for its product POU2F1 (Oct-1), a transcription factor. Interspecies homology in these regions was found to exceed 90% among the human, mouse, rat, pig, and dog genomes, almost all of the Oct-1 binding sites being identical. Some of the sites cluster in the vicinity of each of the two alternative promoters, while others are in the 5' noncoding region 6 kb upstream of the transcription start site. The presence of Oct-1 at the sites was demonstrated by chromatin immunoprecipitation and the electrophoretic mobility shift assay (EMSA). A POU2F1 knockdown activated the U promoter and downregulated the L promoter in Namalwa cells, while Oct-1 overexpression exerted an opposite effect. Thus, Oct-1 acts via negative feedback to autoregulate the U promoter through low-affinity Oct-1 binding sites and positive feedback to autoregulate the L promoter through high-affinity canonical (oct) sites when increasing in concentration in a natural context.

NPR3, transcriptionally regulated by POU2F1, inhibits osteosarcoma cell growth through blocking the PI3K/AKT pathway.

Natriuretic peptide receptor 3 (NPR3), mediates natriuretic peptides degradation, was reported to act as a tumor suppressor or promoter in some types of cancer. Previous studies showed that NPR3 was significantly decreased in osteosarcoma (OS) samples. However, the function and potential regulatory mechanism of NPR3 in OS development are unknown. By analyzing the protein expression of NPR3 in OS cell lines (n = 5) and human osteoblast cell line hFOB 1.19, we found that NPR3 expression was also significantly decreased in OS cells. The loss/gain-of-function analysis indicated that NPR3 overexpression observably decreased OS cell viability, arrested cell cycle, and induced apoptosis. However, NPR3 knockdown further enhanced the malignant phenotype of OS cells. Furthermore, NPR3 downregulation activated the PI3K/AKT pathway in OS cells, and the effects of NPR3 silencing on cell proliferation were reversed by the blockade of PI3K/AKT pathway. Of note, dual-luciferase reported assay and site-directed mutagenesis assay indicated that transcription factor POU domain class 2 transcription factor 1 (POU2F1) was proved to suppress NPR3 promoter activity by mainly binding to the -900 to -800 bp region of NPR3 promoter. Moreover, NPR3 overexpression inversed the promotion effect of POU2F1 on cell proliferation. In vivo experiments confirmed that NPR3 overexpression suppressed the growth of xenograft tumors. Taken together, the present study demonstrates that NPR3 may serve as a novel tumor suppressive factor through blocking the PI3K/AKT pathway and transcriptionally regulated by POU2F1.

LncRNA NEAT1 Promotes Inflammatory Response in Sepsis via the miR-31-5p/POU2F1 Axis.

Sepsis is considered to be a systemic inflammatory response, which results in organ dysfunction. LncRNA nuclear-enriched abundant transcript 1 (NEAT1) involved in sepsis progression has been reported. However, the underlying mechanism of NEAT1 in sepsis-induced inflammatory response remains to be revealed. In this study, NEAT1 and POU domain class 2 transcription factor 1 (POU2F1) were highly expressed in LPS-induced septic RAW264.7 cells, opposite to miR-31-5p expression. Furthermore, we found that NEAT1 silencing inhibited LPS-induced inflammatory response and cell proliferation, and promoted cell apoptosis. Subsequently, we found that miR-31-5p interacted with NEAT1 and targeted the 3'UTR of POU2F1, and in LPS-induced RAW264.7 cells, the inhibition of NEAT1 silencing was reversed by miR-31-5p knockdown, while POU2F1 downregulation could cover the functions of miR-31-5p knockdown. In a word, this study indicates that NEAT1 inhibits the LPS-induced progression of sepsis in RAW264.7 cells by modulating miR-31-5p/POU2F1 axis, suggesting that NEAT1 will be the potential therapeutic target for sepsis.

GSK3 Kinase Inhibitor, CHIR, Suppress Transcription of Tissue Specific POU2F1 Isoform in Burkitt Namalwa Lymphoma Cells.

POU2F1 (Oct-1) is a transcription factor, the overexpression of which is found in many human malignant tumors; a significant increase in its level in cells determines the malignant potential of the tumor. POU2F1 is represented in cells by several isoforms that are transcribed from alternative promoters. In Burkitt's B-cell lymphoma Namalwa, the concentration of tissue-specific isoform Oct-1L is several times higher than in normal B cells. We tested the potential to inhibit the transcription of individual Oct-1 isoforms using the GSK3 kinase inhibitor CHIR, an aminopyrimidine derivative. We have shown that CHIR specifically affects the expression of the tissue-specific isoform Oct-1L, significantly reducing the level of mRNA and Oct-1L protein. However, CHIR does not change the amount of mRNA and protein of the ubiquitous isoform Oct-1A in Namalwa tumor cells. The results obtained show that it is possible to develop a system for selective inhibition of Oct-1 transcription factor isoforms in human cells to suppress drug resistance of tumor cells with a high POU2F1 content.

