IGF2BP2 Maintains Retinal Pigment Epithelium Homeostasis by Stabilizing PAX6 and OTX2.

Purpose: N6-methyladenosine (m6A) methylation is a chemical modification that occurs on RNA molecules, where the hydrogen atom of adenine (A) nucleotides is replaced by a methyl group, forming N6-methyladenosine. This modification is a dynamic and reversible process that plays a crucial role in regulating various biological processes, including RNA stability, transport, translation, and degradation. Currently, there is a lack of research on the role of m6A modifications in maintaining the characteristics of RPE cells. m6A readers play a crucial role in executing the functions of m6A modifications, which prompted our investigation into their regulatory roles in the RPE. Methods: Phagocytosis assays, immunofluorescence staining, flow cytometry experiments, ß-galactosidase staining, and RNA sequencing (RNA-seq) were conducted to assess the functional and cellular characteristics changes in retinal pigment epithelium (RPE) cells following short-hairpin RNA-mediated knockdown of insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2). RNA-seq and ultraviolet crosslinking immunoprecipitation with high-throughput sequencing (HITS-CLIP) were employed to identify the target genes regulated by IGF2BP2. adeno-associated virus (AAV) subretinal injection was performed in 6- to 8-week-old C57 mice to reduce IGF2BP2 expression in the RPE, and the impact of IGF2BP2 knockdown on mouse visual function was assessed using immunofluorescence, quantitative real-time PCR, optical coherence tomography, and electroretinography. Results: IGF2BP2 was found to have a pronounced effect on RPE phagocytosis. Subsequent in-depth exploration revealed that IGF2BP2 modulates the mRNA stability of PAX6 and OTX2, and the loss of IGF2BP2 induces inflammatory and aging phenotypes in RPE cells. IGF2BP2 knockdown impaired RPE function, leading to retinal dysfunction in vivo. Conclusions: Our data suggest a crucial role of IGF2BP2 as an m6A reader in maintaining RPE homeostasis by regulating the stability of PAX6 and OTX2, making it a potential target for preventing the occurrence of retinal diseases related to RPE malfunction.

PAX6 promotes neuroendocrine phenotypes of prostate cancer via enhancing MET/STAT5A-mediated chromatin accessibility.

BACKGROUND: Neuroendocrine prostate cancer (NEPC) is a lethal subset of prostate cancer which is characterized by neuroendocrine differentiation and loss of androgen receptor (AR) signaling. Growing evidence reveals that cell lineage plasticity is crucial in the failure of NEPC therapies. Although studies suggest the involvement of the neural transcription factor PAX6 in drug resistance, its specific role in NEPC remains unclear. METHODS: The expression of PAX6 in NEPC was identified via bioinformatics and immunohistochemistry. CCK8 assay, colony formation assay, tumorsphere formation assay and apoptosis assay were used to illustrate the key role of PAX6 in the progression of in vitro. ChIP and Dual-luciferase reporter assays were conducted to confirm the binding sequences of AR in the promoter region of PAX6, as well as the binding sequences of PAX6 in the promoter regions of STAT5A and MET. For in vivo validation, the xenograft model representing NEPC subtype underwent pathological analysis to verify the significant role of PAX6 in disease progression. Complementary diagnoses were established through public clinical datasets and transcriptome sequencing of specific cell lines. ATAC-seq was used to detect the chromatin accessibility of specific cell lines. RESULTS: PAX6 expression was significantly elevated in NEPC and negatively regulated by AR signaling. Activation of PAX6 in non-NEPC cells led to NE trans-differentiation, while knock-down of PAX6 in NEPC cells inhibited the development and progression of NEPC. Importantly, loss of AR resulted in an enhanced expression of PAX6, which reprogramed the lineage plasticity of prostate cancer cells to develop NE phenotypes through the MET/STAT5A signaling pathway. Through ATAC-seq, we found that a high expression level of PAX6 elicited enhanced chromatin accessibility, mainly through attenuation of H4K20me3, which typically causes chromatin silence in cancer cells. CONCLUSION: This study reveals a novel neural transcription factor PAX6 could drive NEPC progression and suggest that it might serve as a potential therapeutic target for the management of NEPC.

