POU Class Research Articles

Establishment and Comprehensive Analysis of Underlying microRNA-mRNA Interactive Networks in Ovarian Cancer.

Background The rate of ovarian cancer (OC) is one of the highest in women's reproductive systems. An improperly expressed microRNA (miRNA) has been discovered to have a vital role in the pathophysiology of OC. However, more research into OC's miRNA-message RNA (mRNA) gene interaction network is required. Methods Firstly, the microarray data sets GSE25405 and GSE119055 from the GEO (Gene Expression Omnibus) database were downloaded and then analyzed with the GEO2R tool aiming at identifying DEMs (differential expressed miRNAs) between ovarian malignant tissue and ovarian normal tissue. The whole consistently changed miRNAs were then screened out to be candidate DEMs. For estimating underlying upstream transcription factors, FunRich was employed. miRNet was utilized to determine putative DEMs' downstream target genes. The R program was then used to do the GO annotation as well as the analysis of KEGG pathway enrichment for target genes. The PPI (protein-protein interaction), as well as the DEM-hub gene networks, were created by the Cytoscape software and STRING database. Finally, we chose the GSE74448 dataset to test the precision of hub gene expressions. Results We have screened out six (five upregulated and one downregulated) DEMs. The majority of upregulated and downregulated DEMs are likely regulated by SP1 (specificity protein 1). SP4 (s protein 4), POU2F1 (POU class 2 homeobox 1), MEF2A (myocyte-specific enhancer factor 2A), ARID3A (AT-rich interaction domain 3A), and EGR1 (early growth response 1) can regulate upregulated and downregulated DEMs. We have found 807 target genes (656 upregulated and 151 downregulated DEM), being generally enriched in focal adhesion and proteoglycans in cancer, gastric cancer, hepatocellular carcinoma, as well as breast cancer. The majority of hub genes are projected to be controlled by hsa-miR-429, hsa-miR-140-5p, hsa-miR-199a-5p, and hsa-miR-199a-3p after the DEM-hub gene network was built. VEGFA (vascular endothelial growth factor A), EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit), and HIF1A (hypoxia inducible factor 1 subunit alpha) expressions are consistent with the GSE74448 dataset in the first 18 hub genes. Conclusion We have built an underlying miRNA-mRNA interacting network in OC, giving us unparalleled insight into the disease's diagnosis and treatment.

Small activating RNA activation of ATOH1 promotes regeneration of human inner ear hair cells

ABSTRACT The loss of inner ear hair cells leads to irreversible acoustic injury in mammals, and regeneration of inner ear hair cells to restore hearing loss is challenging. ATOH1 is a key gene in the development and regeneration of hair cells. Small activating RNAs (saRNAs) can target a gene to specifically upregulate its expression. This study aimed to explore whether small activating RNAs could induce the differentiation of human adipose-derived mesenchymal stem cells into hair cell-like cells with a combination of growth factors in vitro and thus provide a new strategy for hair cell regeneration and the treatment of sensorineural hearing loss. Fifteen small activating RNAs targeting the human ATOH1 gene were designed and screened in 293 T and human adipose-derived mesenchymal stem cells, and 3 of these candidates were found to be capable of effectively and stably activating ATOH1 gene expression. The selected small activating RNAs were then transfected into hair cell progenitor cells, and hair cell markers were examined 10 days after transfection. After transfection of the selected small activating RNAs, the expression of the characteristic markers of inner ear hair cells, POU class 4 homeobox 3 (POU4F3) and myosin VIIA (MYO7A), was detected. Human adipose-derived mesenchymal stem cells have the potential to differentiate into human hair cell progenitor cells. In vitro, small activating RNAs were able to induce the differentiation of hair cell progenitor cells into hair cell-like cells. Therefore, RNA activation technology has the potential to provide a new strategy for the regeneration of hair cells.