POU2F1 induces the immune escape in lung cancer by up-regulating PD-L1.

PURPOSE: The aim was to research the POU2F1 related genes and mechanism during the progress of immune escape of lung cancer. METHODS: Lung cancer cell lines (H1993, HCC827, A549, H2228, H3122 and H1975) and Human normal lung epithelial cell line (BEAS-2B) were involved in this study. Overexpression or knockdown of POU2F1 was processed in lung cancer cells. POU2F1, PD-L1 and CRK expression in cells were detected by WB and RT-PCR. Flow cytometry and immunofluorescence was used to detect PD-L1 expression on the cell surface. Luciferase reporter detected the promoter activity of CRK. C57BL/6 mice models with knocked down of of POU2F1 were constructed. After tumor formation, anti-PD-1 was administered to detect tumor suppressing ability. IHC assay showed the number of intratumoral CD3+, CD8+, GranzB+ T cells. RESULTS: POU2F1 and PD-L1 were positively correlated in lung cancer cell lines. Overexpression of POU2F1 promoted the expression level of PD-L1 in lung cancer cells. POU2F1 transcription activated the expression of CRK, and further promoted the expression of PD-L1. Knockdown of POU2F1 promoted the efficacy of Anti-PD-1. In addition, tumor growth ability decreased after POU2F1 was knocked down. Cytotoxic effector cytokines levels, tumor suppressive chemokines and interleukin increased, while IL17a level decreased when POU2F1 was knocked down. CONCLUSION: POU2F1 activates the expression of CRK, further promotes the expression of PD-L1, and finally improves the immune escape in lung cancer.

Pathological matrix stiffness promotes cardiac fibroblast differentiation through the POU2F1 signaling pathway.

Cardiac fibroblast (CF) differentiation into myofibroblasts is a crucial cause of cardiac fibrosis, which increases in the extracellular matrix (ECM) stiffness. The increased stiffness further promotes CF differentiation and fibrosis. However, the molecular mechanism is still unclear. We used bioinformatics analysis to find new candidates that regulate the genes involved in stiffness-induced CF differentiation, and found that there were binding sites for the POU-domain transcription factor, POU2F1 (also known as Oct-1), in the promoters of 50 differentially expressed genes (DEGs) in CFs on the stiffer substrate. Immunofluorescent staining and Western blotting revealed that pathological stiffness upregulated POU2F1 expression and increased CF differentiation on polyacrylamide hydrogel substrates and in mouse myocardial infarction tissue. A chromatin immunoprecipitation assay showed that POU2F1 bound to the promoters of fibrosis repressors IL1R2, CD69, and TGIF2. The expression of these fibrosis repressors was inhibited on pathological substrate stiffness. Knockdown of POU2F1 upregulated these repressors and attenuated CF differentiation on pathological substrate stiffness (35 kPa). Whereas, overexpression of POU2F1 downregulated these repressors and enhanced CF differentiation. In conclusion, pathological stiffness upregulates the transcription factor POU2F1 to promote CF differentiation by inhibiting fibrosis repressors. Our work elucidated the crosstalk between CF differentiation and the ECM and provided a potential target for cardiac fibrosis treatment.

The POU2F1/miR-4490/USP22 axis regulates cell proliferation and metastasis in gastric cancer.

PURPOSE: Growing evidence indicates that aberrant expression of microRNAs contributes to tumor development. However, the biological role of microRNA-4490 (miR-4490) in gastric cancer (GC) remains to be clarified. METHODS: To explore the function of miR-4490 in GC, we performed colony formation, EdU incorporation, qRT-PCR, Western blotting, in situ hybridization (ISH), immunohistochemistry (IHC), flow cytometry, ChIP and dual-luciferase reporter assays. In addition, the growth, migration and invasion capacities of GC cells were evaluated. RESULTS: We found that miR-4490 was significantly downregulated in primary GC samples and in GC-derived cell lines compared with normal controls, and that this expression level was negatively correlated with GC malignancy. Exogenous miR-4490 expression not only reduced cell cycle progression and proliferation, but also significantly inhibited GC cell migration, invasion and epithelial-mesenchymal transition (EMT) in vitro. Mechanistically, we found that miR-4490 directly targets USP22, which mediates inhibition of GC cell proliferation and EMT-induced metastasis in vitro and in vivo. Moreover, we found through luciferase and ChIP assays that transcription factor POU2F1 can directly bind to POU2F1 binding sites within the miR-4490 and USP22 promoters and, by doing so, modulate their transcription. Spearman's correlation analysis revealed a positive correlation between USP22 and POU2F1 expression and negative correlations between miR-4490 and USP22 as well as miR-4490 and POU2F1 expression in primary GC tissues. CONCLUSION: Based on our results we conclude that miR-4490 acts as a tumor suppressor, and that the POU2F1/miR-4490/USP22 axis plays an important role in the regulation of growth, invasion and EMT of GC cells.