PAX6/CXCL14 regulatory axis promotes the repair of corneal injury by enhancing corneal epithelial cell proliferation.

BACKGROUND: Corneal injuries, often leading to severe vision loss or blindness, have traditionally been treated with the belief that limbal stem cells (LSCs) are essential for repair and homeostasis, while central corneal epithelial cells (CCECs) were thought incapable of such repair. However, our research reveals that CCECs can fully heal and maintain the homeostasis of injured corneas in rats, even without LSCs. We discovered that CXCL14, under PAX6's influence, significantly boosts the stemness, proliferation, and migration of CCECs, facilitating corneal wound healing and homeostasis. This finding introduces CXCL14 as a promising new drug target for corneal injury treatment. METHODS: To investigate the PAX6/CXCL14 regulatory axis's role in CCECs wound healing, we cultured human corneal epithelial cell lines with either increased or decreased expression of PAX6 and CXCL14 using adenovirus transfection in vitro. Techniques such as coimmunoprecipitation, chromatin immunoprecipitation, immunofluorescence staining, western blot, real-time PCR, cell colony formation, and cell cycle analysis were employed to validate the axis's function. In vivo, a rat corneal epithelial injury model was developed to further confirm the PAX6/CXCL14 axis's mechanism in repairing corneal damage and maintaining corneal homeostasis, as well as to assess the potential of CXCL14 protein as a therapeutic agent for corneal injuries. RESULTS: Our study reveals that CCECs naturally express high levels of CXCL14, which is significantly upregulated by PAX6 following corneal damage. We identified SDC1 as CXCL14's receptor, whose engagement activates the NF-κB pathway to stimulate corneal repair by enhancing the stemness, proliferative, and migratory capacities of CCECs. Moreover, our research underscores CXCL14's therapeutic promise for corneal injuries, showing that recombinant CXCL14 effectively accelerates corneal healing in rat models. CONCLUSION: CCECs play a critical and independent role in the repair of corneal injuries and the maintenance of corneal homeostasis, distinct from that of LSCs. The PAX6/CXCL14 regulatory axis is pivotal in this process. Additionally, our research demonstrates that the important function of CXCL14 in corneal repair endows it with the potential to be developed into a novel therapeutic agent for treating corneal injuries.

A conserved molecular logic for neurogenesis to gliogenesis switch in the cerebral cortex.

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1, Egfr, and Olig2. The increased Ascl1 expression and appearance of Egfr+ and Olig2+ cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.

Experimental interventions attenuate a conjunctival epidermal metaplasia model.

The conjunctiva is a non-keratinized, stratified columnar epithelium with characteristics different from the cornea and eyelid epidermis. From development to adulthood, a distinguishing feature of ocular versus epidermal epithelia is the expression of the master regulator PAX6. A conditionally immortalized conjunctival epithelial cell line (iHCjEC) devoid of stromal or immune cells established in our laboratory spontaneously manifested epidermal metaplasia and upregulated expression of the keratinization-related genes SPRR1A/B and the epidermal cytokeratins KRT1 and KRT10 at the expense of the conjunctival trait. In addition, iHCjEC indicated a significant decrease in PAX6 expression. Dry eye syndrome (DES) and severe ocular surface diseases, such as Sjögren's syndrome and Stevens-Johnson syndrome, cause the keratinization of the entire ocular surface epithelia. We used iHCjECs as a conjunctiva epidermal metaplasia model to test PAX6, serum, and glucocorticoid interventions. Reintroducing PAX6 to iHCjECs resulted in upregulating genes related to cell adhesion and tight junctions, including MIR200CHG and CLDN1. The administration of glucocorticoids or serum resulted in the downregulation of epidermal genes (DSG1, SPRR1A/B, and KRT1) and partially corrected epidermal metaplasia. Our results using an isolated conjunctival epidermal metaplasia model point toward the possibility of rationally "repurposing" clinical interventions, such as glucocorticoid, serum, or PAX6 administration, for treating epidermal metaplasia of the conjunctiva.

PAX6 Expression Patterns in the Adult Human Limbal Stem Cell Niche.