Clinical characteristics and patient outcomes of molecular subtypes of small cell lung cancer (SCLC)

BackgroundRecent studies have shown that according to the expression levels of achaete-scute homolog 1 (ASCL1), neurogenic differentiation factor 1 (NEUROD1), and POU class 2 homeobox 3 (POU2F3), small cell lung cancer (SCLC) can be divided into four subtypes: SCLC-A (ASCL1-dominant), SCLC-N (NEUROD1-dominant), SCLC-P (POU2F3-dominant), and SCLC-I (triple negative or SCLC-inflamed). However, there are limited data on the clinical characteristics and prognosis of molecular subtypes of SCLC.MethodsImmunohistochemistry (IHC) was used to detect the expression levels of ASCL1, NEUROD1, and POU2F3 in 53 patient samples of resectable SCLC. The subtype was defined by the differential expression of the transcription factors for ASCL1, NEUROD1, and POU2F3 or the low expression of all three factors with an inflamed gene signature (SCLC-A, SCLC-N, SCLC-P, and SCLC-I, respectively). The clinicopathological characteristics, immunological features (programmed death ligand 1 [PD-L1] expression and CD8+ tumor infiltrating lymphocyte [TIL] density), and patient outcomes of the four subtypes of SCLC were analyzed.ResultsPositive ASCL1, NEUROD1, and POU2F3 staining was detected in 43 (79.2%), 27 (51.0%), and 17 (32.1%) SCLC specimens by IHC. According to the results of IHC analysis, SCLC was divided into four subtypes: SCLC-A (39.6%), SCLC-N (28.3%), SCLC-P (17.0%), and SCLC-I (15.1%). The 5-year overall survival (OS) rates of these four subtypes were 61.9%, 69.3%, 41.7%, and 85.7%, respectively (P=0.251). There were significant differences in smoking status among different subtypes of SCLC (P= 0.031). However, we did not confirm the correlation between subtypes of SCLC and other clinicopathological factors or immune profiles. Cox multivariate analysis showed that N stage (P=0.025), CD8+ TILs (P=0.024), Ki-67 level (P=0.040), and SCLC-P (P=0.023) were independent prognostic factors for resectable SCLC.ConclusionsOur IHC-based study validated the proposed classification of SCLC using the expression patterns of key transcriptional regulatory factors. We found that SCLC-P was associated with smokers and was one of the poor prognostic factors of limited-stage SCLC. In addition, no correlation was found between PD-L1 expression or CD8+ TIL density and SCLC subtypes.

Rs9390123 and rs9399451 influence the DNA repair capacity of lung cancer by regulating PEX3 and PHACTR2‑AS1 expression instead of PHACTR2.

Lung cancer is a common cancer type, and has the highest mortality rate in the world. A genome‑wide association study suggests that the genetic marker rs9390123 is significantly associated with DNA repair capacity (DRC) in lung cancer. Analysis of the data derived from the 1000 Genomes Project indicated that there is another single nucleotide polymorphism (SNP), rs9399451, in strong linkage disequilibrium with rs9390123 in Caucasian individuals, thus suggesting that this SNP could be associated with DRC. However, the causal SNP and mechanism of DRC remain unclear. In the present study, dual luciferase assay results indicated that both SNPs are functional in lung cells. Through chromosome conformation capture, an enhancer containing the two functional SNPs was observed to bind the promoter of peroxisomal biogenesis factor 3 and phosphatase and actin regulator2 antisense RNA 1 (PHACTR2‑AS1). Knockdown of PHACTR2‑AS1 could significantly influence lung cell proliferation, colony formation, migration and wound healing, which verified that PHACTR2‑AS1 is a novel oncogene for lung cancer. Through chromatin immunoprecipitation, the transcription factor POU class2 homeobox 1 (POU2F1) was identified to bind to the surrounding segments of these two SNPs, and their interaction was investigated. The present study identified the mechanism via which rs9390123 and rs9399451 could influence DRC.

Cochlear implantation in a Chinese patient with a novel frameshift variant in POU3F4 gene and incomplete partition type III: a case report

Variations in the POU Class 3 Homeobox 4 (POU3F4) gene are associated with X-linked mixed deafness. Here, the identification of a novel variant of POU3F4 in a male paediatric patient (the proband) with incomplete partition type III (IP-III) hearing impairment, is described. Clinical data were collected from the proband and his biological parents. Whole exome sequencing of the proband revealed a novel frameshift insertion mutation in POU3F4 (c.717_718ins GTGCCTTGCAG: p.Leu240Valfs*5) in a hemizygous state. This variant likely truncates the protein within the POU-specific domain, and the proband’s biological mother was found to be a carrier of this variant. After excluding all contraindications, the proband underwent cochlear implantation in the right ear in June 2020. Cerebrospinal fluid (CSF) gushing was observed during surgery, but there were no postoperative complications, such as CSF leak, meningitis, or facial nerve stimulation. A novel pathogenic frameshift variant of POU3F4 was identified, enriching the known mutation spectrum of POU3F4. Effective perioperative prevention and response measures should be taken to reduce the incidence of CSF gushing and meningitis in patients receiving IP-III cochlear implantation.