Characterization of POU2F1 Gene and Its Potential Impact on the Expression of Genes Involved in Fur Color Formation in Rex Rabbit.

The naturally colorful fur of the Rex rabbit is becoming increasingly popular in the modern textile market. Our previous study found that POU class 2 homeobox 1 gene (POU2F1) potentially affects the expression of genes involved in fur color formation in the Rex rabbit, but the function and regulation of POU2F1 has not been reported. In this study, the expression patterns of POU2F1 in Rex rabbits of various colors, as well as in different organs, were analyzed by RT-qPCR. Interference and overexpression of POU2F1 were used to identify the potential effects of POU2F1 on other genes related to fur color formation. The results show that the levels of POU2F1 expression were significantly higher in the dorsal skin of the brown and protein yellow Rex rabbits, compared with that of the black one. POU2F1 mRNAs were widespread in the tissues examined in this study and showed the highest level in the lungs. By transfecting rabbit melanocytes with an POU2F1-overexpression plasmid, we found that the POU2F1 protein was located at the nucleus, and the protein showed the classic characteristics of a transcription factor. In addition, abnormal expression of POU2F1 significantly affected the expression of pigmentation-related genes, including SLC7A11, MITF, SLC24A5, MC1R, and ASIP, revealing the regulatory roles of POU2F1 on pigmentation. The results provide the basis for further exploration of the role of POU2F1 in fur color formation of the Rex rabbit.

lncRNA CRNDE promotes the proliferation and metastasis by acting as sponge miR-539-5p to regulate POU2F1 expression in HCC.

BACKGROUND: This article focuses on the roles and mechanism of lncRNA CRNDE on the progression of HCC. METHODS: We used qRT-PCR to detect the expression of lncRNA CRNDE in HCC cells, normal cells and clinical tissues. MTT assay, FCM analysis, Transwell migration and invasion assay were used to detect the effects of lncRNA CRNDE on cell viability, apoptosis, migration and invasion of HCC cells. The expression of apoptosis-related proteins Bcl-2, Bax, Cleaved Caspase 3, Cleaved Caspase 9, EMT epithelial marker E-cadherin and mesothelial marker Vimentin were analyzed by Western blot. Online prediction software was used to predict the binding sites between lncRNA CRNDE and miR-539-5p, or miR-539-5p and POU2F1 3'UTR. Dual luciferase reporter assay, qRT-PCR and RNA pulldown were used to detect target-relationship between lncRNA CRNDE and miR-539-5p. Dual luciferase reporter assay, qRT-PCR, Western blot and Immunofluorescence were used to detect target-relationship between miR-539-5p and POU2F1. qRT-PCR was used to detect the expression of miR-539-5p and POU2F1 in clinical tissues. Rescue experiments was used to evaluate the association among lncRNA CRNDE, miR-539-5p and POU2F1. Finally, we used Western blot to detect the effects of lncRNA CRNDE, miR-539-5p and POU2F1 on NF-κB and AKT pathway. RESULTS: lncRNA CRNDE was highly expressed in HCC cells and HCC tissues compared with normal cells and the corresponding adjacent normal tissues. lncRNA CRNDE promoted the cell viability, migration and invasion of HCC cells, while inhibited the apoptosis and promoted the EMT process of HCC cells. lncRNA CRNDE adsorbed miR-539-5p acts as a competitive endogenous RNA to regulate POU2F1 expression indirectly. In HCC clinical tissues, miR-539-5p expression decreased and POU2F1 increased compared with the corresponding adjacent normal tissues. lncRNA CRNDE/miR-539-5p/POU2-F1 participated the NF-κB and AKT pathway in HCC. CONCLUSION: lncRNA CRNDE promotes the expression of POU2F1 by adsorbing miR-539-5p, thus promoting the progression of HCC.