Paired box 6 (PAX6), a nuclear transcription factor, determines the fate of limbal epithelial progenitor cells (LEPC) and maintains epithelial cell identity. However, the expression of PAX6 in limbal niche cells, primarily mesenchymal stromal cells (LMSC), and melanocytes is scarce and not entirely clear. To distinctly assess the PAX6 expression in limbal niche cells, fresh and organ-cultured human corneoscleral tissues were stained immunohistochemically. Furthermore, the expression of PAX6 in cultured limbal cells was investigated. Immunostaining revealed the presence of PAX6-negative cells which were positive for vimentin and the melanocyte markers Melan-A and human melanoma black-45 in the basal layer of the limbal epithelium. PAX6 staining was not observed in the limbal stroma. Moreover, the expression of PAX6 was observed by Western blot in cultured LEPC but not in cultured LMSC or LM. These data indicate a restriction of PAX6 expression to limbal epithelial cells at the limbal stem cell niche. These observations warrant further studies for the presence of other PAX isoforms in the limbal stem cell niche.

GSK126 an inhibitor of epigenetic regulator EZH2 suppresses cardiac fibrosis by regulating the EZH2-PAX6-CXCL10 pathway.

Myocardial fibrosis is a common pathological companion of various cardiovascular diseases. To date, the role of enhancer of zeste homolog 2 (EZH2) in cancer has been well demonstrated including in renal carcinoma and its inhibitors have entered the stage of phase I/II clinical trials. However, the precise mechanism of EZH2 in cardiac diseases is largely unclear. In the current study, we first found that EZH2 expression was increased in Ang-II-treated cardiac fibroblasts (CFs) and mouse heart homogenates following isoproterenol (ISO) administration for 21 days, respectively. Ang-II induces CFs activation and increased collagen-I, collagen-III, α-SMA, EZH2, and trimethylates lysine 27 on histone 3 (H3K27me3) expressions can be reversed by EZH2 inhibitor (GSK126) and EZH2 siRNA. The ISO-induced cardiac hypertrophy, and fibrosis in vivo which were also related to the upregulation of EZH2 and its downstream target, H3K27me3, could be recovered by GSK126. Furthermore, the upregulation of EZH2 induces the decrease of paired box 6 (PAX6) and C-X-C motif ligand 10 (CXCL10) "which" were also reversed by GSK126 treatment. In summary, the present evidence strongly suggests that GSK126 could be a therapeutic intervention, blunting the development and progression of myocardial fibrosis in an EZH2-PAX6-CXCL10-dependent manner.

Co-expression and Interaction of Pax6 with Genes and Proteins of Immunological Surveillance in the Brain of Mice.

The Pax6 binds to promoter sequence elements of genes involved in immunological surveillance and interacts with Iba1, p53, Ras-GAP, and Sparc in the brain of mice. The Pax6 also affects the expression pattern of genes involved in neurogenesis and neurodegeneration. However, the expression and association of Pax6 in the brain under immunologically challenged conditions are still elusive. Therefore, it has been intended to analyze the association of Pax6 in the immunity of the brain using the immune-challenged Dalton's lymphoma (DL) mice model. The expressions of Pax6, Iba1, and Tmem119 decreased, but expressions of Ifn-γ, Tnf-α, Bdnf, and Tgf-ß increased in the brain of immune-challenged mice as compared to the control. The level of co-expression of Pax6 decreased in dual positive cells with Iba1, Tmem119, Sparc, p53, Bdnf, and Tgf-ß in the brain of immune-challenged mice. Binding of Pax6 to multiple sites of the promoter sequences of Bdnf and Tgf-ß indicates their Pax6-associated differential expression and association with immune responsive gene. The levels of binding of Pax6 to Tmem119, Iba1, Ifn-γ, and Tnf-α got altered during the immune-challenged state as compared to control. Results provide the first evidence of the association of Pax6 in brain-specific immunity.

Thirty Years' History since the Discovery of Pax6: From Central Nervous System Development to Neurodevelopmental Disorders.