Role of prostate cancer stem-like cells in the development of antiandrogen resistance.

Androgen deprivation therapy (ADT) is the standard of care treatment for advance stage prostate cancer. Treatment with ADT develops resistance in multiple ways leading to the development of castration-resistant prostate cancer (CRPC). Present research establishes that prostate cancer stem-like cells (CSCs) play a central role in the development of treatment resistance followed by disease progression. Prostate CSCs are capable of self-renewal, differentiation, and regenerating tumor heterogeneity. The stemness properties in prostate CSCs arise due to various factors such as androgen receptor mutation and variants, epigenetic and genetic modifications leading to alteration in the tumor microenvironment, changes in ATP-binding cassette (ABC) transporters, and adaptations in molecular signaling pathways. ADT reprograms prostate tumor cellular machinery leading to the expression of various stem cell markers such as Aldehyde Dehydrogenase 1 Family Member A1 (ALDH1A1), Prominin 1 (PROM1/CD133), Indian blood group (CD44), SRY-Box Transcription Factor 2 (Sox2), POU Class 5 Homeobox 1(POU5F1/Oct4), Nanog and ABC transporters. These markers indicate enhanced self-renewal and stemness stimulating CRPC evolution, metastatic colonization, and resistance to antiandrogens. In this review, we discuss the role of ADT in prostate CSCs differentiation and acquisition of CRPC, their isolation, identification and characterization, as well as the factors and pathways contributing to CSCs expansion and therapeutic opportunities.

RNA binding protein DDX5 directs tuft cell specification and function to regulate microbial repertoire and disease susceptibility in the intestine

ObjectiveTuft cells residing in the intestinal epithelium have diverse functions. In the small intestine, they provide protection against inflammation, combat against helminth and protist infections, and serve as entry portals...

Positive feedback loop of lncRNA FAM201A/miR‑146a‑5p/POU2F1 regulates IL‑1β‑induced chondrocyte injury invitro.

Numerous studies have previously demonstrated that long non-coding RNAs (lncRNAs) serve an important regulatory role in osteoarthritis (OA). In particular, the lncRNA family with sequence similarity 201 member A (FAM201A) was previously found to be downregulated in necrotic femoral head samples. However, the role of FAM201A in IL-1β-induced chondrocyte injury remains unclear. It was hypothesized that FAM201A may exert a protective effect on IL-1β-induced chondrocyte injury in OA by sponging microRNAs (miRNAs/miRs). The purpose of the present study was to explore the role and molecular mechanism of FAM201A in IL-1β-induced chondrocyte injury. A model of OA was established by stimulation C-28/I2 cell with IL-1β in vitro. The expression levels of FAM201A following IL-1β-induced chondrocyte injury were detected via reverse transcription-quantitative PCR. Luciferase reporter assay was used to assess the possible associations among FAM201A, miR-146a-5p and POU class 2 homeobox 1 (POU2F1). Chromatin immunoprecipitation assay was performed to analyze the interaction between POU2F1 and miR-146a-5p. ELISA, TUNEL and western blotting were performed to measure the level of inflammation, lactate dehydrogenase release, apoptosis and the expression of apoptosis-related proteins (Bcl-2, Bax, cleaved caspase 3 and cleaved caspase 9), respectively. The expression levels of FAM201A were found to be downregulated following IL-1β-induced chondrocyte injury. Overexpression of FAM201A exerted a protective effect against IL-1β-induced chondrocyte injury. In addition, FAM201A could upregulate the expression levels of POU2F1 by sponging miR-146a-5p. Further experiments revealed that POU2F1 could bind to the promoter region of FAM201A and subsequently regulate the expression levels of POU2F1, indicating a role for the FAM201A/miR-146a-5p/POU2F1 positive feedback loop in IL-1β-induced chondrocyte injury. The present study revealed the protective effects of the FAM201A/miR-146a-5p/POU2F1 positive feedback loop on IL-1β-induced chondrocyte injury and provided a potential therapeutic target for OA.

POU5F1 Protein and Gene Expression Analysis in Neonate and Adult Mouse Testicular Germ Cells by Immunohistochemistry and Immunocytochemistry.