Pax6 is a sequence-specific DNA binding transcription factor that positively and negatively regulates transcription and is expressed in multiple cell types in the developing and adult central nervous system (CNS). As indicated by the morphological and functional abnormalities in spontaneous Pax6 mutant rodents, Pax6 plays pivotal roles in various biological processes in the CNS. At the initial stage of CNS development, Pax6 is responsible for brain patterning along the anteroposterior and dorsoventral axes of the telencephalon. Regarding the anteroposterior axis, Pax6 is expressed inversely to Emx2 and Coup-TF1, and Pax6 mutant mice exhibit a rostral shift, resulting in an alteration of the size of certain cortical areas. Pax6 and its downstream genes play important roles in balancing the proliferation and differentiation of neural stem cells. The Pax6 gene was originally identified in mice and humans 30 years ago via genetic analyses of the eye phenotypes. The human PAX6 gene was discovered in patients who suffer from WAGR syndrome (i.e., Wilms tumor, aniridia, genital ridge defects, mental retardation). Mutations of the human PAX6 gene have also been reported to be associated with autism spectrum disorder (ASD) and intellectual disability. Rodents that lack the Pax6 gene exhibit diverse neural phenotypes, which might lead to a better understanding of human pathology and neurodevelopmental disorders. This review describes the expression and function of Pax6 during brain development, and their implications for neuropathology.

lncRNA ZFPM2-AS1 promotes retinoblastoma progression by targeting microRNA miR-511-3p/paired box protein 6 (PAX6) axis.

Long non-coding RNAs (lncRNAs) have been shown to play crucial roles in retinoblastoma progression. In this study, we aimed to investigate the mechanism of lncRNA ZFPM2-AS1 (ZFPM2-AS1) in retinoblastoma progression. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blotting assays were performed to determine the expression of lncRNA, microRNA (miRNA), mRNA, and protein. The changes in cell proliferation, apoptosis, and cell migration were assessed by functional experiments. The interaction between ZFPM2-AS1, miR-511-3p, and paired box protein 6 (PAX6) was confirmed by a luciferase assay. Our study found that ZFPM2-AS1 and PAX6 were upregulated, whereas miR-511-3p was downregulated in retinoblastoma. ZFPM2-AS1 inhibition decreased the viability and migration of retinoblastoma cells. We also found that ZFPM2-AS1 targets miR-511-3p to upregulate PAX6 in Y79 and SO-RB50 cells. Moreover, we demonstrated that inhibiting miR-511-3p reversed the negative effects of silencing ZFPM2-AS1 and PAX6 on retinoblastoma cell viability and migration. In conclusion, retinoblastoma development is regulated by the ZFPM2-AS1/511-3p/PAX6 axis.

G-quadruplex DNA structures in human stem cells and differentiation.

The establishment of cell identity during embryonic development involves the activation of specific gene expression programmes and is underpinned by epigenetic factors including DNA methylation and histone post-translational modifications. G-quadruplexes are four-stranded DNA secondary structures (G4s) that have been implicated in transcriptional regulation and cancer. Here, we show that G4s are key genomic structural features linked to cellular differentiation. We find that G4s are highly abundant in human embryonic stem cells and are lost during lineage specification. G4s are prevalent in enhancers and promoters. G4s that are found in common between embryonic and downstream lineages are tightly linked to transcriptional stabilisation of genes involved in essential cellular functions as well as transitions in the histone post-translational modification landscape. Furthermore, the application of small molecules that stabilise G4s causes a delay in stem cell differentiation, keeping cells in a more pluripotent-like state. Collectively, our data highlight G4s as important epigenetic features that are coupled to stem cell pluripotency and differentiation.

Unilateral zebrafish corneal injury induces bilateral cell plasticity supporting wound closure.

The cornea, transparent and outermost structure of camera-type eyes, is prone to environmental challenges, but has remarkable wound healing capabilities which enables to preserve vision. The manner in which cell plasticity impacts wound healing remains to be determined. In this study, we report rapid wound closure after zebrafish corneal epithelium abrasion. Furthermore, by investigating the cellular and molecular events taking place during corneal epithelial closure, we show the induction of a bilateral response to a unilateral wound. Our transcriptomic results, together with our TGF-beta receptor inhibition experiments, demonstrate conclusively the crucial role of TGF-beta signaling in corneal wound healing. Finally, our results on Pax6 expression and bilateral wound healing, demonstrate the decisive impact of epithelial cell plasticity on the pace of healing. Altogether, our study describes terminally differentiated cell competencies in the healing of an injured cornea. These findings will enhance the translation of research on cell plasticity to organ regeneration.