POU5F1 (POU class 5 homeobox 1) is a transcription factor that is critically involved in the self-renewal of undifferentiated embryonic stem cells. In this present study, we have developed our study to analyze the expression of the POU5F1 in the neonatal and adult mice testis section and isolated spermatogonial stem cells (SSCs). We also examine POU5F1 protein localization by three various kinds of antibodies. In this experimental research, to enhance our understanding of the POU5F1 expression levels, protein localization, and function in testicular germ cell, we used immunohistochemistry, immunocytochemistry, and Fluidigm real-time polymerase chain reaction (RT-PCR) analysis in the mouse testis section and neonatal and adult SSCs, and also we used protein-protein network analysis and gene enrichment analysis for genes involved in testicular development. Counting POU5F1-positive cells represented significantly higher expression (p < 0.05) of POU5F1 in the adult testis in comparison to the neonate. Finally, Fluidigm RT-PCR showed a significant expression (p < 0.05) level of germ cells gene POU5F1 in neonate SSCs (1-2 week) than 16-24 week SSCs. The illustrated results identify POU5F1 as a necessary transcription factor of testicular germ cells and can be supportive for the investigation of the development and differentiation of SSCs.

HN1L promotes stem cell-like properties by regulating TGF-β signaling pathway through targeting FOXP2 in prostate cancer.

Dysregulated hematological and neurological expressed 1-like (HN1L) has been implicated in carcinogenesis of difference cancers, including hepatocellular carcinoma and breast cancer. However, the role of HN1L in the progression of prostate cancer (PCA) remains unknown. Therefore, we aimed to investigate the role of HN1L in stemness and progression of PCA. The expression of HN1L in PCA tissues and cells was determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR), western blot analysis, and/or immunohistochemistry (IHC). CD133+ cells were sorted from PCA cells using magnetic fluorescence cell sorting technology and were considered as cancer stem cells (CSCs). Sphere formation assays, transwell assays, and animal experiments were conducted to assess cell stemness, migration, invasion, and in vivo tumorigenesis, respectively. The results showed that HN1L expression was higher in PCA tissues and cells as compared with normal tissues and cells, as well as in CD133+ cells as compared with CD133- cells. HN1L knockdown significantly decreased the expression levels of CSC markers including OCT4 (POU class 5 homeobox 1), CD44, and SRY-box transcription factor 2, inhibited cell migration, invasion, and tumorigenesis and decreased the number of tumor spheroids and CD133+ cell population. Furthermore, we found that HN1L could bind to forkhead box P2 (FOXP2) and positively regulated transforming growth factor-β (TGF-β) expression via upregulation of FOXP2. In addition, the overexpression of TGF-β in HN1L-knockdown PCA cells increased the number of tumor spheroids and CD133+ cell population, as well as enhanced cell migration and invasion. Collectively, this study demonstrates that HN1L promotes stem cell-like properties and cancer progression by targeting FOXP2 through TGF-β signaling pathway in PCA.

High-Throughput Sequencing Reveals the Differential MicroRNA Expression Profiles of Human Gastric Cancer SGC7901 Cell Xenograft Nude Mouse Models Treated with Traditional Chinese Medicine Si Jun Zi Tang Decoction.

Objective. The present study aimed to investigate the potential mechanism underlying the antitumor effect of Si Jun Zi Tang (SJZT) decoction on gastric cancer. Methods. Twelve human gastric cancer SGC7901 cell xenograft nude mouse models were established. The mice were randomly divided into the Model group and SJZT group. SJZT exerted significant antitumor effects after 21 days of decoction administration. High-throughput sequencing was used to analyze the microRNA (miRNA) expression profiles of tumor tissues. Bioinformatics analysis was performed to provide further information regarding the differentially expressed miRNAs. Five representative differentially expressed miRNAs and four predicted target genes were further validated using quantitative real-time reverse transcription PCR (qRT-PCR). Results. We identified 33 miRNAs that were differentially expressed in the SJZT group compared with the Model group. Among them, 32 miRNAs were upregulated and 1 miRNA was downregulated. Bioinformatic analysis showed that most of miRNAs acted as tumor suppressors and their target genes participated in multiple signaling pathways, including the PI3K/Akt signaling pathway, microRNAs in cancer, and Wnt signaling pathway. The qRT-PCR result confirmed that miR-223-3p, miR-205-5p, miR-147b-3p, and miR-223-5p were overexpressed and their respective paired target genes FUT9, POU2F1, MUC4, and RAB14 mRNA were obviously downregulated in the SJZT group compared with those in the Model group. Network analysis revealed that miR-223-3p and miR-205-5p shared two targets POU2F1 (encoding POU class 2 homeobox 1) and FUT9 (encoding fucosyltransferase 9), suggesting they have a common role in certain pathways. Conclusion. This study provided novel insights into the anticancer mechanism of SJZT against gastric cancer, which might be partly related to the modulation of miRNA expression and their target pathways in tumors.

LncRNA CASC21 induces HGH1 to mediate colorectal cancer cell proliferation, migration, EMT and stemness

ABSTRACT Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. Long non-coding RNAs (lncRNAs) have been increasingly reported to serve vital parts in malignancies including CRC. Although cancer susceptibility 21 (CASC21) has been uncovered to play a part in CRC, its mechanism still needs further explanation. Thus, our study aimed to further explore the influence and mechanism of CASC21 in CRC progression. Quantitative real-time RT-PCR and western blot were performed to detect gene expression; a series of functional assays were performed to investigate the effect of CASC21 on CRC cells; in vivo tumour growth was evaluated via the nude mice xenograft model. The results revealed that CASC21 facilitated CRC cell proliferation, migration, epithelial-mesenchymal transition (EMT) and stemness. In addition, CASC21 was co-expressed with and bound to transcription factor POU5F1B (POU class 5 homeobox 1B). CASC21 recruited POU5F1B to HGH1 promoter to activate the transcription of HGH1 homolog. Also, CASC21 served as a competitive endogenous RNA (ceRNA) to up-regulate HGH1 via endogenously sponging miR-485-5p. Moreover, HGH1 overexpression counteracted the suppression of CASC21 deficiency on CRC tumour growth. In summary, our study indicated that CASC21 enhanced the expression of HGH1 to promote the malignancy of CRC by recruiting POU5F1B and sponging miR-485-5p, suggesting a key role of CASC21 in CRC progression.

Reporter gene systems for the identification and characterization of cancer stem cells.

Cancer stem cells (CSCs) are tumor cells that share functional characteristics with normal and embryonic stem cells. CSCs have increased tumor-initiating capacity and metastatic potential and lower sensitivity to chemo- and radiotherapy, with important roles in tumor progression and the response to therapy. Thus, a current goal of cancer research is to eliminate CSCs, necessitating an adequate phenotypic and functional characterization of CSCs. Strategies have been developed to identify, enrich, and track CSCs, many of which distinguish CSCs by evaluating the expression of surface markers, the initiation of specific signaling pathways, and the activation of master transcription factors that control stemness in normal cells. We review and discuss the use of reporter gene systems for identifying CSCs. Reporters that are under the control of aldehyde dehydrogenase 1A1, CD133, Notch, Nanog homeobox, Sex-determining region Y-box 2, and POU class 5 homeobox can be used to identify CSCs in many tumor types, track cells in real time, and screen for drugs. Thus, reporter gene systems, in combination with in vitro and in vivo functional assays, can assess changes in the CSCs pool. We present relevant examples of these systems in the evaluation of experimental CSCs-targeting therapeutics, demonstrating their value in CSCs research.

Exosomal miR-1246 from glioma patient body fluids drives the differentiation and activation of myeloid-derived suppressor cells

Glioma is a heterogeneous cellular environment in which immune cells play critical roles in tumor progression. Myeloid-derived suppressor cells (MDSCs) contribute to the formation of the immunosuppressive microenvironment of glioma; however, how glioma cells interact with MDSCs and how this interaction affects the function of other immune cells are unclear. Glioma cells can systemically communicate with immune cells via the secretion of exosomes, which contain microRNAs (miRNAs). Leveraging miRNA sequencing of exosomes, we identified enrichment of miR-1246 in glioma-derived exosomes and exosomes isolated from the cerebrospinal fluid (CSF) of glioma patients. We demonstrated that miR-1246 drives the differentiation and activation of MDSCs in a dual specificity phosphatase 3 (DUSP3)/extracellular signal‑regulated kinase (ERK)-dependent manner. In addition, postoperative CSF exosomal miR-1246 expression was found to be associated with the glioma recurrence rate. Hypoxia, a well-recognized feature of the glioblastoma microenvironment, increased miR-1246 levels in glioma-derived exosomes by enhancing miR-1246 transcription and selective packaging via upregulation of POU class 5 homeobox 1 (POU5F1) and heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1). Importantly, we identified a mechanism of 2-methoxyestradiol, a microtubule inhibitor currently undergoing clinical trials for glioblastoma. 2-Methoxyestradiol suppresses MDSC activation by inhibiting hypoxia-driven exosomal miR-1246 expression in glioma cells and PD-L1 expression in MDSCs.