KLF3 and PAX6 are candidate driver genes in late-stage, MSI-hypermutated endometrioid endometrial carcinomas.

Endometrioid endometrial carcinomas (EECs) are the most common histological subtype of uterine cancer. Late-stage disease is an adverse prognosticator for EEC. The purpose of this study was to analyze EEC exome mutation data to identify late-stage-specific statistically significantly mutated genes (SMGs), which represent candidate driver genes potentially associated with disease progression. We exome sequenced 15 late-stage (stage III or IV) non-ultramutated EECs and paired non-tumor DNAs; somatic variants were called using Strelka, Shimmer, SomaticSniper and MuTect. Additionally, somatic mutation calls were extracted from The Cancer Genome Atlas (TCGA) data for 66 late-stage and 270 early-stage (stage I or II) non-ultramutated EECs. MutSigCV (v1.4) was used to annotate SMGs in the two late-stage cohorts and to derive p-values for all mutated genes in the early-stage cohort. To test whether late-stage SMGs are statistically significantly mutated in early-stage tumors, q-values for late-stage SMGs were re-calculated from the MutSigCV (v1.4) early-stage p-values, adjusting for the number of late-stage SMGs tested. We identified 14 SMGs in the combined late-stage EEC cohorts. When the 14 late-stage SMGs were examined in the TCGA early-stage data, only Krüppel-like factor 3 (KLF3) and Paired box 6 (PAX6) failed to reach significance as early-stage SMGs, despite the inclusion of enough early-stage cases to ensure adequate statistical power. Within TCGA, nonsynonymous mutations in KLF3 and PAX6 were, respectively, exclusive or nearly exclusive to the microsatellite instability (MSI)-hypermutated molecular subgroup and were dominated by insertions-deletions at homopolymer tracts. In conclusion, our findings are hypothesis-generating and suggest that KLF3 and PAX6, which encode transcription factors, are MSI target genes and late-stage-specific SMGs in EEC.

miR-130a-3p Enhances the Chemosensitivity of Y79 Retinoblastoma Cells to Vincristine by Targeting PAX6 Expression.

PURPOSE: Chemoresistance remains the primary obstacle threatening the prognosis of retinoblastoma (RB). microRNAs (miRNAs) are acknowledged as critical regulators of drug resistance. This study explored the molecular mechanism of miR-130a-3p affecting the chemosensitivity of RB to vincristine (VCR). METHODS: miR-130a-3p expression of human retinal astrocytes and RB cell lines (Y79, WERI-Rb-1, SO-Rb50, and SO-Rb70) was detected using RT-qPCR. VCR-resistant RB cell line Y79/VCR was induced. miR-130a-3p expression of Y79/VCR cell line and its corresponding parental cell line was detected. Y79/VCR cells were subjected to miR-130a-3p overexpression treatment. The cell proliferation was measured using MTT assay, and the IC50 value and drug resistance index were examined using CCK-8 assay. The targeting relationship between miR-130a-3p and PAX6 was predicted through bioinformatics analysis and verified using dual-luciferase assay. Functional rescue experiments were conducted to confirm the role of PAX6 in chemosensitivity of RB cells. The effect of miR-130a-3p on tumorigenesis and VCR sensitivity was observed in vivo. RESULTS: miR-130a-3p was downregulated in VCR-resistant RB cells. Overexpression of miR-130a-3p repressed the proliferation of Y79/VCR cells and enhanced chemosensitivity. miR-130a-3p targeted PAX6 expression. Overexpression of PAX6 reversed the effect of miR-130a-3p on chemosensitivity of Y79/VCR cells. Overexpression of miR-130a-3p suppressed tumor growth and reduced VCR resistance in vivo. CONCLUSIONS: miR-130a-3p enhanced the chemosensitivity of Y79 RB cells to VCR by targeting PAX6 expression.