A Missense POU4F3 Variant Associated with Autosomal Dominant Midfrequency Hearing Loss Alters Subnuclear Localization and Transcriptional Capabilities.

Background The pathogenic variant, POU class 4 transcription factor 3 (POU4F3), is reported to cause autosomal dominant nonsyndromic hearing loss (ADNSHL). Previously, we have examined a four-generation midfrequency sensorineural hearing loss (MFSNHL) family (no. 6126) and established POU4F3 c.602T>C (p.Leu201Pro) as a potential disease-causing variant. Objectives We explored the structural and functional alterations that the c.602T>C (p.Leu201Pro) variant enforces on the POU4F3 protein. Methods We utilized wild-type (WT) and mutant (MUT) POU4F3 c.602T>C plasmid incorporation into HeLa cells to assess functional changes, by immunofluorescence and luciferase assays. To predict protein structural alterations in the MUT versus WT POU4F3, we also generated 3D structures to compare both types of POU4F3 proteins. Results The WT POU4F3 is ubiquitously present in the nucleus, whereas the MUT form of POU4F3 exhibits a more restricted nuclear presence. This finding is different from other publications, which report a cytoplasmic localization of the MUT POU4F3. We also demonstrated that, as opposed to WT POU4F3, the MUT POU4F3 had 40% reduced luciferase activity. Conclusions The reduced nuclear presence, combined with reduced transcriptional activity, suggests that the POU4F3 c.602T>C variant alters cellular activity and may contribute to the pathogenicity of POU4F3-related hearing loss. It, also, provides more evidence of the pathophysiological characteristics of MFSNHL.

Long Non-Coding RNA ARAP1-AS1 Facilitates the Progression of Cervical Cancer by Regulating miR-149-3p and POU2F2

Background: Emerging research has demonstrated that long non-coding RNAs (lncRNAs) attach great importance to the progression of cervical cancer (CC). LncRNA ARAP1-AS1 was involved in the development of several cancers; however, its role in CC is far from being elucidated. Methods: Real-time PCR (RT-PCR) was employed to detect ARAP1-AS1 and miR-149-3p expression in CC samples. CC cell lines (HeLa and C33A cells) were regarded as the cell models. The biological effect of ARAP1-AS1 on cancer cells was measured using CCK-8 assay, colony formation assay, flow cytometry, Transwell assay and wound healing assay in vitro, and subcutaneous xenotransplanted tumor model and tail vein injection model in vivo. Furthermore, interactions between ARAP1-AS1 and miR-149-3p, miR-149-3p and POU class 2 homeobox 2 (POU2F2) were determined by bioinformatics analysis, qRT-PCR, Western blot, luciferase reporter and RNA immunoprecipitation assay, respectively. Results: The expression of ARAP1-AS1 was enhanced in CC samples, while miR-149-3p was markedly suppressed. Additionally, ARAP1-AS1 overexpression enhanced the viability, migration, and invasion of CC cells. ARAP1-AS1 downregulated miR-149-3p via sponging it. ARAP1-AS1 and miR-149-3p exhibited a negative correlation in CC samples. On the other hand, ARAP1-AS1 enhanced the expression of POU2F2, which was validated as a target gene of miR-149-3p. Conclusion: ARAP1-AS1 was abnormally upregulated in CC tissues and indirectly modulated the POU2F2 expression via reducing miR-149-3p expression. Our study identified a novel axis, ARAP1-AS1/miR-149-3p/POU2F2, in CC tumorigenesis.

POU2F2 regulates glycolytic reprogramming and glioblastoma progression via PDPK1-dependent activation of PI3K/AKT/mTOR pathway

The POU Class Homeobox 2 (POU2F2) is a member of POU transcription factors family, which involves in cell immune response by regulating B cell proliferation and differentiation genes. Recent studies have shown that POU2F2 acts as tumor-promoting roles in some cancers, but the underlying mechanism remains little known. Here, we identified that the highly expressed POU2F2 significantly correlated with poor prognosis of glioblastoma (GBM) patients. POU2F2 promoted cell proliferation and regulated glycolytic reprogramming. Mechanistically, the AKT/mTOR signaling pathway played important roles in the regulation of POU2F2-mediated aerobic glycolysis and cell growth. Furthermore, we demonstrated that POU2F2 activated the transcription of PDPK1 by directly binding to its promoter. Reconstituted the expression of PDPK1 in POU2F2-knockdown GBM cells reactivated AKT/mTOR pathway and recovered cell glycolysis and proliferation, whereas this effect was abolished by the PDPK1/AKT interaction inhibitor. In addition, we showed that POU2F2-PDPK1 axis promoted tumorigenesis by regulating glycolysis in vivo. In conclusion, our findings indicate that POU2F2 leads a metabolic shift towards aerobic glycolysis and promotes GBM progression in PDPK1-dependent activation of PI3K/AKT/mTOR pathway.

Generation of a common marmoset embryonic stem cell line CMES40-OC harboring a POU5F1 (OCT4)-2A-mCerulean3 knock-in reporter allele

POU class 5 homeobox 1 (POU5F1, also known as OCT4) is critical for maintenance of pluripotency, germ cell fate, reprogramming into a pluripotent state, and early embryogenesis. We generated an embryonic stem cell (ESC) line of the common marmoset (Callithrix jacchus) harboring a heterozygous knock-in allele of OCT4-P2A-mCerulean-T2A-pac. The ESC line (CMES40-OC) will be valuable for investigation of primed/naïve pluripotency and germ cell fate. Homozygous OCT4 knock-in clones were generated but could not be sustained in an undifferentiated state in long-term culture. The OCT4 knock-in system facilitated simultaneous knock-in of a reporter construct at another locus, DDX4 (VASA).

Disturbed progesterone signalling in an advanced preclinical model of endometriosis

Research questionDo human endometriosis organoids recapitulate aberrant progesterone signalling in the disease to serve as advanced experimental models for uncovering epigenetic mechanisms involved in attenuated progesterone response in endometriosis? DesignInitially, the organoids were established from acquired biopsies (women with and without endometriosis) and characterized by morphological, histological and immunostaining analyses. Results: A panel of endometriosis-related genes showed a pattern of expressions in cytochrome c oxidase subunit II (COX2), matrix metalloproteinase 2 (MMP2), MMP9, tissue inhibitor of metalloproteinase-3 (TIMP3), transforming growth factor beta 1 (TGF-β1), and zinc finger E-box binding homeobox 1 (ZEB1), and a contradictory expression pattern for cadherin (CDH1), POU class 5 homeobox 1 (POU5F1; also known as OCT4), and Nanog homeobox (NANOG) in the endometriosis organoids that is concordant with published research. These endometriosis organoids failed to upregulate 17β-Hydroxysteroid dehydrogenase 2 (17HSDβ2), progestogen associated endometrial protein (PAEP), secreted phosphoprotein 1 (SPP1), and leukaemia inhibitory factor (LIF) in response to progesterone at the level observed in control endometrium organoids. Progesterone receptor B (PRB) gene expression significantly decreased in both eutopic and ectopic organoids compared with control endometrium organoids. DNA hypermethylation, as an epigenetic mechanism for suppression of transcription, was detected at the PRB promoter in the eutopic, but not ectopic, organoids. Therefore, other epigenetic mechanisms, such as histone modifications and microRNAs, may be responsible for PRB downregulation in ectopic organoids. ConclusionsEndometriosis organoids are powerful preclinical models that can be used to investigate the molecular mechanisms involved in endometriosis-associated progesterone resistance.

Comparative analysis of mouse and human preimplantation development following POU5F1 CRISPR/Cas9 targeting reveals interspecies differences