Decreased FABP5 and DSG1 protein expression following PAX6 knockdown of differentiated human limbal epithelial cells.

PAX6 haploinsufficiency related aniridia is characterized by disorder of limbal epithelial cells (LECs) and aniridia related keratopathy. In the limbal epithelial cells of aniridia patients, deregulated retinoic acid (RA) signaling components were identified. We aimed to visualize differentiation marker and RA signaling component expression in LECs, combining a differentiation triggering growth condition with a small interfering RNA (siRNA) based aniridia cell model (PAX6 knock down). Primary LECs were isolated from corneoscleral rims of healthy donors and cultured in serum free low Ca2+ medium (KSFM) and in KSFM supplemented with 0.9 mmol/L Ca2+. In addition, LECs were treated with siRNA against PAX6. DSG1, PAX6, KRT12, KRT 3, ADH7, RDH10, ALDH1A1, ALDH3A1, STRA6, CYP1B1, RBP1, CRABP2, FABP5, PPARG, VEGFA and ELOVL7 expression was determined using qPCR and western blot. DSG1, FABP5, ADH7, ALDH1A1, RBP1, CRABP2 and PAX6 mRNA and FABP5 protein expression increased (p ≤ 0.03), PPARG, CYP1B1 mRNA expression decreased (p ≤ 0.0003) and DSG1 protein expression was only visible after Ca2+ supplementation. After PAX6 knock down and Ca2+ supplementation, ADH7 and ALDH1A1 mRNA and DSG1 and FABP5 protein expression decreased (p ≤ 0.04), compared to Ca2+ supplementation alone. Using our cell model, with Ca2+ supplementation and PAX6 knockdown with siRNA treatment against PAX6, we provide evidence that haploinsufficiency of the master regulatory gene PAX6 contributes to differentiation defect in the corneal epithelium through alterations of RA signalling. Upon PAX6 knockdown, DSG1 differentiation marker and FABP5 RA signaling component mRNA expression decreases. A similar effect becomes apparent at protein level though differentiation triggering Ca2+ supplementation in the siRNA-based aniridia cell model. Expression data from this cell model and from our siRNA aniridia cell model strongly indicate that FABP5 expression is PAX6 dependent. These new findings may lead to a better understanding of differentiation processes in LECs and are able to explain the insufficient cell function in AAK.

Transcriptional network orchestrating regional patterning of cortical progenitors.

We uncovered a transcription factor (TF) network that regulates cortical regional patterning in radial glial stem cells. Screening the expression of hundreds of TFs in the developing mouse cortex identified 38 TFs that are expressed in gradients in the ventricular zone (VZ). We tested whether their cortical expression was altered in mutant mice with known patterning defects (Emx2, Nr2f1, and Pax6), which enabled us to define a cortical regionalization TF network (CRTFN). To identify genomic programming underlying this network, we performed TF ChIP-seq and chromatin-looping conformation to identify enhancer-gene interactions. To map enhancers involved in regional patterning of cortical progenitors, we performed assays for epigenomic marks and DNA accessibility in VZ cells purified from wild-type and patterning mutant mice. This integrated approach has identified a CRTFN and VZ enhancers involved in cortical regional patterning in the mouse.

Exosomal circular RNA hsa_circ_007293 promotes proliferation, migration, invasion, and epithelial-mesenchymal transition of papillary thyroid carcinoma cells through regulation of the microRNA-653-5p/paired box 6 axis.

Circular RNAs (circRNAs) or exosomes have been reported to exert key regulatory and/or communication functions in human cancer. Nevertheless, current literature on the effects of exosomal circRNAs on tumor invasion and metastasis in thyroid cancer is incomplete. The role of tumor-derived exosomes in driving in vitro papillary thyroid carcinoma (PTC) progression and metastasis requires further investigation. In our study, Exosomes were harvested from PTC patient serum and PTC cell culture medium. Gene expression analysis in PTC cell lines and exosomes was performed with quantitative reverse-transcription polymerase chain reaction. Transwell, wound healing, Western blot assays, and the cell counting kit-8 were applied for functional analysis. Dual-luciferase reporter assay was used to examine the interaction between hsa_circ_007293 (circ007293), microRNA (miR)-653-5p, and paired box 6 (PAX6). Results showed that circ007293 was enriched in exosomes derived from PTC patient serum and cell culture media. Moreover, circ007293 could enter PTC cells through exosomes, and exosomal circ007293 promoted PTC cell epithelial-mesenchymal transition, invasion, migration, and proliferation. circ007293 knockdown reversed the malignant phenotype of PTC cells in vitro. Additionally, circ007293 could competitively bind with miR-653-5p to regulate PAX6 expression. Notably, miR-653-5p overexpression or PAX6 inhibition suppressed the malignant effects of exosomal circ007293. These results evidenced that exosomal circ007293 induced EMT and augmented the invasive and migratory abilities of PTC cells via the miR-653-5p/PAX6 axis, suggesting that it may serve as a promising biomarker for cancer progression.

Transcriptional cooperation of PBX1 and PAX6 in adult neural progenitor cells.

PAX6 is a highly conserved transcription factor and key regulator of several neurogenic processes, including the continuous generation of dopaminergic/GABAergic interneurons in the adult ventricular-subventricular (V-SVZ) neurogenic system in mice. Here we report that PAX6 cooperates with the TALE-homeodomain transcription factor PBX1 in this context. Chromatin-immunoprecipitation showed that PBX1 and PAX6 co-occupy shared genomic binding sites in adult V-SVZ stem- and progenitor cell cultures and mouse embryonic stem cells, while depletion of Pbx1 revealed that association of PAX6 with these sites requires the presence of PBX1. Expression profiling together with viral overexpression or knockdown of Pax6 or Pbx1 identified novel PBX1-PAX6 co-regulated genes, including several transcription factors. Computational modeling of genome wide expression identified novel cross-regulatory networks among these very transcription factors. Taken together, the results presented here highlight the intimate link that exists between PAX6 and TALE-HD family proteins and contribute novel insights into how the orchestrated activity of transcription factors shapes adult V-SVZ neurogenesis.

Extracellular vesicles from neurons promote neural induction of stem cells through cyclin D1.

Extracellular vesicles (EVs) are thought to mediate the transport of proteins and RNAs involved in intercellular communication. Here, we show dynamic changes in the buoyant density and abundance of EVs that are secreted by PC12 cells stimulated with nerve growth factor (NGF), N2A cells treated with retinoic acid to induce neural differentiation, and mouse embryonic stem cells (mESCs) differentiated into neuronal cells. EVs secreted from in vitro differentiated cells promote neural induction of mESCs. Cyclin D1 enriched within the EVs derived from differentiated neuronal cells contributes to this induction. EVs purified from cells overexpressing cyclin D1 are more potent in neural induction of mESC cells. Depletion of cyclin D1 from the EVs reduced the neural induction effect. Our results suggest that EVs regulate neural development through sorting of cyclin D1.

Long noncoding RNA PM maintains cerebellar synaptic integrity and Cbln1 activation via Pax6/Mll1-mediated H3K4me3.

Recent studies have shown that long noncoding RNAs (lncRNAs) are critical regulators in the central nervous system (CNS). However, their roles in the cerebellum are currently unclear. In this work, we identified the isoform 204 of lncRNA Gm2694 (designated as lncRNA-Promoting Methylation (lncRNA-PM)) is highly expressed in the cerebellum and derived from the antisense strand of the upstream region of Cerebellin-1 (Cbln1), a well-known critical cerebellar synaptic organizer. LncRNA-PM exhibits similar spatiotemporal expression pattern as Cbln1 in the postnatal mouse cerebellum and activates the transcription of Cbln1 through Pax6/Mll1-mediated H3K4me3. In mouse cerebellum, lncRNA-PM, Pax6/Mll1, and H3K4me3 are all associated with the regulatory regions of Cbln1. Knockdown of lncRNA-PM in cerebellum causes deficiencies in Cbln1 expression, cerebellar synaptic integrity, and motor function. Together, our work reveals an lncRNA-mediated transcriptional activation of Cbln1 through Pax6-Mll1-H3K4me3 and provides novel insights of the essential roles of lncRNA in the cerebellum.