What is the role of POU class 5 homeobox 1 (POU5F1) in human preimplantation development and how does it compare with the mouse model? POU5F1 is required for successful development of mouse and human embryos to the blastocyst stage as knockout embryos exhibited a significantly lower blastocyst formation rate, accompanied by lack of inner cell mass (ICM) formation. Clustered regularly interspaced short palindromic repeats-CRISPR associated genes (CRISPR-Cas9) has previously been used to examine the role of POU5F1 during human preimplantation development. The reported POU5F1-targeted blastocysts always retained POU5F1 expression in at least one cell, because of incomplete CRISPR-Cas9 editing. The question remains of whether the inability to obtain fully edited POU5F1-targeted blastocysts in human results from incomplete editing or the actual inability of these embryos to reach the blastocyst stage. The efficiency of CRISPR-Cas9 to induce targeted gene mutations was first optimized in the mouse model. Two CRISPR-Cas9 delivery methods were compared in the B6D2F1 strain: S-phase injection (zygote stage) (n = 135) versus metaphase II-phase (M-phase) injection (oocyte stage) (n = 23). Four control groups were included: non-injected media-control zygotes (n = 43)/oocytes (n = 48); sham-injected zygotes (n = 45)/oocytes (n = 47); Cas9-protein injected zygotes (n = 23); and Cas9 protein and scrambled guide RNA (gRNA)-injected zygotes (n = 27). Immunofluorescence analysis was performed in Pou5f1-targeted zygotes (n = 37), media control zygotes (n = 19), and sham-injected zygotes (n = 15). To assess the capacity of Pou5f1-null embryos to develop further in vitro, additional groups of Pou5f1-targeted zygotes (n = 29) and media control zygotes (n = 30) were cultured to postimplantation stages (8.5 dpf). Aiming to identify differences in developmental capacity of Pou5f1-null embryos attributed to strain variation, zygotes from a second mouse strain-B6CBA (n = 52) were targeted. Overall, the optimized methodology was applied in human oocytes following IVM (metaphase II stage) (n = 101). The control group consisted of intracytoplasmically sperm injected (ICSI) IVM oocytes (n = 33). Immunofluorescence analysis was performed in human CRISPR-injected (n = 10) and media control (n = 9) human embryos. A gRNA-Cas9 protein mixture targeting exon 2 of Pou5f1/POU5F1 was microinjected in mouse oocytes/zygotes or human IVM oocytes. Reconstructed embryos were cultured for 4 days (mouse) or 6.5 days (human) in sequential culture media. An additional group of mouse-targeted zygotes was cultured to postimplantation stages. Embryonic development was assessed daily, with detailed scoring at late blastocyst stage. Genomic editing was assessed by immunofluorescence analysis and next-generation sequencing. Genomic analysis in mouse revealed very high editing efficiencies with 95% of the S-Phase and 100% of the M-Phase embryos containing genetic modifications, of which 89.47% in the S-Phase and 84.21% in the M-Phase group were fully edited. The developmental capacity was significantly compromised as only 46.88% embryos in the S-Phase and 19.05% in the M-Phase group reached the blastocyst stage, compared to 86.36% in control M-Phase and 90.24% in control S-Phase groups, respectively. Immunofluorescence analysis confirmed the loss of Pou5f1 expression and downregulation of the primitive marker SRY-Box transcription factor (Sox17). Our experiments confirmed the requirement of Pou5f1 expression for blastocyst development in the second B6CBA strain. Altogether, our data obtained in mouse reveal that Pou5f1 expression is essential for development to the blastocyst stage. M-Phase injection in human IVM oocytes (n = 101) similarly resulted in 88.37% of the POU5F1-targeted embryos being successfully edited. The developmental capacity of generated embryos was compromised from the eight-cell stage onwards. Only 4.55% of the microinjected embryos reached the late blastocyst stage and the embryos exhibited complete absence of ICM and an irregular trophectoderm cell layer. Loss of POU5F1 expression resulted in absence of SOX17 expression, as in mouse. Interestingly, genetic mosaicism was eliminated in a subset of targeted human embryos (9 out of 38), three of which developed into blastocysts. One of the major hurdles of CRISPR-Cas9 germline genome editing is the occurrence of mosaicism, which may complicate phenotypic analysis and interpretation of developmental behavior of the injected embryos. Furthermore, in this study, spare IVM human oocytes were used, which may not recapitulate the developmental behavior of in vivo matured oocytes. Comparison of developmental competency following CRISPR-Cas-mediated gene targeting in mouse and human may be influenced by the selected mouse strain. Gene targeting by CRISPR-Cas9 is subject to variable targeting efficiencies. Therefore, striving to reduce mosaicism can provide novel molecular insights into mouse and human embryogenesis. The research was funded by the Ghent University Hospital and Ghent University and supported by the FWO-Vlaanderen (Flemish fund for scientific research, Grant no. G051516N), and Hercules funding (FWO.HMZ.2016.00.02.01). The authors declare no competing interests. N/A.